Identification of elongation factor g as the conserved cellular target of argyrin B

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.     

A shortcut from plasma to chromatographic analysis: Straightforward and fast sample preparation for analysis of green tea catechins in human plasma

This paper describes a new and straightforward method for determination of the green tea catechins epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate in human plasma. Sample preparation includes addition only of dimethylformamide and trichloroacetic acid. After centrifugation, the supernatant can be injected into the HPLC. If required, the glucuronides and sulphates of the catechins can be enzymatically hydrolysed before extraction. Recovery ranges from 92.9 to 98.2%; limits of detection, from 2.4 to 5.0 ng/mL; and relative standard deviations, from 3.1 to 8.6%. Twelve samples can be processed within 45 min, and are then ready to be injected into the HPLC. The method was successfully applied to human plasma. This method is suitable for studies on absorption, bioavailability, and kinetics of green tea catechins in plasma. Since manual work and time consumption are minimal, the procedure is especially useful for large numbers of samples.     

A comparative genomics study of genetic products potentially encoding ladderane lipid biosynthesis

Background  

The fatty acids of anaerobic ammonium oxidizing (anammox) bacteria contain linearly concatenated cyclobutane moieties, so far unique to biology. These moieties are under high ring strain and are synthesised by a presently unknown biosynthetic pathway.     

Patterning the fly eye: the role of apoptosis

Early development in many tissues is characterized by a rapid expansion in cell number. Excess cells are removed through activation of their intrinsic apoptotic machinery. This over-expansion followed by selective removal is important for the sculpting of these tissues, and how specific cells are selected to die is one of the central questions in development. The Drosophila eye is a unique example of such patterning through cell death. Because of its remarkable reiterative design, the fly eye lends itself to studies of mutants with increased or decreased apoptosis. We know that the process of elimination of lattice cells is highly regulated. And we have learned that each ommatidial unit is involved in the life-death decision of lattice cells through cell-cell signaling. But, we have yet to understand how this signaling is regulated spatially to result in such precision. In this article, we describe and speculate on the role of selective cell death during maturation of the fly eye.     

Selection of chloroplasts by laser microbeam microdissection for single-chloroplast PCR

Laser microbeam microdissection and laser pressure catapulting offer the possibility of separating cell compartments, thus allowing for contamination-free analysis. Using these methods, we were able to select single chloroplasts of Nicotiana tabacum. Starting from homogenized leaf material, chloroplasts were purified by differential centrifugation and applied directly onto a poly-ethylene-naphthalate membrane that was mounted on a microscope slide. Single chloroplasts were dissected under microscopic control and catapulted into a PCR tube. Subsequent PCR of a spacer region between the trnT and trnF genes verified the successful amplification of DNA from a single chloroplast. The advantage of this method compared to the use of capillaries or optical tweezers is that one is able to prepare high numbers of samples in a short time.     

Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.     

Analysis of cardiac signals using spatial filling index and time-frequency domain

Background  

Analysis of heart rate variation (HRV) has become a popular noninvasive tool for assessing the activities of the autonomic nervous system (ANS). HRV analysis is based on the concept that fast fluctuations may specifically reflect changes of sympathetic and vagal activity. It shows that the structure generating the signal is not simply linear, but also involves nonlinear contributions. These signals are essentially non-stationary; may contain indicators of current disease, or even warnings about impending diseases. The indicators may be present at all times or may occur at random in the time scale. However, to study and pinpoint abnormalities in voluminous data collected over several hours is strenuous and time consuming.     

Phosphoinositide 3-kinase catalytic subunit deletion and regulatory subunit deletion have opposite effects on insulin sensitivity in mice

Studies ex vivo have shown that phosphoinositide 3-kinase (PI3K) activity is necessary but not sufficient for insulin-stimulated glucose uptake. Unexpectedly, mice lacking either of the PI3K regulatory subunits p85alpha or p85beta exhibit increased insulin sensitivity. The insulin hypersensitivity is particularly unexpected in p85alpha-/- p55alpha-/- p50alpha-/- mice, where a decrease in p110alpha and p110beta catalytic subunits was observed in insulin-sensitive tissues. These results raised the possibility that decreasing total PI3K available for stimulation by insulin might circumvent negative feedback loops that ultimately shut off insulin-dependent glucose uptake in vivo. Here we present results arguing against this explanation. We show that p110alpha+/- p110beta+/- mice exhibit mild glucose intolerance and hyperinsulinemia in the fasted state. Unexpectedly, p110alpha+/- p110beta+/- mice showed a approximately 50% decrease in p85 expression in liver and muscle. Consistent with this in vivo observation, knockdown of p110 by RNA interference in mammalian cells resulted in loss of p85 proteins due to decreased protein stability. We propose that insulin sensitivity is regulated by a delicate balance between p85 and p110 subunits and that p85 subunits mediate a negative role in insulin signaling independent of their role as mediators of PI3K activation.     

Activation of the p70 S6 kinase and phosphorylation of the 4E-BP1 repressor of mRNA translation by type I interferons

The Type I IFN receptor-generated signals required for initiation of mRNA translation and, ultimately, induction of protein products that mediate IFN responses, remain unknown. We have previously shown that IFNalpha and IFNbeta induce phosphorylation of insulin receptor substrate proteins and downstream engagement of the phosphatidylinositol (PI) 3'-kinase pathway. In the present study we provide evidence for the existence of a Type I IFN-dependent signaling cascade activated downstream of PI 3'-kinase, involving p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated on threonine 421 and serine 424 and is activated during treatment of cells with IFNalpha or IFNbeta. Such activation of p70 S6K is blocked by pharmacological inhibitors of the PI 3'-kinase or the FKBP 12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR). Consistent with this, the Type I IFN-dependent phosphorylation/activation of p70 S6K is defective in embryonic fibroblasts from mice with targeted disruption of the p85alpha and p85beta subunits of the PI 3'-kinase (p85alpha-/-beta-/-). Treatment of sensitive cell lines with IFNalpha or IFNbeta also results in phosphorylation/inactivation of the 4E-BP-1 repressor of mRNA translation. Such 4E-BP1 phosphorylation is also PI3'-kinase-dependent and rapamycin-sensitive, indicating that the Type I IFN-inducible activation of PI3'-kinase and FRAP/mTOR results in dissociation of 4E-BP1 from the eukaryotic initiation factor-4E (eIF4E) complex. Altogether, our data establish that the Type I IFN receptor-activated PI 3'-kinase pathway mediates activation of the p70 S6 kinase and inactivation of 4E-BP1, to regulate mRNA translation and induction of Type I IFN responses.