The Design and Synthesis of Bis-[4′-Azido-2,2′:6′,2″-Terpyridine Platinum(II)] Complexes with Rigid and Extended Linkers for Studying the Topology of DNA by Photoaffinity Labeling

A family of bis-[4′-Azido-2,2′:6′,2″-terpyridine platinum(II)] complexes with linear linkers of varying length have been synthesized. They have been designed to bis-intercalate into two DNA duplexes in close proximity, the azido groups allowing the sites of intercalation to be photoaffinity labeled. The linker to Pt(II) bonds are susceptible to cleavage by thiols and cyanide ion, which is a requirement for the intended method of analysis by 2D gel electrophoresis.     

Eucalyptus gunnii CCR and CAD2 promoters are active in lignifying cells during primary and secondary xylem formation in Arabidopsis thaliana

Cell-specific expression patterns of the Eucalyptus gunnii cinnamoyl coenzymeA reductase (EgCCR) and cinnamyl alcohol dehydrogenase (EgCAD2) promoters were analyzed by promoter-GUS histochemistry in the primary and secondary xylem tissues from floral stems and roots of Arabidopsis thaliana. Expression patterns indicated that the EgCCR and EgCAD2 genes were expressed in a coordinated manner in primary and secondary xylem tissues of the Arabidopsis floral stem and root. Both genes were expressed in all lignifying cells (vessel elements, xylem fibers and paratracheal parenchyma cells) of xylem tissues. The capacity for long-term monolignol production appeared to be related to the cell-specific developmental processes and biological roles of different cell types. Our results suggested that lignification of short-lived vessel elements was achieved by a two-step process involving (i) monolignol production by vessel elements prior to vessel programmed cell death and (ii) subsequent monolignol production by vessel-associated living paratracheal parenchyma cells following vessel element cell death. EgCCR and EgCAD2 gene expression patterns suggested that the process of xylem cell lignification was similar in both primary and secondary xylem tissues in Arabidopsis floral stems and roots.     

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method''s potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges.A protein''s function is largely mediated through its interactions with other proteins, hence the critical importance of protein-protein interaction (PPI)¹ maps for understanding cellular mechanisms of action in health and disease. Whereas many proteins are organized in stable multi-protein complexes, the majority of cellular processes are governed by transient protein encounters, the dynamics of which are directed by a diversity of both intra- and extracellular signals. Our view of protein networks is still, however, mainly a static one (1). Current interactomes consist mainly of data generated by yeast 2-hybrid (Y2H) (2) and (tandem) affinity purification combined with mass spectrometry (3) and should be interpreted as scaffolds of potential PPIs that might occur at a certain time and place in the cell or as snapshots of PPIs taking place under a specific cellular condition. Although very robust and highly efficient, these approaches do not allow studying PPI modulation because they do not offer the proper context for mammalian PPI analysis, e.g. they operate in yeast cells (Y2H) or make use of cell lysates (affinity purification-based methods). Moreover, because these interactome mapping tools are biased against interactions that involve transmembrane proteins, the latter are underrepresented in current interactome network versions (4). Yet, membrane-associated proteins constitute around one third of the entire proteome and their significance is underscored by the fact that over half of currently marketed drugs target membrane proteins (5). These observations support the need for approaches that allow PPIs, including those involving transmembrane proteins, to be assayed in their native cellular environment.Apart from the high-throughput methods mentioned above, a diverse arsenal of other PPI technologies has been developed, a number of which actually operate in mammalian cells. FRET and BRET, which rely on fluorescence or bioluminescence energy transfer between interacting fusion proteins, make assays with high spatiotemporal resolution (6, 7). A variety of PCAs have been reported, including split fluorescent protein or reporter enzyme technologies, that are able to capture aspects of PPI dynamics in a mammalian background (8, 9). A recent addition is an infrared fluorescent PCA that, unlike previous fluorescent PCAs, exhibits reversible complementation, thus enabling spatiotemporal analysis of dynamic PPIs (10). Another binary interaction assay, luminescence-based mammalian interactome mapping (LUMIER), has been applied to map TGFβ induced modulation of PPIs with components of the TGFβ signaling pathway (11). MaMTH, a mammalian version of the split ubiquitin approach, was designed particularly for the analysis of PPIs involving integral membrane proteins, also allowing the detection of functional PPI modulation (12). Efforts to apply purification-based methods for detecting context-dependent PPI modulation recently resulted in the development of AP-SRM (13) and AP-SWATH (14).Our group previously conceived mammalian protein-protein interaction trap (MAPPIT) (supplemental Fig. S1A) (15, 16), a mammalian two-hybrid approach based on complementation of a cytokine receptor that was developed into a broad platform for PPI analysis (17, 18), screening for small molecule PPI disruptors (19, 20) and drug target profiling (21, 22). Although MAPPIT operates in intact human cells, thus providing the natural environment for human protein analysis, the interaction sensor is anchored to the plasma membrane, precluding the analysis of PPIs at their native subcellular localization. In addition, MAPPIT is incompatible with full size transmembrane proteins. Here we describe KInase Substrate Sensor (KISS), a novel binary PPI mapping approach that enables in situ analysis in living mammalian cells of protein interactions and their responses to physiological or pharmacological challenges.     

