Plant regeneration in vitro from immature embryos of lesser burnet (Sanguisorba minor Scop.)

Efficient and reproducible shoot regeneration has been established from immature zygotic embryos of lesser burnet on MS medium containing 4μM BAP and 10μM NAA. Regenerated shoots were best rooted in half-strength MS medium supplemented with 10μM NAA and later established in the greenhouse

Regulation of mTOR complex 1 in response to growth factors and nutrients

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections."June 2010." Cataloged from student submitted PDF version of thesis.Includes bibliographical references.In multicellular organisms, cells ensure the simultaneous availability of growth factors and nutrients before they invest in cellular processes that lead to growth. The TOR kinase is a master regulator of cellular growth and nucleates two distinct protein complexes known as TOR complex 1 and 2 (TORC1 and 2). The activity of TORC2 is mainly regulated by growth factors, whereas TORC1 activity is responsive to both growth factors and nutrients, and thus acts as a detector of favorable growth conditions. The consequence of TORC1 activation is increased protein translation through its substrates S6K and 4E-BP1. Many of the upstream signals that lead to activation of TOR pathway are tumor suppressors and deregulation of the pathway leads to disease. We aim to better understand the regulation of mammalian TORC1 (mTORC1) by upstream signals. In the work described here, we identified PRAS40 as a new component of mTORC1. PRAS40 is phosphorylated in response to growth factors and this phosphorylation event leads to mTORC1 activation. We also showed that the small GTPase Rheb, a major upstream activator of mTORC1, activates it by directly interacting with mTORC1. Both PRAS40 and Rheb relay information from growth factors to mTORC1, and do not seem to be regulated by nutrients, particularly amino acids. In this work, we also investigated how amino acids lead to mTORC1 activation. We showed that amino acids activate mTORC1 by recruiting the complex to lysosomal surface through the action of evolutionarily conserved Rag GTPases. We also indentified a complex of three proteins (p14, p18 and MP1), that we call the "Ragulator" complex, as Rag GTPase interacting proteins. The Ragulator itself localizes to lysosomes through lipid modifications on p18, and tethers the Rag GTPases on the lysosomal surface. We speculate that amino acid-induced lysosomal localization of mTORC1 enables its encounter with Rheb. Through this work, we propose a model for growth factor and amino acid-induced mTORC1 activation.by Yasemin S. Sancak.Ph.D

DEPTOR Is an mTOR Inhibitor Frequently Overexpressed in Multiple Myeloma Cells and Required for Their Survival

The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. We identify DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of multiple myelomas harboring cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, we identify a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival.Howard Hughes Medical Institute (Investigator)Dana-Farber Cancer Institute (High-Tech Research Fund)National Cancer Institute (U.S.)National Institutes of Health (U.S.) (Intramural Research Program)American Cancer SocietyCanadian Institutes of Health Research (Fellowship)American Diabetes Association (Fellowship)W. M. Keck FoundationNational Institutes of Health (U.S.) (R01 CA103866)National Institutes of Health (U.S.) (NIH; R01 AI47389

Analyses of the mechanical, electrical and electromagnetic shielding properties of thermoplastic composites doped with conductive nanofillers

The purpose of this study is to observe effect of incorporating vapor-grown carbon nanofibers with various amounts in polyvinylidene fluoride matrix in terms of mechanical strength and electromagnetic shielding effectiveness. Thermoplastic conductive nanocomposites were prepared by heat-pressed compression molding. Vapor-grown carbon nanofibers were utilized at various weight ratios (1 wt.%, 3 wt.%, 5 wt.%, and 8 wt.%) as conductive and reinforcing materials. Polyvinylidene fluoride was used as a thermoplastic polymer matrix. Scanning electron microscopic analysis was conducted in order to characterize the morphology and structural properties of the nanocomposites and results revealed well dispersion of carbon nanofibers within the matrix for all concentrations. Mechanical characteristics were investigated according to standards. Findings proved that overall increments of 16%, 37.5%, and 56% were achieved in terms of tensile strength, elasticity modulus, and impact energy, respectively, where a total reduction of 44.8% was observed in terms of elongation for 8 wt.% vapor-grown nanofiber matrix compared to that of 0 wt.%. Electromagnetic shielding effectiveness's of the nanocomposites were determined by standard protocol using coaxial transmission line measurement technique in the frequency range of 15–3000 MHz. It was observed that resistance, sheet resistance, and resistivity of nanocomposites depicted substantial reduction with the increment in nanofiber content. Nevertheless, it was observed that nanofiber content, dispersion, and network formation within the composites were highly influent on the electromagnetic shielding effectiveness performance of the structures

