RESUMO
Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By now, only supportive therapies have been available. However, promising specific therapies are currently being developed based on different mechanisms to increase the level of SMN protein; in particular, intrathecal antisense oligonucleotides that prevent the skipping of exon 7 during SMN2 transcription, and intravenous SMN1 insertion using viral vector. These therapeutic perspectives open a new era in the natural history of the disease. In this review, we intend to discuss the most recent and promising therapeutic strategies, with special consideration to the pathogenesis of the disease and the mechanisms of action of such therapies.
Assuntos
DNA Antissenso/administração & dosagem , Terapia Genética/métodos , Atrofia Muscular Espinal/terapia , Oligonucleotídeos/administração & dosagem , Proteína 1 de Sobrevivência do Neurônio Motor/administração & dosagem , Humanos , Injeções Espinhais , Mutação , FenótipoRESUMO
ABSTRACT Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By now, only supportive therapies have been available. However, promising specific therapies are currently being developed based on different mechanisms to increase the level of SMN protein; in particular, intrathecal antisense oligonucleotides that prevent the skipping of exon 7 during SMN2 transcription, and intravenous SMN1 insertion using viral vector. These therapeutic perspectives open a new era in the natural history of the disease. In this review, we intend to discuss the most recent and promising therapeutic strategies, with special consideration to the pathogenesis of the disease and the mechanisms of action of such therapies.
RESUMO A atrofia muscular espinhal (AME) é uma grave doença dos neurônios motores, de grande variabilidade clínica e causada na maioria dos casos por mutação em homozigose no gene SMN1. Pelo menos quatro fenótipos clínicos distintos são reconhecidos com base na idade de início e no grau de envolvimento motor. Tal variabilidade clínica é em parte relacionada com o número de cópias do gene SMN2. Até recentemente, apenas terapias de suporte estavam disponíveis. Atualmente, terapias especificas estão sendo desenvolvidas com base em diferentes mecanismos para aumentar o nível de proteína SMN; em particular oligonucleotídeos antissenso por via intratecal e inserção de cópia do gene SMN1, via endovenosa, usando vetor viral. Nesta revisão, objetivamos discutir as mais recentes e promissoras estratégias terapêuticas, com consideração especial aos aspectos patogênicos da doença e aos mecanismos de ação de tais terapias.
Assuntos
Humanos , Oligonucleotídeos/administração & dosagem , Atrofia Muscular Espinal/terapia , Terapia Genética/métodos , DNA Antissenso/administração & dosagem , Proteína 1 de Sobrevivência do Neurônio Motor/administração & dosagem , Fenótipo , Injeções Espinhais , MutaçãoRESUMO
Thyrotropin-releasing hormone (TRH) has anorexigenic and anxiolytic functions when injected intraventricularly. Nucleus accumbens (NAcc) is a possible brain region involved, since it expresses proTRH. TRH from hypothalamic paraventricular nucleus (PVN) has a food intake-regulating role. TRHergic pathways of NAcc and PVN are implicated in anxiety and feeding. Both behaviors depend on cAMP and phosphorylated-cAMP response element binding protein (pCREB) intracellular levels. Intracellular levels of cAMP are controlled by the degrading activity of phosphodiesterases (PDEs). Since TRH transcription is activated by pCREB, a specific inhibitor of PDE7B may regulate TRH-induced effects on anxiety and feeding. We evaluated the effectiveness of an intra-accumbal and intraperitoneal (i.p.) administration of a PDE7 inhibitor (BRL-50481) on rats' anxiety-like behavior and food intake; also on TRH mRNA and protein expression in NAcc and PVN to define its mediating role on the PDE7 inhibitor-induced behavioral changes. Accumbal injection of 4µg/0.3µL of PDE7 inhibitor decreased rats' anxiety. The i.p. injection of 0.2mg/kg of the inhibitor was able to increase the PVN TRH mRNA expression and to decrease feeding but did not change animals' anxiety levels; in contrast, 2mg/kg b.w inhibitor enhanced accumbal TRH mRNA, induced anxiolysis with no change in food intake. PDE7 inhibitor induced anxiolytic and anorexigenic like behavior depending on the dose used. Results supported hypothalamic TRH mediated feeding-reduction effects, and accumbal TRH mediation of inhibitor-induced anxiolysis. Thus, an i.p dose of this inhibitor might be reducing anxiety with no change in feeding, which could be useful for obese patients.
