RESUMEN
BACKGROUND: Atopic dermatitis (AD) is a common, complex, and highly heritable inflammatory skin disease. Genome-wide association studies offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between 2 candidate genes, EMSY and LRRC32 (leucine-rich repeat-containing 32) but the functional mechanisms affecting risk of AD remain unclear. OBJECTIVES: We sought to apply a combination of genomic and molecular analytic techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease. METHODS: We used interrogation of available genomic and chromosome conformation data in keratinocytes, small interfering RNA (siRNA)-mediated knockdown in skin organotypic culture and functional assessment of barrier parameters, mass spectrometric global proteomic analysis and quantitative lipid analysis, electron microscopy of organotypic skin, and immunohistochemistry of human skin samples. RESULTS: Genomic data indicate active promoters in the genome-wide association study locus and upstream of EMSY; EMSY, LRRC32, and intergenic variants all appear to be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, overexpression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, which is consistent with an increased functional effect of this transcriptional control protein. CONCLUSION: Our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach.
Asunto(s)
Dermatitis Atópica/inmunología , Regulación de la Expresión Génica/inmunología , Proteínas de Neoplasias/inmunología , Proteínas Nucleares/inmunología , Proteínas Represoras/inmunología , Piel/inmunología , Transcripción Genética/inmunología , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 11/inmunología , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Femenino , Proteínas Filagrina , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Represoras/genética , Piel/patologíaRESUMEN
MicroRNAs (miRNAs) are short (21-23nt long) RNAs that post-transcriptionally regulate gene expression in plants and animals. They are key regulators in all biological processes. In mammalian cells miRNAs are loaded into one of the four members of the Argonaute (Ago) protein family to form the RNA-induced silencing complex (RISC). RISCs inhibit the translation of mRNAs that share sequence complementarity with their loaded miRNAs. miRNA processing and miRNA-mediated gene regulation are highly regulated processes and involve many RNA-binding proteins as auxiliary factors. Here we show that the two RNA-binding proteins, p72 and KHSRP, both with known roles in promoting miRNA biogenesis, regulate the protein level of human Ago2 in transformed human cells. We determined that p72 and KHSRP influence Ago2 stability by regulating miRNA levels in the cell and that loss of p72/KHSRP results in a decrease of unloaded Ago2.
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Proteínas Argonautas/genética , ARN Helicasas DEAD-box/genética , MicroARNs/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Transactivadores/genética , Proteínas Argonautas/metabolismo , Línea Celular Tumoral , ARN Helicasas DEAD-box/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Transducción de Señal , Transactivadores/metabolismo , TransfecciónRESUMEN
Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , MicroARNs/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/fisiología , Animales , Secuencia de Bases , Benzamidas/farmacología , Northern Blotting , Factor Neurotrófico Derivado del Encéfalo/farmacología , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Datos de Secuencia Molecular , Neuronas/metabolismo , Neuronas/microbiología , Técnicas de Amplificación de Ácido Nucleico , Fosforilación/genética , Fosforilación/fisiología , Reacción en Cadena de la Polimerasa , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.
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Janus Quinasa 3/genética , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Técnicas de Sustitución del Gen , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Janus Quinasa 3/química , Janus Quinasa 3/metabolismo , Ratones , Modelos Genéticos , Dominios Proteicos , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg8 > Arg6 â« Arg4. The conjugate with Arg8 exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp-Arg8-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp-Arg8-1 and 17-desacetylvinblastineTrp-Arg8-2 are epimers at the tryptophan α carbon atom.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Arginina/análogos & derivados , Arginina/farmacología , Vinblastina/química , Antineoplásicos/química , Arginina/química , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Células HeLa , Humanos , Estructura Molecular , EstereoisomerismoRESUMEN
The conjugate of acid labile cis-aconityl-daunomycin (cAD) with branched chain polypeptide, poly[Lys(Glui-DL-Alam)] (EAK) was very effective against L1210 leukemia in mice. However, Dau attached to a polycationic polypeptide, poly[Lys(Seri-DL-Alam)] (SAK) exhibited no in vivo antitumor effect. In order to understand this difference we have performed comparative in vitro studies to dissect properties related to interaction with the whole body (e.g., biodistribution) from those present at cellular or even molecular level. We report here (a) the kinetics of acid-induced Dau liberation, (b) interaction with DPPC phospholipid bilayer, (c) in vitro cytotoxic effect on different tumor cells, and (d) intracellular distribution in HL-60 cells of polycationic (cAD-SAK) and amphoteic (cAD-EAK) conjugates. Fluorescence properties of the two conjugates are also reported. Our findings demonstrate that the kinetics of the drug release, intracellular distribution and in vitro cytotoxic effect are rather similar, while the effect on DPPC phospholipid bilayer and fluorescence properties of the two conjugates are not the same. We also found that the in vitro cytotoxicity is cell line dependent. These observations suggest that the structure of the polypeptide carrier could have marked influence on drug uptake related events.
