Global Approaches to the Role of miRNAs in Drug-Induced Changes in Gene Expression

Neurons modulate gene expression with subcellular precision through excitation-coupled local protein synthesis, a process that is regulated in part through the involvement of microRNAs (miRNAs), a class of small non-coding RNAs. The biosynthesis of miRNAs is reviewed, with special emphasis on miRNA families, the subcellular localization of specific miRNAs in neurons, and their potential roles in the response to drugs of abuse. For over a decade, DNA microarrays have dominated genome-wide gene expression studies, revealing widespread effects of drug exposure on neuronal gene expression. We review a number of recent studies that explore the emerging role of miRNAs in the biochemical and behavioral responses to cocaine. The more powerful next-generation sequencing technology offers certain advantages and is supplanting microarrays for the analysis of complex transcriptomes. Next-generation sequencing is unparalleled in its ability to identify and quantify low-abundance transcripts without prior sequence knowledge, facilitating the accurate detection and quantification of miRNAs expressed in total tissue and miRNAs localized to postsynaptic densities (PSDs). We previously identified cocaine-responsive miRNAs, synaptically enriched and depleted miRNA families, and confirmed cocaine-induced changes in protein expression for several bioinformatically predicted target genes. The miR-8 family was found to be highly enriched and cocaine-regulated at the PSD, where its members may modulate expression of cell adhesion molecules. An integrative approach that combines mRNA, miRNA, and protein expression profiling in combination with focused single gene studies and innovative behavioral paradigms should facilitate the development of more effective therapeutic approaches to treat addiction

Older women\u27s perceived independence in post-widowhood repartnership

This study investigated older women\u27s perceived independence in late-life post-widowhood repartnerships and sought to better understand the ways in which repartnership impacted these women\u27s sense of independence. Using a qualitative, exploratory design, the study adds to this relatively unexplored topic. Heterosexual women who had lost a spouse and become re-partnered at the age of 65 or older were recruited from across Massachusetts. Ten repartnered women ages 73-93 participated in this study. In face to face interviews they were asked a series of questions focused on the following themes: 1) negotiation of shared and separate time, activities, space, resources, tasks and friendships in re-partnership; 2) decision making and compromise in re-partnership; 3) perceived independence in re-partnership; 4) perceived independence in previous marriage; and 5) perceived independence during widowhood and time spent alone. Participants had multiple subjective definitions of independence. The majority felt independence could be maintained while choosing to make compromises in order to have companionate romantic relationships that impacted aspects of their autonomy. Others created stricter boundaries within romantic relationships to protect the autonomy gained during widowhood. Nearly all participants described a strong sense of perceived independence in their repartnerships and reported that this was stronger than in their previous marriages

O sistema produtivo de flores e plantas ornamentais e sua relação com a cidade de Corupá e a micro-região do Norte do Estado de SC

TCC (graduação) - Universidade Federal de Santa Catarina. Centro Tecnológico. ArquiteturaTCC sem resumo

Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation

<p>Abstract</p> <p>Background</p> <p>Many studies implicate Arf6 activity in Rac-mediated membrane ruffling and cytoskeletal reorganization. Although Arf6 facilitates the trafficking of Rac1 to the plasma membrane and in many cases Arf6 activation leads to the activation of Rac1, the details of how Arf6 influences Rac function remain to be elucidated.</p> <p>Results</p> <p>We demonstrate in binding assays and by co-immunoprecipitation that GDP-bound Arf6 binds to Kalirin5, a Rho family guanine nucleotide exchange factor, through interaction with the spectrin repeat region. In cells, expression of wild type Arf6 recruits spectrin repeat 5 and Kalirin to the plasma membrane and leads to enhanced Kalirin5-induced ruffling. By contrast, expression of an Arf6 mutant that cannot become activated, Arf6 T27N, still recruits spectrin repeat 5 and Kalirin to membranes but inhibits Kalirin5-induced ruffling in HeLa cells. Kalirin5-induced Rac1 activation is increased by the expression of wild type Arf6 and decreased by Arf6T27N. Furthermore, expression of a catalytically-inactive mutant of Kalirin5 inhibits cytoskeletal changes observed in cells expressing EFA6, an Arf6 guanine nucleotide exchange factor that leads to activation of Rac.</p> <p>Conclusion</p> <p>We show here with over-expressed proteins that the GDP-bound form of Arf6 can bind to the spectrin repeat regions in Kalirin Rho family GEFs thereby recruiting Kalirin to membranes. Although Kalirin is recruited onto membranes by Arf6-GDP, subsequent Rac activation and membrane ruffling requires Arf6 activation. From these results, we suggest that Arf6 can regulate through its GTPase cycle the activation of Rac.</p

