Dephosphorylation of clustered phosphoserine residues in human Grb14 by protein phosphatase 1 and its effect on insulin receptor complex formation

The physical interaction of the human growth factor receptor‐bound protein 14 (hGrb14) and the insulin receptor (IR) represses insulin signaling. With respect to the recruiting mechanism of hGrb14 to IR respond to insulin stimulus, our previous reports have suggested that phosphorylation of Ser³⁵⁸, Ser³⁶², and Ser³⁶⁶ in hGrb14 by glycogen synthase kinase‐3 repressed hGrb14–IR complex formation. In this study, we investigated phosphatase‐mediated dephosphorylation of the hGrb14 phosphoserine residues. An in vitro phosphatase assay with hGrb14‐derived synthetic phosphopeptides suggested that protein phosphatase 1 (PP1) is involved in the dephosphorylation of Ser³⁵⁸ and Ser³⁶². Furthermore, coimmunoprecipitation experiments suggested that insulin‐induced hGrb14–IR complex formation was repressed by the substitution of Ser³⁵⁸ or Ser³⁶² with glutamic acid. These findings suggested that phosphate groups on Ser³⁵⁸ and Ser³⁶² in hGrb14 are dephosphorylated by PP1, and the dephosphorylation facilitates hGrb14–IR complex formation.     

Characterization and systematic implications of the diversity in timing of programmed cell death of the suspensors in Leguminosae

? Premise of the study: In angiosperm seeds, the developing embryo acquires nutrients via a suspensor that typically undergoes programmed cell death (PCD) at the early cotyledon stage. However, in Leguminosae (the third largest angiosperm family), the suspensors can disappear at the heart-shaped stage (i.e., prior to the cotyledon stage) or still persist at the cotyledon stage. Here, in a comprehensive survey of legume suspensors and embryos, the variation and the evolutionary direction of timing of suspensor PCD in Leguminosae were characterized, and systematic implications were evaluated. ? Methods: Suspensor development and morphology for 66 leguminous species from 49 genera, 21 tribes, and 3 subfamilies were comparatively studied using standard paraffin sectioning and light microscopy. ? Key results: Three patterns of suspensor PCD were observed at the early cotyledon stage. (A) The suspensor persisted. (B) The suspensor separated from the wall of the embryo sac and persisted as a vestige at the radicle apex. (C) The suspensor disappeared completely, and the absorption of nutrients by embryo was carried out via a "contact zone" between the embryo and the endosperm. Pattern C of early suspensor PCD was found only in the tribe Fabeae. An ancestral character reconstruction revealed that the long-lived suspensors of pattern A represented a plesiomorphic condition in Leguminosae and that the suspensors of pattern C evolved only once in the common ancestor of Fabeae. ? Conclusions: In Leguminosae, short-lived suspensors have thus evolved multiple times from long-lived suspensors. It remains largely unknown, however, how the embryo acquires nutrients after the early suspensor PCD.     

Venestatin from parasitic helminths interferes with receptor for advanced glycation end products (RAGE)-mediated immune responses to promote larval migration

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca²⁺-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca²⁺-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.     

Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes

Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.     

Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia

In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. Here, we show the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerated as development proceeded, and completed a full 360° rotation. This acceleration was impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that formed a ring shape and surrounded the male genitalia: the inner ring rotated with the genitalia and the outer ring rotated later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlated with the movement of the outer ring, and upregulation of the apoptotic signal increased the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame.     

