Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

Metabolism and specific benzopyrene metabolite modification of DNA in early S by human lung epithelial and fibroblast cells leading to the expression of an abnormal phenotype

Examination of the high pressure liquid chromatographic profiles of ethyl acetate extractable benzo [a] pyrene (B(a)P)-metabolites from human lung fibroblast and type II epithelial cells after S phase entry indicated that B(a)P-7,8-diol and 9,10-diol species were produced following the oxygenation of B(a)P. These metabolites were detected intracellularly and in the extracellular growth medium. Both cell types appeared to release extracellularly, elevated amounts of the B(a)P-7,8-diol species. It was interesting to note of the 4 pmol of oxygenated metabolites localized intracellularly, in the fibroblast, that we identified two major metabolites, B(a)P-9,10 and -7,8-diol species. Lung epithelial cells metabolize intracellular B(a)P extensively, greater than or equal to 93% of the parent B(a)P. No tetrols were detected intracellularly or extracellularly in the treated fibroblast cells. The treated epithelial cells produced both tetrols and sulfate conjugates. The extent of observed modification of early S phase nuclear DNA of lung epithelial cells was 7.5 +/- 4.9 adducts per 10(6) bases and 4.2 +/- 2.7 adducts per 10(6) bases in lung fibroblasts. The major adduct formed in both cell types was 7 beta-BPDE-I-dG. Under conditions for transformation, both the B(a)P treated lung epithelial cells and lung fibroblasts treated in early S with either B(a)P or BPDE-I yielded populations that exhibited properties of anchorage independent growth and cellular invasiveness. Metabolism and the presence internally of metabolites did not correlate with the extent of modification of DNA in early S.     

Inhibition of carcinogen-induced cellular transformation of human fibroblasts by drugs that interact with the poly(ADP-ribose) polymerase system. Initial evidence for the development of transformation resistance

Two types of interactions of 13 drugs with human fibroblasts were determined: I50 of nuclear poly(ADP-ribose) polymerase, as assayed with isolated nuclei in vitro, and the non-toxic concentration of drugs that prevented carcinogen-induced cell transformation of intact fibroblasts (RCF1). In general, RCF1 was much lower than I50, and one antitransformer did not inhibit the enzyme in vitro, indicating that low-affinity enzyme inhibitory sites appear to play no role in the mechanism of prevention of cell transformation. Two enzyme inhibitors, caffeine and 1-methylnicotinamide, exhibited no antitransforming activity. Benzamide when applied in population doubling 1 induced resistance to cell transformation in population doubling 6 by carcinogens added at this stage.     

Induction of anchorage-independent growth in human diploid fibroblasts by the cyclopenta-polycyclic aromatic hydrocarbon, benz[l]aceanthrylene

Cyclopenta-fused polycyclic aromatic hydrocarbons are a class of environmental PAH that have been recently identified. Many of these chemicals have been found to be more active than benzo[a]pyrene in tests for genetic toxicity using bacterial and rodent cells. Benz[l]aceanthrylene, a cyclopenta-polycyclic aromatic hydrocarbon related to benz[a]anthracene, and benzo[a]pyrene were compared for their activity to induce cytotoxicity and anchorage-independent growth with normal human diploid fibroblasts. Both benz[l]aceanthrylene and benzo[a]pyrene were relatively non-cytotoxic to normal human diploid fibroblasts. However, benz[l]aceanthrylene was twice as active compared to benzo[a]pyrene over the concentration range examined as an inducer of anchorage-independent growth. The ability of benz[l]aceanthrylene to induce anchorage-independent colony growth in normal human cells, in combination with its demonstrated ability as a mouse-skin tumorigen, suggests this PAH to be a potential multi-species carcinogen.     

Ecological and methodological drivers of non-stationarity in tree growth response to climate

Radial tree growth is sensitive to environmental conditions, making observed growth increments an important indicator of climate change effects on forest growth. However, unprecedented climate variability could lead to non-stationarity, that is, a decoupling of tree growth responses from climate over time, potentially inducing biases in climate reconstructions and forest growth projections. Little is known about whether and to what extent environmental conditions, species, and model type and resolution affect the occurrence and magnitude of non-stationarity. To systematically assess potential drivers of non-stationarity, we compiled tree-ring width chronologies of two conifer species, Picea abies and Pinus sylvestris, distributed across cold, dry, and mixed climates. We analyzed 147 sites across the Europe including the distribution margins of these species as well as moderate sites. We calibrated four numerical models (linear vs. non-linear, daily vs. monthly resolution) to simulate growth chronologies based on temperature and soil moisture data. Climate–growth models were tested in independent verification periods to quantify their non-stationarity, which was assessed based on bootstrapped transfer function stability tests. The degree of non-stationarity varied between species, site climatic conditions, and models. Chronologies of P. sylvestris showed stronger non-stationarity compared with Picea abies stands with a high degree of stationarity. Sites with mixed climatic signals were most affected by non-stationarity compared with sites sampled at cold and dry species distribution margins. Moreover, linear models with daily resolution exhibited greater non-stationarity compared with monthly-resolved non-linear models. We conclude that non-stationarity in climate–growth responses is a multifactorial phenomenon driven by the interaction of site climatic conditions, tree species, and methodological features of the modeling approach. Given the existence of multiple drivers and the frequent occurrence of non-stationarity, we recommend that temporal non-stationarity rather than stationarity should be considered as the baseline model of climate–growth response for temperate forests.     

多元方差分析中的单一自由度独立比较

本文根据一元方差分析的单一自由度比较原理提出了多元方差分析中的单一自由度比较方法,以用于各种平衡试验设计中具多个变量的处理间多重比较．     

Role of agonist and antagonist muscle strength in performance of rapid movements

Six subjects performed rapid self-terminated elbow movements under different mechanical conditions prior to, and 5 weeks after an elbow extensor strengthening programme. Despite the large difference in the strengths of elbow flexors and extensors, the pretest did not demonstrate significant differences between the movement time of flexion and extension movements performed under the same mechanical conditions. The results obtained in the posttest demonstrated a decrease in movement time (i.e. an increase in movement speed) in both elbow flexion and extension movements under some mechanical conditions. In addition, flexion movements demonstrated a relative increase in the acceleration time (acceleration time as a proportion of the movement time). It was concluded that the strength of both the agonist and antagonist muscles was important for the performance of rapid movements. Stronger agonists could increase the acceleration of the limb being moved, while stronger antagonists could facilitate the arrest of the limb movement in a shorter time, providing a longer time for acceleration.     

Growth characteristics,morphology, and phospholipid composition of human type II pulmonary alveolar cells grown in a collagen-free microenvironment

Summary Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro. This work was supported in part by grants from EPA, R 806638-01 and 131-640-1599A1     

Growth characteristics, morphology, and phospholipid composition of human type II pulmonary alveolar cells grown in a collagen-free microenvironment

Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture. Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting materials and subsequent population doublings. Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung (age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated, epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under conditions of growth in vitro.