Assuntos
Ansiedade/induzido quimicamente , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/antagonistas & inibidores , Comportamento Alimentar/efeitos dos fármacos , Nitrocompostos/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Sulfonamidas/farmacologia , Hormônio Liberador de Tireotropina/metabolismo , Animais , Ansiedade/tratamento farmacológico , AMP Cíclico/metabolismo , DNA Antissenso/farmacologia , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Nitrocompostos/uso terapêutico , Ratos , Ratos Wistar , Sulfonamidas/uso terapêutico , Hormônio Liberador de Tireotropina/genética , Fatores de Tempo , Iodotironina Desiodinase Tipo IIRESUMO
UDP-glucuronate decarboxylase (UDP-xylose synthase; UXS, EC 4.1.1.35) is an essential enzyme of the non-cellulosic polysaccharide biosynthetic pathway. In the present study, using transient expression of fluorescently labeled Gossypium hirsutum UXS (GhUXS3) protein in onion epidermal cells, we observed that this protein was distributed in the cytoplasm. The GhUXS3 cDNA of cotton was expressed in an antisense orientation in Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. Homozygous plants showing down-regulation of UXS were analyzed with northern blots. Compared to the untransformed control, transgenic plant showed shorter roots, earlier blossom formation, and delayed senescence. Biochemical analysis indicated that levels of rhamnose, mannose, galactose, glucose, xylose, and cellulose were reduced in some of the down-regulated antisense plants. These results suggest that GhUXS3 regulates the conversion of non-cellulosic polysaccharides and modulates their composition in plant cell walls. We also discuss a possible cellular function for GhUXS in determining the quality of cotton fibers.
Assuntos
Arabidopsis/genética , Metabolismo dos Carboidratos/genética , Carboxiliases/genética , Parede Celular/metabolismo , DNA Antissenso , Gossypium/enzimologia , Envelhecimento , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Parede Celular/química , DNA de Plantas , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Gossypium/genética , Raízes de Plantas/anatomia & histologia , Plantas Geneticamente ModificadasRESUMO
We investigated the effects of hepatitis B virus (HBV) S/C double gene loci antisense locked nucleic acid on replication and expression of HBV in hepatitis transgenic mice. HBV mice (N = 30) were randomly divided into five groups of six mice: 5% glucose solution control, empty liposome control, single-target S, single-target C, and dual-target SC groups. An antisense locked nucleic acid fragment was injected into the mice. Serum HBsAg, serum HBV DNA, HBV C-mRNA expression in liver tissue, HbsAg and HbcAg expression in hepatocytes, serum albumin, alanine transaminase (ALT), urea nitrogen, and creatinine were detected. Liver and kidney sections were examined for the effects of antisense locked nucleic acid. The expression of HBsAg was markedly inhibited; the inhibition rates of the S, C, and SC target groups were 36.63, 31.50, and 54.87%, respectively; the replication of HBV DNA was also inhibited: 23.97, 21.13, and 35.83%, respectively. After injection at 1, 3, and 5 days, the corresponding rates for HBsAg inhibition were 14.40, 25.61, and 31.33%, and for HBV DNA inhibition they were 11.04, 19.24, and 24.13%. Compared with the control group, the differences in serum albumin, ALT, urea nitrogen, and creatinine in each group were not statistically significant, and the number of HbsAg- and HBcAg-positive cells in the mouse liver was significantly reduced. The liver and kidney tissues were normal. The gene therapy had significant inhibitory effects on the replication and expression of HBV in transgenic mice, and double-gene targeting was better than single-gene targeting.