Accuracy of Ultrasonography and Magnetic Resonance Imaging in the Diagnosis of Placenta Accreta

Purpose

To evaluate the accuracy of ultrasonography and magnetic resonance imaging (MRI) in the diagnosis of placenta accreta and to define the most relevant specific ultrasound and MRI features that may predict placental invasion.

Material and Methods

This study was approved by the institutional review board of the French College of Obstetricians and Gynecologists. We retrospectively reviewed the medical records of all patients referred for suspected placenta accreta to two university hospitals from 01/2001 to 05/2012. Our study population included 42 pregnant women who had been investigated by both ultrasonography and MRI. Ultrasound images and MRI were blindly reassessed for each case by 2 raters in order to score features that predict abnormal placental invasion.

Results

Sensitivity in the diagnosis of placenta accreta was 100% with ultrasound and 76.9% for MRI (P = 0.03). Specificity was 37.5% with ultrasonography and 50% for MRI (P = 0.6). The features of greatest sensitivity on ultrasonography were intraplacental lacunae and loss of the normal retroplacental clear space. Increased vascularization in the uterine serosa-bladder wall interface and vascularization perpendicular to the uterine wall had the best positive predictive value (92%). At MRI, uterine bulging had the best positive predictive value (85%) and its combination with the presence of dark intraplacental bands on T2-weighted images improved the predictive value to 90%.

Conclusion

Ultrasound imaging is the mainstay of screening for placenta accreta. MRI appears to be complementary to ultrasonography, especially when there are few ultrasound signs.     

Alternatives to the Six-Minute Walk Test in Pulmonary Arterial Hypertension

Introduction

The physiological response during the endurance shuttle walk test (ESWT), the cycle endurance test (CET) and the incremental shuttle walk test (ISWT) remains unknown in PAH. We tested the hypothesis that endurance tests induce a near-maximal physiological demand comparable to incremental tests. We also hypothesized that differences in respiratory response during exercise would be related to the characteristics of the exercise tests.

Methods

Within two weeks, twenty-one PAH patients (mean age: 54(15) years; mean pulmonary arterial pressure: 42(12) mmHg) completed two cycling exercise tests (incremental cardiopulmonary cycling exercise test (CPET) and CET) and three field tests (ISWT, ESWT and six-minute walk test (6MWT)). Physiological parameters were continuously monitored using the same portable telemetric device.

Results

Peak oxygen consumption (VO_2peak) was similar amongst the five exercise tests (p = 0.90 by ANOVA). Walking distance correlated markedly with the VO_2peak reached during field tests, especially when weight was taken into account. At 100% exercise, most physiological parameters were similar between incremental and endurance tests. However, the trends overtime differed. In the incremental tests, slopes for these parameters rose steadily over the entire duration of the tests, whereas in the endurance tests, slopes rose sharply from baseline to 25% of maximum exercise at which point they appeared far less steep until test end. Moreover, cycling exercise tests induced higher respiratory exchange ratio, ventilatory demand and enhanced leg fatigue measured subjectively and objectively.

