Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.     

Operational concept for the improved synthesis of (R)-3,3'-furoin and related hydrophobic compounds with benzaldehyde lyase

Biphasic reaction systems for enzyme catalysis are an elegant way to overcome limited solubility and stability of reactants and facilitate continuous processes. However, many synthetically useful enzymes are not stable in biphasic systems of water and organic solvent. The entrapment in polymer beads of polyvinyl alcohol has been shown to enable the stable operation of enzymes unstable in conventional biphasic reaction systems. We report the extension of this concept to continuous operation in a fluidised bed reactor. The enzyme benzaldehyde lyase was used for the continuous synthesis of enantiopure (R)-3,3'-furoin. The results show enhanced stability with half-life times under operation conditions of more than 100 h, as well as superior enzyme utilisation in terms of productivity. Furthermore, racemisation and oxidation of the product could be successfully prevented under the non-aqueous and inert reaction conditions.     

Oceanic fronts in the Sargasso Sea control the early life and drift of Atlantic eels

Anguillid freshwater eels show remarkable life histories. In the Atlantic, the European eel (Anguilla anguilla) and American eel (Anguilla rostrata) undertake extensive migrations to spawn in the oceanic Sargasso Sea, and subsequently the offspring drift to foraging areas in Europe and North America, first as leaf-like leptocephali larvae that later metamorphose into glass eels. Since recruitment of European and American glass eels has declined drastically during past decades, there is a strong demand for further understanding of the early, oceanic phase of their life cycle. Consequently, during a field expedition to the eel spawning sites in the Sargasso Sea, we carried out a wide range of dedicated bio-physical studies across areas of eel larval distribution. Our findings suggest a key role of oceanic frontal processes, retaining eel larvae within a zone of enhanced feeding conditions and steering their drift. The majority of the more westerly distributed American eel larvae are likely to follow a westerly/northerly drift route entrained in the Antilles/Florida Currents. European eel larvae are generally believed to initially follow the same route, but their more easterly distribution close to the eastward flowing Subtropical Counter Current indicates that these larvae could follow a shorter, eastward route towards the Azores and Europe. The findings emphasize the significance of oceanic physical–biological linkages in the life-cycle completion of Atlantic eels.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

A high–performance compact filter system treating domestic wastewater

A new compact wastewater treatment system for use in single houses has been constructed in eastern Norway. The system is based on the principles of sub-surface flow constructed wetlands using various types of Filtralite as filter media. It consists of a septic tank followed by an aerobic biofilter succeeded by an upflow saturated filter. The aerobic biofilter is essential to remove organic matter and achieve nitrification, while the upflow filter polishes the wastewater and removes microorganisms and phosphorus. During the first 3 years of operation, the system has show stable and high removal with the following average values measured from the outlet of septic tank to the outlet of the upflow filter: 97.0%-BOD₇, 30%-N, 99.4%-P, and 70.8%-SS. No Escherichia coli or somatic coliphages have been detected in the effluent. Due to considerable removal of organic mater, nutrients, and pathogens, the effluent will not negatively affect water and soil ecosystems. The system requires low maintenance and is designed to remove phosphorus for 5 years before renewal of the upflow filter media. When saturated with phosphorus, the media is a suitable fertilizer for plant production.     

Silver ions disrupt K⁺ homeostasis and cellular integrity in intact barley (Hordeum vulgare L.) roots

The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer (42)K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K(+) fluxes. Doses as low as 5 μM AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) were required to produce similar responses. Reduced influx and enhanced efflux of K(+) resulted in a net loss of >40% of root tissue K(+) during a 15 min application of 500 μM AgNO(3), comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K(+) homeostasis by directly inhibiting K(+) influx at lower concentrations, and indirectly inhibiting K(+) influx and enhancing K(+) efflux, via membrane destruction, at higher concentrations. Ni(2+), Cd(2+), and Pb(2+), three heavy metals not generally known to affect aquaporins, did not enhance K(+) efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application.     

A pharmacological analysis of high-affinity sodium transport in barley (Hordeum vulgare L.): a 24Na+/42K+ study

Soil sodium, while toxic to most plants at high concentrations, can be beneficial at low concentrations, particularly when potassium is limiting. However, little is known about Na(+) uptake in this 'high-affinity' range. New information is provided here with an insight into the transport characteristics, mechanism, and ecological significance of this phenomenon. High-affinity Na(+) and K(+) fluxes were investigated using the short-lived radiotracers (24)Na and (42)K, under an extensive range of measuring conditions (variations in external sodium, and in nutritional and pharmacological agents). This work was supported by electrophysiological, compartmental, and growth analyses. Na(+) uptake was extremely sensitive to all treatments, displaying properties of high-affinity K(+) transporters, K(+) channels, animal Na(+) channels, and non-selective cation channels. K(+), NH(4)(+), and Ca(2+) suppressed Na(+) transport biphasically, yielding IC(50) values of 30, 10, and <5 μM, respectively. Reciprocal experiments showed that K(+) influx is neither inhibited nor stimulated by Na(+). Sodium efflux constituted 65% of influx, indicating a futile cycle. The thermodynamic feasibility of passive channel mediation is supported by compartmentation and electrophysiological data. Our study complements recent advances in the molecular biology of high-affinity Na(+) transport by uncovering new physiological foundations for this transport phenomenon, while questioning its ecological relevance.     

Afforestation does not necessarily reduce nitrous oxide emissions from managed boreal peat soils

Pristine peatlands have generally low nitrous oxide (N₂O) emissions but drainage and management practices enhance the microbial processes and associated N₂O emissions. It is assumed that leaving peat soils from intensive management, such as agriculture, will decrease their N₂O emissions. In this paper we report how the annual N₂O emission rates will change when agricultural peat soil is either left abandoned or afforested and also N₂O emissions from afforested peat extraction sites. In addition, we evaluated a biogeochemical model (DNDC) with a view to explaining GHG emissions from peat soils under different land uses. The abandoned agricultural peat soils had lower mean annual N₂O emissions (5.5?±?5.4?kg?N?ha^?1) than the peat soils in active agricultural use in Finland. Surprisingly, N₂O emissions from afforested organic agricultural soils (12.8?±?9.4?kg?N?ha^?1) were similar to those from organic agricultural soils in active use. These emissions were much higher than those from the forests on nutrient rich peat soils. Abandoned and afforested peat extraction sites emitted more N₂O, (2.4?±?2.1?kg?N?ha^?1), than the areas under active peat extraction (0.7?±?0.5?kg?N?ha^?1). Emissions outside the growing season contributed significantly, 40% on an average, to the annual emissions. The DNDC model overestimated N₂O emission rates during the growing season and indicated no emissions during winter. The differences in the N₂O emission rates were not associated with the age of the land use change, vegetation characteristics, peat depth or peat bulk density. The highest N₂O emissions occurred when the soil C:N ratio was below 20 with a significant variability within the measured C:N range (13–27). Low soil pH, high nitrate availability and water table depth (50–70?cm) were also associated with high N₂O emissions. Mineral soil has been added to most of the soils studied here to improve the fertility and this may have an impact on the N₂O emissions. We infer from the multi-site dataset presented in this paper that afforestation is not necessarily an efficient way to reduce N₂O emissions from drained boreal organic fields.