Assuntos
DNA Antissenso/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/genética , Hepatite B/virologia , Animais , DNA Antissenso/administração & dosagem , DNA Antissenso/toxicidade , Modelos Animais de Doenças , Regulação Viral da Expressão Gênica , Hepatite B/sangue , Hepatite B/patologia , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Humanos , Testes de Função Renal , Testes de Função Hepática , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , Carga Viral , Replicação Viral/genéticaRESUMO
The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with increased lysine, methionine and threonine contents. The objective of the study was to investigate the possible changes in the regulation of key enzymes of the aspartate metabolic pathway and the contents of aspartate-derived amino acids in the nontransgenic line (Hordeum vulgare L. cv. Golden Promise) and five antisense C-hordein transgenic barley lines. Considering the amounts of soluble and protein-bound aspartate-derived amino acids together with the analysis of key enzymes of aspartate metabolic pathway, we suggest that the C-hordein suppression did not only alter the metabolism of at least one aspartate-derived amino acid (threonine), but major changes were also detected in the metabolism of lysine and methionine. Modifications in the activities and regulation of aspartate kinase, dihydrodipicolinate synthase and homoserine dehydrogenase were observed in most transgenic lines. Furthermore the activities of lysine α-ketoglutarate reductase and saccharopine dehydrogenase were also altered, although the extent varied among the transgenic lines.
Assuntos
DNA Antissenso , Glutens , Hordeum/metabolismo , Lisina/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Hordeum/genética , Lisina/genética , Plantas Geneticamente Modificadas/genéticaRESUMO
O splicing alternativo do pré-mRNA de BCL-X produz duas isoformas de mRNAs com funções antagônicas, a pró-apoptótica BCL-XS e a anti-apoptótica BCL-XL, cujo balanço regula a homeostasia celular. Entretanto, o mecanismo que regula esse processamento ainda é desconhecido. Nesse trabalho, nós identificamos e caracterizamos um longo RNA não codificador de proteínas (lncRNA) nomeado INXS, que é transcrito a partir da fita oposta do locus genômico de BCL-X, sendo menos abundante em linhagens celulares tumorais e tecidos tumorais de pacientes quando comparados com os respectivos pares não tumorais. INXS é um RNA unspliced de 1903 nts, é transcrito pela RNA Polimerase II, possui cap 5', está enriquecido na fração nuclear das células e se liga à proteína Sam68 do complexo modulador de splicing. O tratamento de células tumorais 786-O com cada um de três agentes indutores de apoptose aumentou a expressão endógena do INXS, levando ao aumento expressivo da proporção entre os mRNAs de BCL-XS / BCL-XL, e ativação das caspases 3, 7 e 9. Estes efeitos foram anulados na presença do knockdown do INXS. Da mesma forma, a superexpressão ectópica do INXS causou uma mudança no splicing favorecendo a isoforma BCL-XS e ativação das caspases, aumentando os níveis da proteína BCL-XS e conduzindo as células à apoptose. Utilizando um modelo in vivo, cinco injeções intra-tumorais do INXS durante 15 dias causaram uma regressão acentuada no volume dos xenotumores. Portanto, INXS é um lncRNA que induz a apoptose, sugerindo que essa molécula seja um possível alvo a ser explorado na terapia contra o câncer
BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL, whose balance regulates cell homeostasis. However, the mechanism that regulates the splice shifting is incompletely understood. Here, we identified and characterized a long noncoding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was less abundant in tumor cell lines and patient tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA Polymerase II, 5'-capped, nuclear enriched and binds Sam68 splicing-modulator. The treatment of tumor cell line 786-O with each of three apoptosis-inducing agents increased endogenous INXS lncRNA, increased BCL-XS / BCL-XL mRNA ratio, and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing towards BCL-XS and activation of caspases, increasing the levels of BCL-XS protein and then leading the cells to apoptosis. In a mouse xenograft model, five intra-tumor injections of INXS along 15 days caused a marked regression in tumor volume. INXS is an lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies
Assuntos
Apoptose/genética , RNA Longo não Codificante/análise , Processamento Alternativo/genética , Proteína bcl-X , Proteína bcl-X/análise , DNA Antissenso , Expressão Gênica/genética , Neoplasias , RNARESUMO
The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41 percent in HepG2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.