GSK3-mediated raptor phosphorylation supports amino acid-dependent Q2 mTORC1-directed signalling

The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser(859). We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation

Prevalence of daytime urinary incontinence and related risk factors in primary school children in Turkey

Purpose: Urinary incontinence is one of the major urinary symptoms in children and adolescents and can lead to major distress for the affected children and their parents. In accordance with the definitions of the Standardization Committee of the International Children's Continence Society, daytime urinary incontinence (DUI) is uncontrollable leakage of urine during the day. The aim of this cross-sectional study was to investigate the prevalence and associated risk factors of DUI in Turkish primary school children. Materials and Methods: The questionnaire, which covered sociodemographic variables and the voiding habits of the children, was completed by the parents of 2,353 children who were attending primary school in Denizli, a developing city of Turkey. The children's voiding habits were evaluated by use of the Dysfunctional Voiding and Incontinence Symptoms Score, which is a validated questionnaire. Children with a history of neurological or urological diseases were excluded. Results: The participation rate was 91.9% (2,164 people). The overall prevalence of DUI was 8.0%. The incidence of DUI tended to decrease with increasing age and was not significantly different between genders (boys, 8.8%; girls, 7.3%; p=0.062). Age, maternal education level, family history of daytime wetting, settlement (urban/rural), history of constipation, urinary tract infection, and urgency were independent risk factors of DUI. Conclusions: Our findings showed that DUI is a common health problem in primary school children. In an effort to increase awareness of children's voiding problems and the risk factors for urinary dysfunction in the population, educational programs and larger school-based screening should be carried out, especially in regions with low socioeconomic status. © The Korean Urological Association, 2014

Functional characterisation of the TSC1–TSC2 complex to assess multiple TSC2 variants identified in single families affected by tuberous sclerosis complex

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by seizures, mental retardation and the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). METHODS: We have used a combination of different assays to characterise the effects of a number of pathogenic TSC2 amino acid substitutions on TSC1-TSC2 complex formation and mTOR signalling. RESULTS: We used these assays to compare the effects of 9 different TSC2 variants (S132C, F143L, A196T, C244R, Y598H, I820del, T993M, L1511H and R1772C) identified in individuals with symptoms of TSC from 4 different families. In each case we were able to identify the pathogenic mutation. CONCLUSION: Functional characterisation of TSC2 variants can help identify pathogenic changes in individuals with TSC, and assist in the diagnosis and genetic counselling of the index cases and/or other family members

Crystal structure of the Ego1-Ego2-Ego3 complex and its role in promoting Rag GTPase-dependent TORC1 signaling

The target of rapamycin complex 1 (TORC1) integrates various hormonal and nutrient signals to regulate cell growth, proliferation, and differentiation. Amino acid-dependent activation of TORC1 is mediated via the yeast EGO complex (EGOC) consisting of Gtr1, Gtr2, Ego1, and Ego3. Here, we identify the previously uncharacterized Ycr075w-a/Ego2 protein as an additional EGOC component that is required for the integrity and localization of the heterodimeric Gtr1-Gtr2 GTPases, equivalent to mammalian Rag GTPases. We also report the crystal structure of the Ego1-Ego2-Ego3 ternary complex (EGO-TC) at 2.4 Å resolution, in which Ego2 and Ego3 form a heterodimer flanked along one side by Ego1. Structural data also reveal the structural conservation of protein components between the yeast EGO-TC and the human Ragulator, which acts as a GEF for Rag GTPases. Interestingly, however, artificial tethering of Gtr1-Gtr2 to the vacuolar membrane is sufficient to activate TORC1 in response to amino acids even in the absence of the EGO-TC. Our structural and functional data therefore support a model in which the EGO-TC acts as a scaffold for Rag GTPases in TORC1 signaling

The Mitochondrial Ca(2+) Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca(2+) uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca(2+) uptake and our current understanding of mitochondrial Ca(2+) homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca(2+) uniporter complex