Kalirin12 interacts with dynamin

<p>Abstract</p> <p>Background</p> <p>Guanine nucleotide exchange factors (GEFs) and their target Rho GTPases regulate cytoskeletal changes and membrane trafficking. Dynamin, a large force-generating GTPase, plays an essential role in membrane tubulation and fission in cells. Kalirin12, a neuronal RhoGEF, is found in growth cones early in development and in dendritic spines later in development.</p> <p>Results</p> <p>The IgFn domain of Kalirin12, not present in other Kalirin isoforms, binds dynamin1 and dynamin2. An inactivating mutation in the GTPase domain of dynamin diminishes this interaction and the isolated GTPase domain of dynamin retains the ability to bind Kalirin12. Co-immunoprecipitation demonstrates an interaction of Kalirin12 and dynamin2 in embryonic brain. Purified recombinant Kalirin-IgFn domain inhibits the ability of purified rat brain dynamin to oligomerize in response to the presence of liposomes containing phosphatidylinositol-4,5-bisphosphate. Consistent with this, expression of exogenous Kalirin12 or its IgFn domain in PC12 cells disrupts clathrin-mediated transferrin endocytosis. Similarly, expression of exogenous Kalirin12 disrupts transferrin endocytosis in cortical neurons. Expression of Kalirin7, a shorter isoform which lacks the IgFn domain, was previously shown to inhibit clathrin-mediated endocytosis; the GTPase domain of dynamin does not interact with Kalirin7.</p> <p>Conclusion</p> <p>Kalirin12 may play a role in coordinating Rho GTPase-mediated changes in the actin cytoskeleton with dynamin-mediated changes in membrane trafficking.</p

Kalirin Dbl-Homology Guanine Nucleotide Exchange Factor 1 Domain Initiates New Axon Outgrowths via RhoG-Mediated Mechanisms

The large multidomain Kalirin and Trio proteins containing dual Rho GTPase guanine nucleotide exchange factor (GEF) domains have been implicated in the regulation of neuronal fiber extension and pathfinding during evelopment. In mammals, Kalirin is expressed predominantly in the nervous system, whereas Trio, broadly expressed throughout the body, is expressed at a lower level in the nervous system. To evaluate the role of Kalirin in fiber initiation and outgrowth, we microinjected cultured sympathetic neurons with vectors encoding Kalirin or with Kalirin antisense oligonucleotides, and we assessed neuronal fiber growth in a serum-free, satellite cell-free environment. Kalirin antisense oligonucleotides blocked the continued extension of preexisting axons. Kalirin overexpression induced the prolific sprouting of new axonal fibers that grew at the normal rate; the activity of Kalirin was entirely dependent on the activity of the first GEF domain. KalGEF1-induced sprouting of new fibers from lamellipodial structures was accompanied by extensive actin cytoskeleton reorganization. The kalGEF1 phenotype was mimicked by constitutively active RhoG and was blocked by RhoG inhibitors. Constitutively active Rac1, RhoA, and Cdc42 were unable to initiate new axons, whereas dominant-negative Rac1, RhoA, and Cdc42 failed to block axon sprouting. Thus Kalirin, acting via RhoG in a novel manner, plays a central role in establishing the morphological phenotypic diversity that is essential to the connectivity of the developing nervous system

Coordination of peroxide to the CuM center of peptidylglycine α-hydroxylating monooxygenase (PHM): Structural and computational study

Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine a-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction-the hydroxylation of peptidylglycine substrates at the Ca position of the terminal glycine. The hydroxylation reaction is copper- and O2-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests thatO2 is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (CuH and CuM). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-A° -resolution structure (hydro)peroxide binds solely to CuM in a slightly asymmetric side-on mode. The O-O interatomic distance of the copperbound ligand is 1.5 A ° , characteristic of peroxide/hydroperoxide species, and the Cu-O distances are 2.0 and 2.1 A ° . Density functional theory calculations using the first coordination sphere of the CuM active site as a model system show that the computed energies of the side-on L3CuM(II)-O2 2- species and its isomeric, end-on structure L3CuM(I)-O2 - are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L3CuM(II)O2 2- is in good agreement with the results of a hybrid quantum mechanical-molecular mechanical optimization of this species.Fil: Rudzka, Katarzyna. University Johns Hopkins; Estados UnidosFil: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; Argentina. Universidad de Buenos Aires; ArgentinaFil: Eipper, Betty. University Of Connecticut; Estados UnidosFil: Mains, Richard. University Of Connecticut; Estados UnidosFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires; ArgentinaFil: Amzel, L. Mario. University Johns Hopkins; Estados Unido

The role of the Bcl-2 family in proliferation and apoptosis and in mediating the development of lymphatic diseases

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.Includes bibliographical references (leaves [54]-[66]).The development of the immune system is a highly dynamic process, characterized by quickly and frequently changing cell types and numbers. The orchestration of cell growth and proliferation and also of cell death is a necessarily complex process, taking cues from a wide variety of sources. The nematode Caenorhabditus elegans has provided an elegant and simple model of the control of programmed cell death, or apoptosis, in metazoans. Apoptosis in mammals is regulated by pathways related to but more intricate than metazoans. Several key features define the onset of apoptosis in any given cell; these include DNA fragmentation and chromatin condensation, "blebbing" of the plasma membrane, and subsequent phagocytosis of the resulting cell fragments by adjacent cells (Kerr et al. 1972 and Wyllie et al. 1980).by Marcie A. Eipper-Mains.S.M