Assuntos
Animais , Humanos , Camundongos , Carcinoma Hepatocelular/metabolismo , DNA Antissenso/genética , Expressão Gênica , Vetores Genéticos/genética , Proliferação de Células , DNA Antissenso/metabolismo , Células Eucarióticas/metabolismo , Citometria de Fluxo , Vetores Genéticos/metabolismo , /metabolismo , Camundongos Nus , Transplante de Neoplasias , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/análise , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG(2) cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG(2) cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG(2) cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G(1) phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.
Assuntos
Carcinoma Hepatocelular/metabolismo , DNA Antissenso/genética , Expressão Gênica , Vetores Genéticos/genética , Animais , Proliferação de Células , DNA Antissenso/metabolismo , Células Eucarióticas/metabolismo , Citometria de Fluxo , Vetores Genéticos/metabolismo , Células Hep G2/metabolismo , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Plasmídeos/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In the present work, the response of tobacco (Nicotiana tabaccum L.) wild-type SR1 and transgenic CAT1AS plants (with a basal reduced CAT activity) was evaluated after exposure to the herbicide paraquat (PQ). Superoxide anion (O (2) (.-) ) formation was inhibited at 3 or 21 h of exposure, but H(2)O(2) production and ion leakage increased significantly, both in SR1 or CAT1AS leaf discs. NADPH oxidase activity was constitutively 57% lower in non-treated transgenic leaves than in SR1 leaves and was greatly reduced both at 3 or 21 h of PQ treatment. Superoxide dismutase (SOD) activity was significantly reduced by PQ after 21 h, showing a decrease from 70% to 55%, whereas catalase (CAT) activity decreased an average of 50% after 3 h of treatment, and of 90% after 21 h, in SR1 and CAT1AS, respectively. Concomitantly, total CAT protein content was shown to be reduced in non-treated CAT1AS plants compared to control SR1 leaf discs at both exposure times. PQ decreased CAT expression in SR1 or CAT1AS plants at 3 and 21 h of treatment. The mechanisms underlying PQ-induced cell death were possibly not related exclusively to ROS formation and oxidative stress in tobacco wild-type or transgenic plants.
Assuntos
Catalase/metabolismo , Nicotiana/efeitos dos fármacos , Paraquat/toxicidade , Folhas de Planta/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Western Blotting , Catalase/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , DNA Antissenso/genética , Herbicidas/toxicidade , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Fatores de Tempo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Transposable elements comprise a significant part of genomes and are involved in their evolvability. The hobo element is found as an active class II transposable element in Drosophila melanogaster that is able to induce gonadal dysgenesis. Some hobo-related sequences (hRSs) are thought to be relics of old "hobo" invasions, and are therefore ancient genomic constituents. However, some of these hRSs are still mobile. The present study analyzed the expression pattern of hobo and a particular type of hRSs, hobo(VAHS). Both elements were shown to be expressed as sense and antisense mRNA transcripts. Expression analysis in whole mount embryos revealed a pattern similar to that of some developmental regulatory genes. Here we suggest that cis-regulatory sequences similar to those in developmental genes exist in hobo sequences. Therefore, hobo mobilization may contribute to the development of new regulatory networks during genomic evolution.
Assuntos
Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Transposases/genética , Animais , DNA Antissenso/genética , Drosophila/embriologia , Embrião não Mamífero/metabolismo , Evolução Molecular , Genoma de Inseto , Transcrição GênicaRESUMO
Bacterial resistance to existing antibiotics continues to grow, necessitating the discovery of new compounds of this type. Antisense-based whole-cell target-based screening is a new and highly sensitive antibiotic discovery approach that has led to a number of new natural product antibiotics. Screening with a rpsD-sensitized strain led to the discovery of a number of natural product polyketides from Streptomyces lucensis. Complete workup of the fermentation extract of this strain allowed for the isolation of seven new compounds, lucensimycins A-G (1-3, 4a, 5-7), with varying degrees of antibacterial activities. Lucensimycin E (5) exhibited the best activity and showed MIC values of 32 microg/mL against Staphylococcus aureus and 8 microg/mL against Streptococcus pneumoniae. The isolation, structure elucidation, and antibacterial activities of four new members, lucensimycins D-G, are described. Lucensimycins D (4a) and E (5) are N-acetyl-l-cysteine adducts of lucensimycin A (1). Semisynthesis of lucensimycins D and E from lucensimycin A has also been described. Lucensimycins F and G are myo-inositolyl-alpha-2-amino-2-deoxy-l-idosyl amide derivatives of lucensimycins D and E, respectively. The relative configuration of these compounds was determined, in part, by molecular dynamics simulations.
