Two distinct conformational states define the interaction of human RAD51‐ATP with single‐stranded DNA

An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 k_BT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.     

Influence of low temperatures on the growth and photosynthetic activity of industrial chicory, <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. partim

The cold stress effect on early vigour and photosynthesis efficiency was evaluated for five industrial chicory varieties with contrasting early vigour. The relationships between the growth and physiological parameters were assessed. The varieties were examined at three growth temperatures: 16 (reference), 8 (intermediate) and 4 °C (stress). The effect was measured using physiological processes (growth, photosynthesis, chlorophyll a fluorescence), and pigment content. The analysis of the measured growth parameters (dry leaf and root mass, and leaf area) indicated that temperature had a significant effect on the varieties, but the overall reaction of the varieties was similar with lowering temperatures. The photosynthesis and chlorophyll a fluorescence measurements revealed significant changes for the photosynthesis (maximum net photosynthesis, quantum efficiency, light compensation point and dark respiration) and chlorophyll a fluorescence parameters (photochemical and non-photochemical quenching) with lowering temperatures for Hera and Eva, two extremes in youth growth. No significant differences could be found between the extremes for the different temperatures. The pigment content analysis revealed significant differences at 4 °C in contrast to 16 and 8 °C, especially for the xanthophyll/carotenoid pool, suggesting a protective role. Subsequently, the relationship between the physiological processes was evaluated using principal component analysis. At 4 °C, 2 principal components were detected with high discriminating power for the varieties and similar classification of the varieties as determined in the growth analysis. This provides a preview on the possible relationships between photosynthesis and growth for industrial chicory at low temperatures.     

Evaluation of cold stress of young industrial chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.) by chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence imaging. II. Dark relaxation kinetics

Industrial chicory, Cichorium intybus L., has rather poor early vigour under the typical early spring morning conditions of low temperatures and high light intensity. Screening tools are being developed to assess the cold tolerance/sensitivity of young industrial chicory plants under these conditions. Refinement of such tools requires better understanding of the plants’ physiological responses. In this paper we discuss the effects of growth temperature (GT), measurement temperature (MT), and measuring light intensity (ML) on the relaxation of the Kautsky curve. We chose the chicory variety ‘Hera’, as it is known to possess a good average early vigour. Young plants of the variety ‘Hera’ were grown at three temperatures (GT): 16°C (reference), 8°C (intermediate), and 4°C (cold stress). The dark relaxation kinetics were analyzed at different light intensities (ML) in combination with different measurement temperatures (MT). The three components of the nonphotochemical quenching process (NPQ_E, NPQ_T, and NPQ_I) were determined. NPQ_E was not affected by GT but was significantly affected by MT and ML. NPQ_T and NPQ_I were affected by all factors and their interactions. An acclimation effect for plants grown at low GT was detected. Acclimation resulted in lower NPQ_T and NPQ_I values. The halftime of the inhibition depending on NPQ (NPQ_I) was not affected by any of the factors investigated. Based on the data generated, we conclude that NPQ_I is a valuable parameter for screening the cold sensitivity of young industrial chicory plants.     

Bacterial swimming strategies and turbulence

Most bacteria in the ocean can be motile. Chemotaxis allows bacteria to detect nutrient gradients, and hence motility is believed to serve as a method of approaching sources of food. This picture is well established in a stagnant environment. In the ocean a shear microenvironment is associated with turbulence. This shear flow prevents clustering of bacteria around local nutrient sources if they swim in the commonly assumed "run-and-tumble" strategy. Recent observations, however, indicate a "back-and-forth" swimming behavior for marine bacteria. In a theoretical study we compare the two bacterial swimming strategies in a realistic ocean environment. The "back-and-forth" strategy is found to enable the bacteria to stay close to a nutrient source even under high shear. Furthermore, rotational diffusion driven by thermal noise can significantly enhance the efficiency of this strategy. The superiority of the "back-and-forth" strategy suggests that bacterial motility has a control function rather than an approach function under turbulent conditions.     

Multifactorial approach and superior treatment efficacy in renal patients with the aid of nurse practitioners. Design of The MASTERPLAN Study [ISRCTN73187232]

Background

Patients with chronic kidney disease (CKD) are at a greatly increased risk of developing cardiovascular disease. Recently developed guidelines address multiple risk factors and life-style interventions. However, in current practice few patients reach their targets. A multifactorial approach with the aid of nurse practitioners was effective in achieving treatment goals and reducing vascular events in patients with diabetes mellitus and in patients with heart failure. We propose that this also holds for the CKD population.

Design

MASTERPLAN is a multicenter randomized controlled clinical trial designed to evaluate whether a multifactorial approach with the aid of nurse-practicioners reduces cardiovascular risk in patients with CKD. Approximately 800 patients with a creatinine clearance (estimated by Cockcroft-Gault) between 20 to 70 ml/min, will be included. To all patients the same set of guidelines will be applied and specific cardioprotective medication will be prescribed. In the intervention group the nurse practitioner will provide lifestyle advice and actively address treatment goals. Follow-up will be five years. Primary endpoint is the composite of myocardial infarction, stroke and cardiovascular mortality. Secondary endpoints are cardiovascular morbidity, overall mortality, decline of renal function, change in markers of vascular damage and change in quality of life. Enrollment has started in April 2004 and the study is on track with 700 patients included on October 15th, 2005. This article describes the design of the MASTERPLAN study.     

Genetic ablation of caveolin-2 sensitizes mice to bleomycin-induced injury

Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1^−/−) and Cav-2-deficient mice (Cav-2^−/−) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2^−/− mice, but not Cav-1^−/− mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2^−/− mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2^−/− mice. Since Cav-1^−/− mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.     

Release of growth hormone from ox pituitary slices after pronase treatment

Proteolytic enzymes have been used both to modify properties of the cell membrane and to dissociate cells from many tissues including pituitary (4, 5, 12). Exposure of secretory tissues to pronase can alter their secretory response. Thus incubation of pancreatic islets of Langerhans in the presence of low concentrations of pronase increased the subsequent release of insulin in the presence of stimulatory and nonstimulatory glucose concentrations (7). The purpose of the present investigation was to determine whether low concentrations of pronase have the same stimulatory effect on the release of a pituitary hormone, growth hormone. Such an effect on hormone release could be of some importance in view of the development of dissociated cell systems as models for the study of the control of hormone release (4, 5).