5-Aminomethylbenzimidazoles as potent ITK antagonists

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.     

Synthesis and biological evaluation of a novel 99mTc nitrido radiopharmaceutical with deoxyglucose dithiocarbamate,showing tumor uptake

The deoxyglucose dithiocarbamate (DGDTC) was synthesized and radiolabelled with [^99mTcN]²⁺ intermediate to form the ^99mTcN–DGDTC complex. The radiochemical purity of the ^99mTcN–DGDTC complex was over 90%, as measured by TLC and by HPLC, without any notable decomposition at room temperature over a period of 6 h. The partition coefficient and electrophoresis results indicated that this complex was hydrophilic and neutral. The biodistribution of ^99mTcN–DGDTC in mice bearing S 180 tumor showed that the complex accumulated in the tumor with high uptake and good retention. The tumor/blood and tumor/muscle ratios increased with time and reached 2.32 and 1.68 at 4 h post-injection, suggesting it would be a promising candidate for tumor imaging.     

Tandem affinity purification and identification of protein complex components

As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.     

Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair

The histone variant H2AX is rapidly phosphorylated (denoted gammaH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for gammaH2AX in DNA repair; however, gammaH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on gammaH2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish gammaH2AX functions. We found that integration promotes transient formation of gammaH2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of gammaH2AX in chromatin repair.     

Effects of the Conversion of Native Vegetation to Farmlands on Soil Microarthropod Biodiversity and Ecosystem Functioning in a Desert Oasis

Increased demand for food due to the rapidly growing human population has led to extensive conversion of native steppes at the margins of oases in arid lands of northwest China into intensively managed farmlands. However, the consequences of this land-use change for soil microarthropod biodiversity and ecosystem functioning remain unknown. Here we assessed how conversion of a native steppe to irrigated farmlands of different ages affects the abundance and composition of soil microarthropods and how changes in soil microarthropod biodiversity could scale up to influence soil carbon and nitrogen stocks. We sampled microarthropod communities over two growing seasons from native steppes and cultivated soils of a 27-year-old irrigated farmland and a 90-year-old irrigated farmland, both of which were converted from the native steppe. Topsoil properties and bulk and labile pools of carbon and nitrogen, including soil organic carbon, dissolved organic carbon (DOC), microbial biomass carbon (MBC), total nitrogen (TN), inorganic nitrogen (IN), and microbial biomass nitrogen (MBN), were also measured. The conversion of native steppe to either of the two farmlands significantly increased the abundance and taxa richness of three taxonomic groups (mites, collembolans, and others) and four trophic groups (herbivores, predators, detritivores, and fungivores); this effect was greater in the 90-year-old farmland for the abundance of all taxonomic and trophic groups except for herbivores and was similar between the two farmlands for the richness of all taxonomic and trophic groups. Taxonomic and trophic composition of the microarthropod community showed strong shifts in response to conversion of native steppe to either of the two farmlands. Compositional changes were largely mediated by changes in soil environments. Changes in soil carbon and nitrogen stocks due to conversion of native steppe to farmlands followed similar patterns to soil microarthropod biodiversity, but the greater storage of DOC, MBC, TN, IN, and MBN occurred in the 90-year-old farmland. Our results suggest that soil microarthropod communities are affected positively by native steppe conversion to farmland and farmland age, and that increased microarthropod biodiversity significantly improved the ability of soils to retain carbon and nitrogen.     

A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase

CFTRDeltaF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRDeltaF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRDeltaF508. Nonubiquitinated CFTRDeltaF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRDeltaF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.     

FMRP acts as a key messenger for dopamine modulation in the forebrain

The fragile X mental retardation protein (FMRP) is an RNA-binding protein that controls translational efficiency and regulates synaptic plasticity. Here, we report that FMRP is involved in dopamine (DA) modulation of synaptic potentiation. AMPA glutamate receptor subtype 1 (GluR1) surface expression and phosphorylation in response to D1 receptor stimulation were reduced in cultured Fmr1(-/-) prefrontal cortex (PFC) neurons. Furthermore, D1 receptor signaling was impaired, accompanied by D1 receptor hyperphosphorylation at serine sites and subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in both PFC and striatum of Fmr1(-/-) mice. FMRP interacted with GRK2, and pharmacological inhibition of GRK2 rescued D1 receptor signaling in Fmr1(-/-) neurons. Finally, D1 receptor agonist partially rescued hyperactivity and enhanced the motor function of Fmr1(-/-) mice. Our study has identified FMRP as a key messenger for DA modulation in the forebrain and may provide insights into the cellular and molecular mechanisms underlying fragile X syndrome.     

介绍一种改进的研究气孔运动的方法

本文介绍了以液氮现场固定叶样,扫描电镜直接观察（照相）记录气孔形态、分布、日变化等的方法。从给出的实例可以看到该法能成功地记录气孔在一天的动态变化情况。这种方法特别适合于研究叶表皮密被绒毛的植物。