Assuntos
Antibacterianos , DNA Bacteriano/genética , Compostos de Espiro , Streptomyces/química , Streptomyces/genética , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , DNA Antissenso/genética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Compostos de Espiro/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Índias OcidentaisRESUMO
MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)), which also control stomatal movements. We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression. Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H(2)O(2), both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H(2)O(2) synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H(2)O(2). These results provide clear evidence for the important role of MPK3 in the perception of ABA and H(2)O(2) in guard cells.
Assuntos
Ácido Abscísico/farmacologia , Arabidopsis/fisiologia , Peróxido de Hidrogênio/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Epiderme Vegetal/citologia , Folhas de Planta/citologia , Arabidopsis/citologia , Proteínas de Arabidopsis/metabolismo , DNA Antissenso/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Epiderme Vegetal/fisiologia , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Transdução de Sinais , Transformação GenéticaRESUMO
BACKGROUND: The expression of the thyroid cancer-1(TC-1) gene seems to be related with malignant transformation in the thyroid tissue. OBJECTIVE: We evaluated the potential use of TC-1 gene expression as a marker of malignancy in thyroid nodules. METHODS: A total of 92 frozen thyroid samples were studied, including 46 samples from thyroid nodules (19 papillary carcinomas, 1 follicular carcinoma, 24 adenomatous goiters, and 2 follicular adenomas) and 46 samples from normal surrounding thyroid tissue. Total RNA was extracted and TC-1 expression was assessed by semiquantitative Multiplex PCR. Results were verified using real-time RT-PCR in some of the samples. RESULTS: Overall mean TC-1 gene expression (normalized by the ABL gene) was 1.73 +/- 1.67 (0.33-9.33). There was a significant difference (p < 0.001) between TC-1 gene expression in benign thyroid lesions (1.07 +/- 0.10) and carcinomas (2.73 +/- 0.51). CONCLUSION: Our results suggest that TC-1 gene expression may be useful in the differential diagnosis of goiters and thyroid papillary carcinomas.
Assuntos
Bócio/genética , Proteínas de Neoplasias/genética , Neoplasias da Glândula Tireoide/genética , Adenoma/diagnóstico por imagem , Adenoma/genética , Adolescente , Adulto , Idoso , Carcinoma Papilar/diagnóstico por imagem , Carcinoma Papilar/genética , DNA Antissenso/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276-1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, beta-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and beta-2 microglobulin (beta2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE-beta2m to the apical membrane. The amount of HFE-beta2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or beta2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE-beta2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Mucosa Intestinal/metabolismo , Ferro/farmacocinética , Proteínas de Membrana/metabolismo , Microglobulina beta-2/metabolismo , Células CACO-2 , Proteínas de Transporte de Cátions/metabolismo , DNA Antissenso/genética , Hemocromatose/metabolismo , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoprecipitação/métodos , Mucosa Intestinal/citologia , Ferro/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Membrana/genética , Modelos Biológicos , Transfecção/métodos , Microglobulina beta-2/genéticaRESUMO
To determine the role of Dp71 in neuronal cells, we generated PC12 cell lines in which Dp71 protein levels were controlled by stable transfection with either antisense or sense constructs. Cells expressing the antisense Dp71 RNA (antisense-Dp71 cells) contained reduced amounts of the two endogenous Dp71 isoforms. Antisense-Dp71 cells exhibited a marked suppression of neurite outgrowth upon the induction with NGF or dibutyryl cyclic AMP. Early responses to NGF-induced neuronal differentiation, such as the cessation of cell division and the activation of ERK1/2 proteins, were normal in the antisense-Dp71 cells. On contrary, the induction of MAP2, a late differentiation marker, was disturbed in these cells. Additionally, the deficiency of Dp71 correlated with an altered expression of the dystrophin-associated protein complex (DAPC) members alpha and beta dystrobrevins. Our results indicate that normal expression of Dp71 is essential for neurite outgrowth in PC12 cells and constitute the first direct evidence implicating Dp71 in a neuronal function.