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Daunorrubicina/análogos & derivados , Portadores de Fármacos/química , Péptidos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Daunorrubicina/química , Daunorrubicina/metabolismo , Células HL-60 , Humanos , Péptidos y Proteínas de Señalización Intercelular , Leucemia L1210/tratamiento farmacológico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Liposomas/química , Ratones , Estructura Molecular , Proteínas/química , Células Tumorales CultivadasRESUMEN
The immediate early gene activity-regulated cytoskeletal protein (Arc)/Arg3.1 and the neurotrophin brain-derived neurotrophic factor (BDNF) play important roles in synaptic plasticity and learning and memory in the mammalian brain. However, the mechanisms by which BDNF regulates the expression of Arc/Arg3.1 are unclear. In this study, we show that BDNF acts via the ERK1/2 pathway to activate the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 then induces Arc/Arg3.1 expression via the phosphorylation of histone H3 at the Arc/Arg3.1 promoter. MSK1 can also phosphorylate the transcription factor cyclic-AMP response element-binding protein (CREB) on Ser133. However, this is not required for BDNF-induced Arc.Arg3.1 transcription as a Ser133Ala knockin mutation had no effect on Arc/Arg3.1 induction. In parallel, ERK1/2 directly activates Arc/Arg3.1 mRNA transcription via at least one serum response element on the promoter, which bind a complex of the Serum Response Factor (SRF) and a Ternary Complex Factor (TCF).
RESUMEN
miRNAs are small RNAs that are key regulators of gene expression in eukaryotic organisms. The processing of miRNAs is regulated by structural characteristics of the RNA and is also tightly controlled by auxiliary protein factors. Among them, RNA binding proteins play crucial roles to facilitate or inhibit miRNA maturation and can be controlled in a cell, tissue and species-specific manners or in response to environmental stimuli. In this study we dissect the molecular mechanism that promotes the overexpression of miR-132 in mice over its related, co-transcribed and co-regulated miRNA, miR-212. We have shown that the loop structure of miR-132 is a key determinant for its efficient processing in cells. We have also identified a range of RNA binding proteins that recognize the loop of miR-132 and influence both miR-132 and miR-212 processing. The DEAD box helicase p72/DDX17 was identified as a factor that facilitates the specific processing of miR-132.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , MicroARNs/química , MicroARNs/genética , MicroARNs/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Células HeLa , Humanos , Ratones , Modelos Moleculares , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , Regulación hacia ArribaRESUMEN
PURPOSE: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. EXPERIMENTAL DESIGN: mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. RESULTS: We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. CONCLUSIONS: Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. Clin Cancer Res; 22(23); 5929-38. ©2016 AACR.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Ciclina H/genética , Quinasas Ciclina-Dependientes/genética , Expresión Génica/genética , Receptores de Estrógenos/genética , Adulto , Proteínas de Ciclo Celular , Femenino , Humanos , Persona de Mediana Edad , Fosforilación/genética , Pronóstico , Transducción de Señal/genética , Factores de Transcripción , Transcripción Genética/genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53ß promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53ß increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53ß is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53ß depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53ß induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias del Colon/genética , Recurrencia Local de Neoplasia/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Neoplasias del Colon/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Invasividad Neoplásica/genética , Recurrencia Local de Neoplasia/patología , Isoformas de Proteínas/genética , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Treatment of the squaric acid amide esters (7, 9) of anthracycline glycoside antibiotics with aliphatic and aromatic primary and secondary amines, amino acids, peptides and aminodeoxy sugars furnished the new asymmetric diamides 16-19, 25-30, 32, 34 and 38-40 in stereoselective reactions which do not require protecting group-manipulations. The IC50 = 0.12 microM value measured for daunorubicin (1) on human leukemia (HL-60) cells is comparable to those obtained for the daunomycin-L-leucyl squaric acid diamide (30, IC50 = 0.18 microM) and the corresponding D-galactosamine derivative (40, IC50=0.22 microM).
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Antraciclinas/síntesis química , Antraciclinas/farmacología , Antibióticos Antineoplásicos/síntesis química , Antibióticos Antineoplásicos/farmacología , Ciclobutanos/química , Ciclobutanos/farmacología , Antraciclinas/química , Antibióticos Antineoplásicos/química , Línea Celular , Humanos , Estructura MolecularRESUMEN
Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action.