Conclusion

Endurance tests induce a maximal physiological demand in PAH. Differences in peak respiratory response during exercise are related to the modality (cycling vs. walking) rather than the progression (endurance vs. incremental) of the exercise tests.     

The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase

The activation of monocytes involves a stimulation of glycolysis, release of potent inflammatory mediators, and alterations in gene expression. All of these processes are known to be further increased under hypoxic conditions. The activated monocytes express inducible 6-phosphofructo-2-kinase (iPFK-2), which synthesizes fructose 2,6-bisphosphate, a stimulator of glycolysis. During ischemia, AMP-activated protein kinase (AMPK) activates the homologous heart 6-phosphofructo-2-kinase isoform by phosphorylating its Ser-466. Here, we studied the involvement of AMPK and iPFK-2 in the stimulation of glycolysis in activated monocytes under hypoxia. iPFK-2 was phosphorylated on the homologous serine (Ser-461) and activated by AMPK in vitro. The activation of human monocytes by lipopolysaccharide induced iPFK-2 expression and increased fructose 2,6-bisphosphate content and glycolysis. The incubation of activated monocytes with oligomycin, an inhibitor of oxidative phosphorylation, or under hypoxic conditions activated AMPK and further increased iPFK-2 activity, fructose 2,6-bisphosphate content, and glycolysis. In cultured human embryonic kidney 293 cells, the expression of a dominant-negative AMPK prevented both the activation and phosphorylation of co-transfected iPFK-2 by oligomycin. It is concluded that the stimulation of glycolysis by hypoxia in activated monocytes requires the phosphorylation and activation of iPFK-2 by AMPK.     

Agrin binds to the N-terminal region of Lrp4 protein and stimulates association between Lrp4 and the first immunoglobulin-like domain in muscle-specific kinase (MuSK)

Neuromuscular synapse formation depends upon coordinated interactions between motor neurons and muscle fibers, leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. Synapse formation begins as motor axons approach muscles that are prepatterned in the prospective synaptic region in a manner that depends upon Lrp4, a member of the LDL receptor family, and muscle-specific kinase (MuSK), a receptor tyrosine kinase. Motor axons supply Agrin, which binds Lrp4 and stimulates further MuSK phosphorylation, stabilizing nascent synapses. How Agrin binds Lrp4 and stimulates MuSK kinase activity is poorly understood. Here, we demonstrate that Agrin binds to the N-terminal region of Lrp4, including a subset of the LDLa repeats and the first of four β-propeller domains, which promotes association between Lrp4 and MuSK and stimulates MuSK kinase activity. In addition, we show that Agrin stimulates the formation of a functional complex between Lrp4 and MuSK on the surface of myotubes in the absence of the transmembrane and intracellular domains of Lrp4. Further, we demonstrate that the first Ig-like domain in MuSK, which shares homology with the NGF-binding region in Tropomyosin Receptor Kinase (TrKA), is required for MuSK to bind Lrp4. These findings suggest that Lrp4 is a cis-acting ligand for MuSK, whereas Agrin functions as an allosteric and paracrine regulator to promote association between Lrp4 and MuSK.     

The effects of a change in gravity on the dynamics of prehension

The objective of this study was to measure the forces applied on an object manipulated in different gravitational fields attained during parabolic flights. Eight subjects participated flights (ES) and four were inexperienced (NES). They had to move continuously an instrumented object up and down in three different gravitational conditions (1 g, 1.8 g, 0 g). In 1 g, the grip force precisely anticipated the fluctuations of load force which was maximum and minimum at the bottom and at the top of the arm trajectory respectively. When the gravity changed (0 g and 1.8 g), the grip-load force coupling persisted for all the subjects from the first parabola. While the ES immediately exerted a grip force appropriate to the gravity, the NES dramatically increased their grip when faced with hyper and microgravity for the first time. Then, they progressively released their grip until a continuous grip-load force relationship with regard to 1 g was established after the fifth parabola. We suggest that each new gravitational field is rapidly incorporated into an internal model within the CNS which can then be reused as required by the occasion.