Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

Protein networks supporting AP-3 function in targeting lysosomal membrane proteins

The AP-3 adaptor complex targets selected transmembrane proteins to lysosomes and lysosome-related organelles. We reconstituted its preferred interaction with liposomes containing the ADP ribosylation factor (ARF)-1 guanosine triphosphatase (GTPase), specific cargo tails, and phosphatidylinositol-3 phosphate, and then we performed a proteomic screen to identify new proteins supporting its sorting function. We identified ≈30 proteins belonging to three networks regulating either AP-3 coat assembly or septin polymerization or Rab7-dependent lysosomal transport. RNA interference shows that, among these proteins, the ARF-1 exchange factor brefeldin A-inhibited exchange factor 1, the ARF-1 GTPase-activating protein 1, the Cdc42-interacting Cdc42 effector protein 4, an effector of septin-polymerizing GTPases, and the phosphatidylinositol-3 kinase IIIC3 are key components regulating the targeting of lysosomal membrane proteins to lysosomes in vivo. This analysis reveals that these proteins, together with AP-3, play an essential role in protein sorting at early endosomes, thereby regulating the integrity of these organelles.     

The BMP-binding protein Crossveinless 2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila

In Drosophila, the secreted BMP-binding protein Short gastrulation (Sog) inhibits signaling by sequestering BMPs from receptors, but enhances signaling by transporting BMPs through tissues. We show that Crossveinless 2 (Cv-2) is also a secreted BMP-binding protein that enhances or inhibits BMP signaling. Unlike Sog, however, Cv-2 does not promote signaling by transporting BMPs. Rather, Cv-2 binds cell surfaces and heparan sulfate proteoglygans and acts over a short range. Cv-2 binds the type I BMP receptor Thickveins (Tkv), and we demonstrate how the exchange of BMPs between Cv-2 and receptor can produce the observed biphasic response to Cv-2 concentration, where low levels promote and high levels inhibit signaling. Importantly, we show also how the concentration or type of BMP present can determine whether Cv-2 promotes or inhibits signaling. We also find that Cv-2 expression is controlled by BMP signaling, and these combined properties enable Cv-2 to exquisitely tune BMP signaling.     

Sim4cc: a cross-species spliced alignment program

Advances in sequencing technologies have accelerated the sequencing of new genomes, far outpacing the generation of gene and protein resources needed to annotate them. Direct comparison and alignment of existing cDNA sequences from a related species is an effective and readily available means to determine genes in the new genomes. Current spliced alignment programs are inadequate for comparing sequences between different species, owing to their low sensitivity and splice junction accuracy. A new spliced alignment tool, sim4cc, overcomes problems in the earlier tools by incorporating three new features: universal spaced seeds, to increase sensitivity and allow comparisons between species at various evolutionary distances, and powerful splice signal models and evolutionarily-aware alignment techniques, to improve the accuracy of gene models. When tested on vertebrate comparisons at diverse evolutionary distances, sim4cc had significantly higher sensitivity compared to existing alignment programs, more than 10% higher than the closest competitor for some comparisons, while being comparable in speed to its predecessor, sim4. Sim4cc can be used in one-to-one or one-to-many comparisons of genomic and cDNA sequences, and can also be effectively incorporated into a high-throughput annotation engine, as demonstrated by the mapping of 64 000 Fagus grandifolia 454 ESTs and unigenes to the poplar genome.     

Imidazo[1,2-a]pyrazine diaryl ureas: Inhibitors of the receptor tyrosine kinase EphB4

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent^® and Nexavar^®, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.     

Spring migration dynamics and sex-specific patterns in stopover strategy in the Wood Sandpiper Tringa glareola