Asunto(s)
Neoplasias de la Mama/genética , Citidina Desaminasa/genética , Citidina/metabolismo , Reparación del ADN por Unión de Extremidades , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Sitios de Unión , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Citidina Desaminasa/antagonistas & inhibidores , Citidina Desaminasa/metabolismo , ADN/genética , ADN/metabolismo , Daño del ADN , Desaminación , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Antígenos de Histocompatibilidad Menor , Pronóstico , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Análisis de Supervivencia , Transcripción Genética , Factor Trefoil-1 , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The Nuclear Receptor (NR) superfamily of transcription factors comprises 48 members, several of which have been implicated in breast cancer. Most important is estrogen receptor-α (ERα), which is a key therapeutic target. ERα action is facilitated by co-operativity with other NR and there is evidence that ERα function may be recapitulated by other NRs in ERα-negative breast cancer. In order to examine the inter-relationships between nuclear receptors, and to obtain evidence for previously unsuspected roles for any NRs, we undertook quantitative RT-PCR and bioinformatics analysis to examine their expression in breast cancer. While most NRs were expressed, bioinformatic analyses differentiated tumours into distinct prognostic groups that were validated by analyzing public microarray data sets. Although ERα and progesterone receptor were dominant in distinguishing prognostic groups, other NR strengthened these groups. Clustering analysis identified several family members with potential importance in breast cancer. Specifically, RORγ is identified as being co-expressed with ERα, whilst several NRs are preferentially expressed in ERα-negative disease, with TLX expression being prognostic in this subtype. Functional studies demonstrated the importance of TLX in regulating growth and invasion in ERα-negative breast cancer cells.
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Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama/metabolismo , Núcleo Celular/metabolismo , Análisis por Conglomerados , Biología Computacional , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Invasividad Neoplásica , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Nucleares Huérfanos , PronósticoRESUMEN
miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNß induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.
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Hipocampo/metabolismo , MicroARNs/genética , Neocórtex/metabolismo , Plasticidad Neuronal/genética , Receptores AMPA/genética , Transmisión Sináptica/genética , Animales , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/genética , Femenino , Fibroblastos/inmunología , Fibroblastos/virología , Hipocampo/citología , Interferón beta/biosíntesis , Interferón beta/inmunología , Potenciación a Largo Plazo/genética , Masculino , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Neocórtex/citología , Neuronas/citología , Neuronas/metabolismo , Cultivo Primario de Células , Receptores AMPA/metabolismo , Virus Sendai/fisiología , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
MiRNAs can regulate gene expression through versatile mechanisms that result in increased or decreased expression of the targeted mRNA and it could effect the expression of thousands of protein in a particular cell. An increasing body of evidence suggest that miRNAs action can be modulated by proteins that bind to the same 3'UTRs that are targeted by miRNAs, suggesting that other factors apart from miRNAs and their target sites determine miRNA-modulation of gene expression. We applied an affinity purification protocol using biotinylated let-7 miRNA inhibitor to isolate proteins that are involved in let-7 mediated gene regulation that resulted in an affinity purification of Polypyrimidine Tract Binding protein (PTB). Here we show that PTB interacts with miRNAs and human Argonaute 2 (hAgo2) through RNA as well as identified potential mammalian cellular targets that are co-regulated by PTB and hAgo2. In addition, using genetic approach, we have demonstrated that PTB genetically interacts with Caenorhabditis elegans let-7 indicating a conserved role for PTB in miRNA-mediated gene regulation.
