A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3.     

Break-induced loss of heterozygosity in fission yeast: dual roles for homologous recombination in promoting translocations and preventing de novo telomere addition

Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss approximately 25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32(+), rad50(+), nbs1(+), rhp51(+), rad22(+), rhp55(+), rhp54(+), and mus81(+). Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Delta and rhp55Delta backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Delta rhp55Delta strains, were reduced in exo1Delta rhp55Delta and an rhp55Delta strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.     

Engineering a twist in 9,10-diethynylanthracenes by steric interactions.

A series of 9,10-bis(phenylethynyl)anthracenes decorated with sterically demanding tert-butyl substituents have been prepared and spectroscopically characterised. We demonstrate that the introduction of two bulky substituents in the ortho position of the phenyl rings effectively locks the ground state into a conformation in which the three rings are orthogonal. Fluorescence spectroscopy reveals evidence for partial planarisation of this compound in the excited state at ambient temperature, but this is prevented in low temperature solvent glasses.     

Rumen microbial population dynamics in response to photoperiod

AIMS: This work was carried out to determine if there was a difference in the microbial population of the rumen associated with daylength at which sheep are housed. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) was used to study the ciliate and bacterial diversity in the rumen of Soay rams kept in long day (16 h light) or short day (8 h light) photoperiods. Bacterial diversity varied according to the daylength conditions where the host animal was housed, as did total volatile fatty acids (VFA) concentrations. No differences associated with daylength were detected in ciliate diversity, branched VFA concentrations or the ruminal ammonia concentrations. CONCLUSIONS: As diets had identical composition, yet voluntary intakes levels were higher during long days, it is proposed that the differences in bacterial populations arise because of the differences in amount of food consumed. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this study demonstrated that factors beyond dietary composition must be taken into account when trying to study microbial populations, even in what can be considered a fairly constant environment.     

Low-temperature growth of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of approximately 35 degrees C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature ( approximately 22 degrees C) MR-1 grows with a doubling time of about 40 min, but when moved from 22 degrees C to 3 degrees C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of approximately 67 h. In comparison to cells grown at 22 degrees C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22 degrees C.     

The inner ear morphology and hearing abilities of the Paddlefish (Polyodon spathula) and the Lake Sturgeon (Acipenser fulvescens)

Concern regarding the spread of silver carp (Hypopthalmichthys molitrix) and bighead carp (Aristichthysc nobilis) through the Illinois River has prompted the development of an Acoustic Fish Deterrent (AFD) system. The application of this technology has resulted in a need to understand the auditory physiology of fish other than the target species, in order to minimise the effect of the AFD barrier on the ecology of indigenous fish populations. To this end, both the structures involved in sound reception and the hearing abilities of the paddlefish (Polyodon spathula) and the lake sturgeon (Acipenser fulvescens) are studied here using a combination of morphological and physiological approaches, revealing that both fish are responsive to sounds ranging in frequency from 100 to 500 Hz. The lowest hearing thresholds from both species were acquired from frequencies in a bandwidth of between 200 and 300 Hz, with higher thresholds at 100 and 500 Hz. The rationale for studying hearing in P. spathula and A. fulvescens in particular, is the value placed on them by both the commercial caviar producing industry and by the recreational fisheries sector. The hearing abilities of twelve P. spathula and twelve A. fulvescens were tested in sound fields dominated by either sound pressure or particle motion, with the results showing that acipenseriform fish are responsive to the motion of water particles in a sound field, rather than the sound pressure component. In this study, we measure the intensity of the sound field required to evoke threshold responses using a pressure sensitive hydrophone, as pressure dominated sound fields are the most audible acoustic condition for specialists like H. molitrix and A. nobilis (the target species). The results of the auditory examination clearly show that P. spathula and A. fulvescens are not sensitive to sound pressure, and will therefore have a significantly higher deterrent threshold than H. molitrix and A. nobilis in a pressure dominated sound field.     

1-Aryl-3,4-dihydro-1H-quinolin-2-one derivatives, novel and selective norepinephrine reuptake inhibitors

A novel series of 1-aryl-3,4-dihydro-1H-quinolin-2-ones have been discovered as potent and selective norepinephrine reuptake inhibitors. Efficient synthetic routes have been developed which allow for the multi-gram preparation of both final targets and advanced intermediates for SAR expansion.     

Restraining PI3K: mTOR signalling goes back to the membrane

The lipid kinase phosphoinositide 3-kinase (PI3K) is activated in response to various extracellular signals including peptide growth factors such as insulin and insulin-like growth factors (IGFs). Phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] generated by PI3K is central to the diverse responses elicited by insulin, including glucose homeostasis, proliferation, survival and cell growth. The actions of lipid phosphatases have been considered to be the main means of attenuating PI3K signalling, whereby the principal 3-phosphatase - phosphatase and tensin homologue deleted on chromosome 10 (PTEN) - dephosphorylates PtdIns(3,4,5)P(3), reversing the action of PI3K. Recently, however, another pathway of regulation of PI3K has been identified in which activation of PI3K itself is prevented. This finding, together with earlier work, strongly suggests that a major form of negative feedback inhibition of PI3K results from activated growth signalling via mammalian target of rapamycin (mTOR) and the p70 S6 kinase (S6K) - a pathway that could have consequences for the development of type 2 diabetes and tuberous sclerosis complex.     

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra-skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co-localizes with P-selectin and von Willebrand factor (vWF), within the Weibel-Palade bodies (WPB). Treatment of HUVEC with the pro-inflammatory cytokines, tumor necrosis factor (TNF)-alpha and IL-1beta, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF-alpha treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24-h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P-Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP-1), raising the intriguing possibility that OPG may provide a link between TSP-1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.