Due to being a virtually monomorphic wader species, migration dynamics and sex-related migration patterns in the Wood Sandpiper (Tringa glareola) have rarely been investigated. We captured spring migrants at an important stopover site in northeastern Austria. Birds were individually color-marked, and sex was determined by an analysis of DNA from tail feather material. Among temporary residents (birds seen again after day of capture), males migrated on average 3 days earlier than females. However, since sexes did not differ in fat score, the length of stay and the proportion of transients (birds not seen again after day of capture) and temporary residents, we suggest that males and females adopt similar migration strategies in the spring. The large number of transients captured as well as shorter stopover durations in later temporary residents indicate that Wood Sandpipers minimize time at this stage of their northbound migration. Temporary residents earlier in the season exhibited lower fat stores than later ones. Nevertheless, since the fat stores of transients and temporary residents were similar even after the progress of the season had been accounted for, we assume that Wood Sandpipers may afford to exhibit individual flexibility in migration strategy and the use of stopover sites, especially early in the season. This variability may be a necessary adaptation to cope with possible varying environmental conditions at dynamic and unpredictable inland stopover sites. After having reached North Mediterranean regions, mean body mass of spring migrants gradually increases during successive stopovers, indicating that Wood Sandpipers follow a ‘hopping’ migration strategy. This emphasizes the high conservation value of even small artificial mudflat pools as important stepping stones in order to maintain a continuous network of wetland habitats for this continental migrant.     

Cardioprotection by adaptation to ischaemia augments autophagy in association with BAG-1 protein

Autophagy is an intracellular process in which a cell digests its own constituents via lysosomal degradative pathway. Though autophagy has been shown in several cardiac diseases like heart failure, hypertrophy and ischaemic cardiomyopathy, the role and the regulation of autophagy is still largely unknown. Bcl-2-associated athanogene (BAG-1) is a multifunctional pro-survival molecule that binds with Hsp70/Hsc70. In this study, myocardial adaptation to ischaemia by repeated brief episodes of ischaemia and reperfusion (I/R) prior to lethal I/R enhanced the expression of autophagosomal membrane specific protein light chain 3 (LC3)-II, and Beclin-1, a molecule involved in autophagy and BAG-1. Autophagosomes structures were found in the adapted myocardium through electron microscopy. Co-immunoprecipitation and co-immunofluorescence analyses revealed that LC3-II was bound with BAG-1. Inhibition of autophagy by treating rats with Wortmannin (15 μg/kg; intraperitoneally) abolished the ischaemic adaptation-induced induction of LC3-II, Beclin-1, BAG-1 and cardioprotection. Intramyocardial injection of BAG-1 siRNA attenuated the induction of LC3-II, and abolished the cardioprotection achieved by adaptation. Furthermore, hypoxic adaptation in cardiac myoblast cells induced LC3-II and BAG-1. BAG-1 siRNA treatment attenuated hypoxic adaptation-induced LC3-II and BAG-1, and abolished improvement in cardiac cell survival and reduction of cell death. These results clearly indicate that myocardial protection elicited by adaptation is mediated at least in part via up-regulation of autophagy in association with BAG-1 protein.     

Investigation of saturated and aromatic hydrocarbon resistance mechanisms in Pseudomonas aeruginosa IBBML1

Pseudomonas aeruginosa IBB_ML1, isolated from Poeni petroleum sludge, was able to tolerate and degrade both saturated (n-hexane, n-heptane, n-hexadecane, cyclohexane) and aromatic (benzene, ethylbenzene, propylbenzene, xylene isomers, styrene) hydrocarbons. Molecular studies have revealed that the high hydrocarbon resistance of Pseudomonas aeruginosa IBB_ML1 could be due to the action of members of the HAE1 (hydrophobe/amphiphile efflux 1) family of transporters. It is further possible that additional mechanisms may account for the tolerance of Pseudomonas aeruginosa IBB_ML1 to hydrocarbons, and a combination of short-term and long-term mechanisms may act together in the adaptation of Pseudomonas aeruginosa IBB_ML1 cells to saturated and aromatic hydrocarbons. β-galactosidase activity measurements revealed that there was significant induction of the lacZ gene in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of either 5% and 10% (v/v) saturated or aromatic hydrocarbons, compared with control (cells incubated without hydrocarbons). Rhodamine 6G accumulation in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of 5% and 10% (v/v) saturated hydrocarbons was higher than rhodamine 6G accumulation in cells grown in the presence of 5% and 10% (v/v) aromatic hydrocarbons. The study of cellular and molecular modifications to Pseudomonas aeruginosa IBB_ML1 induced by 5% and 10% (v/v) saturated and aromatic hydrocarbons revealed a complex response of bacterial cells to the presence of different hydrophobic substrates in the culture medium.     

Dissecting the Signaling Events That Impact Classical Nuclear Import and Target Nuclear Transport Factors

Background

Signaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined.

Methodology/Principal Findings

To better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant.

Conclusions/Significance

Our studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling.