The DenA/DEN1 Interacting Phosphatase DipA Controls Septa Positioning and Phosphorylation-Dependent Stability of Cytoplasmatic DenA/DEN1 during Fungal Development

DenA/DEN1 and the COP9 signalosome (CSN) represent two deneddylases which remove the ubiquitin-like Nedd8 from modified target proteins and are required for distinct fungal developmental programmes. The cellular DenA/DEN1 population is divided into a nuclear and a cytoplasmatic subpopulation which is especially enriched at septa. DenA/DEN1 stability control mechanisms are different for the two cellular subpopulations and depend on different physical interacting proteins and the C-terminal DenA/DEN1 phosphorylation pattern. Nuclear DenA/DEN1 is destabilized during fungal development by five of the eight CSN subunits which target nuclear DenA/DEN1 for degradation. DenA/DEN1 becomes stabilized as a phosphoprotein at S243/S245 during vegetative growth, which is necessary to support further asexual development. After the initial phase of development, the newly identified cytoplasmatic DenA/DEN1 interacting phosphatase DipA and an additional developmental specific C-terminal phosphorylation site at serine S253 destabilize DenA/DEN1. Outside of the nucleus, DipA is co-transported with DenA/DEN1 in the cytoplasm between septa and nuclei. Deletion of dipA resulted in increased DenA/DEN1 stability in a strain which is unresponsive to illumination. The mutant strain is dysregulated in cytokinesis and impaired in asexual development. Our results suggest a dual phosphorylation-dependent DenA/DEN1 stability control with stabilizing and destabilizing modifications and physical interaction partner proteins which function as control points in the nucleus and the cytoplasm.     

A hydraulic signal in root-to-shoot signalling of water shortage

Photosynthesis and biomass production of plants are controlled by the water status of the soil. Upon soil drying, plants can reduce water consumption by minimizing transpiration through stomata, the closable pores of the leaf. The phytohormone abscisic acid (ABA) mediates stomatal closure, and is the assigned signal for communicating water deficit from the root to the shoot. However, our study does not support ABA as the proposed long-distance signal. The shoot response to limited soil water supply is not affected by the capacity to generate ABA in the root; however, the response does require ABA biosynthesis and signalling in the shoot. Soil water stress elicits a hydraulic response in the shoot, which precedes ABA signalling and stomatal closure. Attenuation of the hydraulic response in various plants prevented long-distance signalling of water stress, consistent with root-to-shoot communication by a hydraulic signal.     

Discovery of potent and selective phenylalanine derived CCR3 receptor antagonists. Part 2

Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists.     

蟾蜍脑突触质膜对精氨酸-催产素(AVT)的酶促转化

本文研究了蟾蜍脑突触质膜对精氨酸-催产素(AVT,Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-GlyNH_2)的酶促转化过程。利用高压液相层析(HPLC)对降解产物进行了分离,并分析了这些产物的氨基酸组成。发现最主要的一个降解产物为AVT_(1-8)(Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg)。其降解机制与大白鼠脑突触质膜降解AVT的不同。还用二种不同类型的蛋白酶抑制剂,即对氯汞苯甲酸(PCMB)和苯甲基磺酰氟(PMSF),对该酶促转化过程的抑制作用进行了比较,结果表明该水解酶的活性中心可能含有丝氨酸。     

The use of flow microfluorimetry in the analysis of the phenotype expression of mouse histocompatibility antigens

Quantitation of the expression of cell surface antigens has hitherto been limited to analysis by either cytotoxicity tests or radioimmune assays (5, 15). We report here the use of a new methodology to analyze and quantitate the expression of mouse histocompabililty antigens (H-2 locus) in hybrid clones and parental cell types. The binding of fluorescein-tagged antibody is measured on a cell-to-cell basis in large viable cell populations using flow microfluorimetric techniques. These techniques have been used to measure hapten and immunoglobulin binding to lymphocyte populations (8, 9, 14). However, this is the first report in which these techniques have been used to examine the expression of the H-2 locus. The advantage of this approach is twofold: first, a large and statistically significant sample population may be analyzed one cell at a time, thus revealing the fine detail of heterogeneity in the expression of the cell surface markers within a population. Second, as has been demonstrated for analysis of specific components of the immune system, this method does permit fluorescence-activated sorting of cell types according to their different surface populations (8, 9, 14).     

Chemically synthesized zinc finger molecules as nano-addressable probes for double-stranded DNAs

Magnetic nanoparticles (Fe₃O₄) were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe₃O₄ nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications.     

Generation of active pools of abscisic acid revealed by in vivo imaging of water-stressed Arabidopsis

A noninvasive, cell-autonomous reporter system was developed to monitor the generation and distribution of physiologically active pools of abscisic acid (ABA). ABA response (abi1-1) and biosynthesis (aba2-1) mutants of Arabidopsis (Arabidopsis thaliana) were used to validate the system in the presence and absence of water stress. In the absence of water stress, low levels of ABA-dependent reporter activation were observed in the columella cells and quiescent center of the root as well as in the vascular tissues and stomata of cotyledons, suggesting a nonstress-related role for ABA in these cell types. Exposure of seedlings to exogenous ABA resulted in a uniform pattern of reporter expression. In marked contrast, reporter expression in response to drought stress was predominantly confined to the vasculature and stomata. Surprisingly, water stress applied to the root system resulted in the generation of ABA pools in the shoot but not in the root. The analysis of the response dynamics revealed a spread of physiologically active ABA from the vascular tissue into the areoles of the cotyledons. Later, ABA preferentially activated gene expression in guard cells. The primary sites of ABA action identified by in planta imaging corresponded to the sites of ABA biosynthesis, i.e. guard cells and cells associated with vascular veins. Hence, water stress recognized by the root system predominantly results in shoot-localized ABA action that culminates in a focused response in guard cells.