Dynein,Lis1 and CLIP‐170 counteract Eg5‐dependent centrosome separation during bipolar spindle assembly

Bipolar spindle assembly critically depends on the microtubule plus‐end‐directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus‐end‐directed motor dynein, or the dynein‐binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5‐dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus‐end‐tracking protein CLIP‐170. This function of CLIP‐170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP‐115, a highly homologous protein that lacks the dynein–dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP‐170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.     

Characterization of the in vivo and in vitro metabolic profile of PAC-1 using liquid chromatography-mass spectrometry

In the present study, the metabolic profile of PAC-1, a potential anticancer drug, was investigated using liquid chromatography-mass spectrometric (LC/MS) techniques. Two different types of mass spectrometers--a quadrupole time-of-flight (Q-TOF) mass spectrometer and an ion trap (IT) mass spectrometer--were employed to acquire structural information on PAC-1 metabolites. A gradient liquid chromatographic system composed of 0.2% formic acid in methanol and 0.2% formic acid in water was used for metabolite separation on an Agilent TC-C(18) column. A total of 16 metabolites were detected. The corresponding product ion spectra were acquired and interpreted, and structures were proposed. Accurate mass measurement using LC-Q-TOF was used to determine the elemental composition of metabolites thereby confirming the proposed structures of these metabolites. Phase I metabolic changes were predominantly observed, including debenzylation, dihydrodiol formation, hydroxylation, and dihydroxylation. The detected phase II metabolites included PAC-1 and hydroxylated PAC-1 glucuronide conjugates. Based on metabolite analysis, several PAC-1 metabolic pathways in rat were proposed.     

Anti-adhesive Property of Maize Leaf Surface Related with Temperature and Humidity

The anti-adhesive surfaces have always aroused great interest of worldwide scientists and engineers.But in practical applications,it often faces the threat and impact of temperature and humidity.In this work,the excellent anti-adhesive performance of maize leaf under high temperature and humidity were investigated in detail.Firstly,the adhesion forces of the maize leaf surface under different temperature and humidity were measured by using Atomic Force Microscopy (AFM).The temperature of the substrate was varied between 23 ℃ to 100 ℃,and the ambient relative humidity is from 18％ to 100％.It was found that the adhesion force of maize leaf decreased with the increase of temperature and humidity.The mechanism of its excellent anti-adhesive performance of maize leaf under high temperature and relative humidity was revealed.The transverse and longitudinal ridges on maize leaf surface interlace with each other,forming small air pockets,which reduces the actual contact area between the object and the maize leaf.With the increase of humidity,the liquid film will be formed in the air pockets gradually and so much water vapor is produced with increase of temperature.Then the air flow rate increases though the wavy top of transverse ridges,inducing the dramatic decrease of adhesion force.Inspired by this mechanism,four samples with this bionic structure were made.This functional "biomimetic structure" would have potential value in the wide medical equipments such as high frequency electric knife with anti-adhesion surface under high temperature and high humidity.     

High‐Performance and Stable All‐Polymer Solar Cells Using Donor and Acceptor Polymers with Complementary Absorption

To explore the advantages of emerging all‐polymer solar cells (all‐PSCs), growing efforts have been devoted to developing matched donor and acceptor polymers to outperform fullerene‐based PSCs. In this work, a detailed characterization and comparison of all‐PSCs using a set of donor and acceptor polymers with both conventional and inverted device structures is performed. A simple method to quantify the actual composition and light harvesting contributions from the individual donor and acceptor is described. Detailed study on the exciton dissociation and charge recombination is carried out by a set of measurements to understand the photocurrent loss. It is unraveled that fine‐tuned crystallinity of the acceptor, matched donor and acceptor with complementary absorption and desired energy levels, and device architecture engineering can synergistically boost the performance of all‐PSCs. As expected, the PBDTTS‐FTAZ:PNDI‐T10 all‐PSC attains a high and stable power conversion efficiency of 6.9% without obvious efficiency decay in 60 d. This work demonstrates that PNDI‐T10 can be a potential alternative acceptor polymer to the widely used acceptor N2200 for high‐performance and stable all‐PSCs.     

Climatic signals in tree ring of Picea schrenkiana along an altitudinal gradient in the central Tianshan Mountains, northwestern China

Tree-ring samples of Picea schrenkiana (Fisch. et Mey) were studied along an altitudinal gradient in the central Tianshan Mountains, and ring-width chronologies were developed for three sites at different altitudes: low-forest border (1600–1700 m a.s.l.), interior forest (2100–2200 m a.s.l.), and upper treeline (2600–2700 m a.s.l.). Annual ring-width variations were similar among the three sites but variability was greatest at the low-forest border site. The statistical characters of the chronologies showed that mean sensitivity (MS) and standard deviation (SD) decreased with increasing elevation. In other words, the response of tree growth to environmental changes decreased with increasing altitude. To understand the differing response of trees at different elevations to the environmental changes, response function analysis was used to study the relationships between tree-ring widths and mean monthly temperature and total monthly precipitation from 1961 to 2000. The results showed that precipitation was the most important factor limiting tree radial growth in the arid central Tianshan Mountains, precipitation in August of the prior growth year played an important role on tree's radial growth across the entire altitudinal gradient even at the cold, high-elevation treeline site. It is expected that with increasing altitude air temperature decreased and precipitation increased, the importance of precipitation on tree growth decreased, and the response of tree growth to environmental changes decreased, too. This conclusion may be helpful to understand and research the relationship between climatic change and tree growth in arid and semiarid area.     

Meeting threats to global health: a call for American leadership

Although the application of major biomedical advances has yielded spectacular results for individual health, there has been little improvement in the health of whole populations. There is a "back to the future" irony in the fact that at the inception of the 21st century, the eruption and spread of a multitude of "old" and "new" infectious diseases has become the most serious global threat to the health of humankind. At this historical juncture, the United States is the country with the most potential for favorably influencing global health and health care. Although there are historical, cultural, economic, and political factors that impede the United States from rising to this challenge, there is both a moral imperative and a rational long-term self-interest basis for the U.S. medical profession and government to exercise leadership in facing the health challenges of tragic and genocidal proportions that threaten everyone in an increasingly interdependent world.     

Expression of Rapeseed Microsomal Lysophosphatidic Acid Acyltransferase Isozymes Enhances Seed Oil Content in Arabidopsis