AS1411 aptamer tagged PLGA-lecithin-PEG nanoparticles for tumor cell targeting and drug delivery

Liposomes and polymers are widely used drug carriers for controlled release since they offer many advantages like increased treatment effectiveness, reduced toxicity and are of biodegradable nature. In this work, anticancer drug‐loaded PLGA‐lecithin‐PEG nanoparticles (NPs) were synthesized and were functionalized with AS1411 anti‐nucleolin aptamers for site‐specific targeting against tumor cells which over expresses nucleolin receptors. The particles were characterized by transmission electron microscope (TEM) and X‐ray photoelectron spectroscopy (XPS). The drug‐loading efficiency, encapsulation efficiency and in vitro drug release studies were conducted using UV spectroscopy. Cytotoxicity studies were carried out in two different cancer cell lines, MCF‐7 and GI‐1 cells and two different normal cells, L929 cells and HMEC cells. Confocal microscopy and flowcytometry confirmed the cellular uptake of particles and targeted drug delivery. The morphology analysis of the NPs proved that the particles were smooth and spherical in shape with a size ranging from 60 to 110 nm. Drug‐loading studies indicated that under the same drug loading, the aptamer‐targeted NPs show enhanced cancer killing effect compared to the corresponding non‐targeted NPs. In addition, the PLGA‐lecithin‐PEG NPs exhibited high encapsulation efficiency and superior sustained drug release than the drug loaded in plain PLGA NPs. The results confirmed that AS1411 aptamer‐PLGA‐lecithin‐PEG NPs are potential carrier candidates for differential targeted drug delivery. Biotechnol. Bioeng. 2012; 109: 2920–2931. © 2012 Wiley Periodicals, Inc.     

Rapamycin inhibits transforming growth factor β-induced peritoneal angiogenesis by blocking the secondary hypoxic response

Patients with end-stage kidney disease on peritoneal dialysis often develop progressive scarring of the peritoneal tissues. This manifests as submesothelial thickening and is associated with increased vascularization that leads to ultrafiltration dysfunction. Hypoxia induces a characteristic series of responses including angiogenesis and fibrosis. We investigated the role of hypoxia in peritoneal membrane damage. An adenovirus expressing transforming growth factor (TGF) β was used to induce peritoneal fibrosis. We evaluated the effect of the mTOR inhibitor rapamycin, which has been previously shown to block hypoxia-inducible factor (HIF) 1α. We also assessed the effect of HIF1α independently using an adenovirus expressing active HIF1α. To identify the TGFβ1-independent effects of HIF1α, we expressed HIF1α in the peritoneum of mice lacking the TGFβ signalling molecule Smad3. We demonstrate that TGFβ-induced fibroproliferative tissue is hypoxic. Rapamycin did not affect the early angiogenic response, but inhibited angiogenesis and submesothelial thickening 21 days after induction of fibrosis. In primary mesothelial cell culture, rapamycin had no effect on TGFβ-induced vascular endothelial growth factor (VEGF) but did suppress hypoxia-induced VEGF. HIF1α induced submesothelial thickening and angiogenesis in peritoneal tissue. The fibrogenic effects of HIF1α were Smad3 dependent. In summary, submesothelial hypoxia may be an important secondary factor, which augments TGFβ-induced peritoneal injury. The hypoxic response is mediated partly through HIF1α and the mTOR inhibitor rapamycin blocks the hypoxic-induced angiogenic effects but does not affect the direct TGFβ-mediated fibrosis and angiogenesis.     

Diversification of seed arrangement induced by ovule rotation and septum formation in Leguminosae

The plants of Leguminosae, which comprises 731 genera, bear monocarpellary flowers. The fruit, known as a pod, generally contains a row of seeds, and the internal structure appears to be simple. In Leguminosae, the seeds are generally arranged longitudinally along the suture of the pod but, in a few genera, namely, Cassia, Indigofera, and Senna, they are arranged transversely. Transverse seed arrangement facilitates the accommodation of a considerable number of seeds and has been considered to be established by rotation of seeds during development. We studied the development both these patterns of seed arrangement, i.e., longitudinal and transverse, in Senna and Indigofera in order to elucidate the structural changes occurring in ovaries (or pods) and determine the forces responsible for rotation of ovules (or seeds) by dissecting ovaries and pods at different developmental stages. Our results showed that at an early developmental stage the ovules were obliquely oriented or transversely arranged; later, the direction of their growth was restricted by the ovary walls or neighboring ovules, and therefore the obliquely oriented ovules rotated inward or outward. Thus, it was clarified that the processes involved in the formation of the internal structure of pods are regulated in a complex manner. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.