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Proteínas Argonautas/metabolismo , Regulación de la Expresión Génica/genética , MicroARNs/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Animales , Secuencia de Bases , Caenorhabditis elegans , Cromatografía de Afinidad , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , MicroARNs/genética , Datos de Secuencia Molecular , Proteína de Unión al Tracto de Polipirimidina/genéticaRESUMEN
BACKGROUND: Skeletal muscle insulin resistance (IR) is considered a critical component of type II diabetes, yet to date IR has evaded characterization at the global gene expression level in humans. MicroRNAs (miRNAs) are considered fine-scale rheostats of protein-coding gene product abundance. The relative importance and mode of action of miRNAs in human complex diseases remains to be fully elucidated. We produce a global map of coding and non-coding RNAs in human muscle IR with the aim of identifying novel disease biomarkers. METHODS: We profiled >47,000 mRNA sequences and >500 human miRNAs using gene-chips and 118 subjects (n = 71 patients versus n = 47 controls). A tissue-specific gene-ranking system was developed to stratify thousands of miRNA target-genes, removing false positives, yielding a weighted inhibitor score, which integrated the net impact of both up- and down-regulated miRNAs. Both informatic and protein detection validation was used to verify the predictions of in vivo changes. RESULTS: The muscle mRNA transcriptome is invariant with respect to insulin or glucose homeostasis. In contrast, a third of miRNAs detected in muscle were altered in disease (n = 62), many changing prior to the onset of clinical diabetes. The novel ranking metric identified six canonical pathways with proven links to metabolic disease while the control data demonstrated no enrichment. The Benjamini-Hochberg adjusted Gene Ontology profile of the highest ranked targets was metabolic (P < 7.4 x 10-8), post-translational modification (P < 9.7 x 10-5) and developmental (P < 1.3 x 10-6) processes. Protein profiling of six development-related genes validated the predictions. Brain-derived neurotrophic factor protein was detectable only in muscle satellite cells and was increased in diabetes patients compared with controls, consistent with the observation that global miRNA changes were opposite from those found during myogenic differentiation. CONCLUSIONS: We provide evidence that IR in humans may be related to coordinated changes in multiple microRNAs, which act to target relevant signaling pathways. It would appear that miRNAs can produce marked changes in target protein abundance in vivo by working in a combinatorial manner. Thus, miRNA detection represents a new molecular biomarker strategy for insulin resistance, where micrograms of patient material is needed to monitor efficacy during drug or life-style interventions.
RESUMEN
We propose a method for performing binary intensity and continuous phase modulation of beams with a spatial light modulator (SLM) and a low-pass spatial filtering 4-f system. With our method it is possible to avoid the use of phase masks in holographic data storage systems or to enhance the phase encoding of the SLM by making it capable of binary amplitude modulation. The data storage capabilities and the limitations of the method are studied.
RESUMEN
Data reported in this article describe the synthesis of Arg-rich oligopeptide conjugates of ferrocenecarboxylic acid on solid support with two different strategies and for the first time, the successful preparation of peptide conjugates of ferrocenylacrylic acid in solution. The antitumor effect of conjugates was analyzed by MTT assay in vitro. We demonstrated that ferrocenylacrylic acid possessing an enone (--CH==CH--CO--) moiety exhibited remarkable antiproliferative effect against human leukemia cells (HL-60) in vitro, but its effect was not improved by conjugation with hexa- or octaarginines. However, we observed highly increased water-solubility. In contrast, the results provide evidence that conjugation of ferrocenecarboxylic acid to Arg(n) (n = 6, 8) improved not only its water-solubility, but also antitumor effect on human leukemia cells in vitro.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Compuestos Ferrosos/síntesis química , Compuestos Ferrosos/farmacología , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Compuestos Ferrosos/química , Células HL-60 , Humanos , Metalocenos , Estructura Molecular , Oligopéptidos/químicaRESUMEN
his review will summarize available information on the ability of macromolecular conjugates containing no specific recognition motifs to deliver anthracyclines (daunomycin, adriamycin) or methotrexate to target cells such as tumour cells or macrophages. Conjugates with natural (proteins, DNA, carbohydrates) and synthetic macromolecules (linear and branched chain poly-alpha-amino acids, non-biodegradable DIVEMA, HPMA etc.) will be reviewed. Experimental data from several laboratories indicate that these conjugates are taken up by cells mainly by fluid-phase or adsorptive endocytosis. It is believed that these processes do not involve 'specific receptors'. Two examples of methotrexate and daunomycin conjugates will be discussed to show the effect of the chemical structure of branched chain polypeptides on the uptake and antitumour or antiparasitic (Leishmania donovani infection) efficacy of conjugates.
Asunto(s)
Antraciclinas/farmacocinética , Antibióticos Antineoplásicos/farmacocinética , Antimetabolitos Antineoplásicos/farmacocinética , Daunorrubicina/farmacocinética , Sistemas de Liberación de Medicamentos , Metotrexato/farmacocinética , Animales , Antraciclinas/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/administración & dosificación , Daunorrubicina/administración & dosificación , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Metotrexato/administración & dosificaciónRESUMEN
Applicability of a commercial twisted-nematic liquid-crystal display is examined at approximately 400 nm. Different modulation modes predicted by Jones-matrix calculus are experimentally tested. High contrast amplitude modulation with negligible loss, high contrast and low loss hybrid ternary modulation, and 1.5pi continuous phase delay without intensity modulation and with low loss are presented. Simulation results of a 4f holographic system prove the usefulness of the high contrast for amplitude modulation, and the importance of pi phase difference between high transmission white levels in a hybrid ternary modulation.