In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.With increasing environmental challenges and concerns, there is renewed interest in deriving plant-based sustainable alternatives for petroleum products, including carburants, lubricants, and industrial feed stocks. Modifying oilseed crops to produce oils of uniform composition containing fatty acids varying in chain length or possessing reactive functional groups is a primary objective (Jaworski and Cahoon, 2003), as is that of increasing the yield of seed oil (Lardizabal et al., 2008; Zheng et al., 2008). Early success in modifying seed oils to produce the more common fatty acids has been tempered by limited success in the production of high levels of unusual fatty acids (UFAs) in cultivated oilseeds (Thelen and Ohlrogge, 2002; Drexler et al., 2003). Such studies have led to the conclusion that in order to achieve levels of UFAs similar to those present in the oil of native species, enzymatic activities additional to fatty acid modification are necessary to optimize the synthesis (Mekhedov et al., 2001), stability (Eccleston and Ohlrogge, 1998), and channeling (Bafor et al., 1990) of the desired fatty acid into triacylglycerol (TAG).The synthesis of glycerolipids occurs in the cytoplasm using de novo-synthesized fatty acids exported from the plastid as acyl-CoA thioesters. The fatty acyl groups are incorporated into membrane and storage lipids by the sequential esterification of glycerol-3-phosphate by the action of glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) at sn-1 to form lysophosphatidic acid followed by lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) at sn-2 to form phosphatidic acid (PA; Somerville et al., 2000). Dephosphorylation of PA results in the formation of diacylglycerol (DAG), which in developing seeds may be directed into the production of TAG by acyl-CoA-independent reactions or by diacylglycerol acyltransferase (DAGAT; EC 2.3.1.20; Roscoe, 2005). The substrate preferences for acyl-thioesters and the selectivities for the acceptor molecules displayed by the microsomal acyltransferases play a crucial role in establishing the acyl composition of lipids (Frentzen, 1998). The TAG synthesized in most oilseeds of agronomic importance contains fatty acids that are the same as those present in cytoplasmic membrane lipids. In contrast, the seeds of species that synthesize TAGs with exotic fatty acid compositions possess microsomal acyltransferases that facilitate the incorporation of UFAs into storage lipids because of their broad GPAT and/or their selective DAGAT specificities (Wiberg et al., 1994; Frentzen, 1998). Furthermore, oilseeds characterized by TAGs that contain UFAs at sn-2 possess additional seed-specific microsomal LPAATs (Brown et al., 1995; Hanke et al., 1995; Knutzon et al., 1995) that exhibit a wide variation in substrate preference and that serve to ensure the channeling of UFAs to this position, thereby segregating incompatible fatty acids away from membrane lipids.Cloning of cDNAs from cultivated and exotic plants and the availability of entirely sequenced genomes from plant and algal species have revealed that a minimum of two classes of genes encoding microsomal LPAATs exist (Frentzen, 1998) within a larger, LPAAT-like gene family containing acyltransferases as yet functionally uncharacterized but distinct from GPATs (Roscoe, 2005). The class A microsomal LPAATs defined by Frentzen (1998) possess substrate preferences for C18:1-CoA typical of enzymes involved in membrane lipid synthesis and are ubiquitously expressed in the plant. In contrast, individual members of the class B LPAATs display preferences for distinct, unusual saturated or unsaturated acyl groups and are normally expressed in storage organs. Although class B LPAATs have been exploited to alter the stereochemical composition of rapeseed (Brassica napus) oil to permit the incorporation of modified fatty acids at sn-2 (Lassner et al., 1995; Knutzon et al., 1999), a significant increase in the total amount of UFAs was not accomplished by the expression of the class B LPAATs alone. In contrast, the transformation of rapeseed and Arabidopsis (Arabidopsis thaliana) with a yeast gene encoding a variant LPAAT, SLC1-1, capable of accepting very long chain fatty acyl (VLCFA)-CoA substrates resulted in an increase in the total VLCFAs and, unexpectedly, in total oil content (Zou et al. 1997).In our efforts to modify the fatty acid composition of oil in rapeseed, in particular to increase the content of VLCFAs, we have addressed the question of optimizing the environment for the correct functioning of LPAATs encoded by transgenes. The above studies using the various LPAAT transgenes indicate that channeling of UFAs into sn-2 of oilseed species remains problematic. The ability to obtain oils with uniform composition strongly depends on the occupancy of sn-2 by UFAs, yet the level of occupancy of sn-2 by fatty acids corresponding to the selectivity of the introduced LPAAT is variable and relatively modest. Occupancy of sn-2 is determined in part by the ability of the LPAAT encoded by the transgene to compete with the endogenous enzyme, a function of the acyl-CoA substrates available to the enzymes and the relative efficiencies of the enzymes to compete for the donor and acceptor substrates. We argued that there is latitude for the reduction of competing activities using an antisense strategy, and although microsomal LPAATs have been cloned from rapeseed, there are no reports of the characterization of the enzyme. Our objectives in this work were to identify and evaluate the potential contribution of LPAAT isozymes to TAG biosynthesis in rapeseed, thereby discerning targets for optimizing efforts to modify oils for industrial purposes. In this study, we catalogue a previously undescribed complexity in microsomal LPAAT diversity and identify a LPAAT isozyme likely to play an important role in TAG synthesis in rapeseed. In contrast to diverged LPAATs of plant origin, we demonstrate a positive effect of the overexpression of microsomal LPAATs on oil content and seed weight.     

G-protein-coupled receptor kinase specificity for beta-arrestin recruitment to the beta2-adrenergic receptor revealed by fluorescence resonance energy transfer

The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.