Assuntos
Distrofina/análogos & derivados , Distrofina/fisiologia , Neuritos/ultraestrutura , Células PC12/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular , DNA Antissenso/farmacologia , Distrofina/genética , Humanos , Cinética , Proteínas Associadas aos Microtúbulos/análise , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/química , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/ultraestrutura , Neuropeptídeos/análise , Isoformas de Proteínas/análise , Ratos , TransfecçãoRESUMO
Histamine and H2 agonists transiently induce an important cAMP response in promonocytic U-937 cells but fail to induce monocytic differentiation because of a rapid receptor desensitization mediated by G protein-coupled receptor kinases (GRKs). The aims of the present study were to investigate the participation of GRK2 in the desensitization mechanism of the H2 receptor in U-937 cells by reducing GRK2 levels through antisense technology and to evaluate the differentiating capacity of cells expressing lower GRK2 level, stimulated by H2 agonists. By stable U-937 cell transfection with a GRK2-antisense cDNA, we obtained D5 and A2 cell clones exhibiting a reduction in GRK2 expression and an H3 clone with no significant difference in GRK2 expression from control cells. The cAMP response induced by the H2 agonist in D5 and A2 but not in H3 cells was higher than in U-937 and persisted for a longer period of time, although the number of H2 receptors in D5 and A2 cells was lower than in U-937. Furthermore, D5 and A2 cells treated with H2 agonist showed patterns of c-Fos and CD88 expression consistent with monocytic differentiated cells. Overall, these results indicate a direct correlation between the expression of GRK2 and the desensitization of natively expressed H2 receptors in U-937 cells, suggesting that GRK2 plays a major role in the regulation of these receptors' response. In turn, desensitization process is a key component of H2 receptor signaling, determining the differentiation capability of promonocytic cells.
Assuntos
Senescência Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores Histamínicos H2/metabolismo , Senescência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , DNA Antissenso/genética , DNA Antissenso/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Células U937 , Quinases de Receptores Adrenérgicos betaRESUMO
In this paper we describe the cloning of rat olfactory bulb tubulin tyrosine ligase (TTL) cDNA, and investigate the physiological role of TTL in cultured CHO-K1 cells. Comparison of the deduced amino acid sequence of rat TTL cDNA with those of bovine and pig showed approximately 90% of identity. Transient transfection of CHO-K1 cells with a dominant negative mutant of TTL that contains the binding site to the substrate (tubulin) but not the catalytic domain, significantly decreased the endogenous TTL activity as determined in vitro. Similar results were obtained using a construction encoding for the antisense sequence of TTL. The reduction in TTL activity is not accompanied by a decrease in the tyrosination levels of microtubules, as judged by immunofluorescence analysis. Strikingly, the number of cells in the plates transfected with the mutant TTL or the antisense TTL cDNA was, after 72 h of culture, two and three times higher, respectively, than the number of cells in the control plates. These results support the hypothesis that TTL may play a role in the regulation of the cell cycle in living cells.
Assuntos
Divisão Celular/genética , Bulbo Olfatório/enzimologia , Peptídeo Sintases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Divisão Celular/efeitos dos fármacos , Clonagem Molecular , Cricetinae , DNA Antissenso/farmacologia , DNA Complementar , Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutação , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , RatosRESUMO
Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. Recently, we described that the TRH precursor gene overexpression induces hypertension in the normal rat. In addition, we published that spontaneously hypertensive rats (SHR) have central extrahypothalamic TRH hyperactivity with increased TRH synthesis and release and an elevated TRH receptor number. In the present study, we report that intracerebroventricular antisense (AS) treatment with a phosphorothioate oligonucleotide against the TRH precursor gene significantly diminished up to 72 hours and in a dose-dependent manner the increased diencephalic TRH content, whereas normalized systolic blood pressure (SABP) was present in the SHR compared with Wistar-Kyoto (WKY) rats. Although basal thyrotropin was higher in SHR compared with WKY rats and this difference disappeared after antisense treatment, no differences were observed in plasma T4 or T3 between strains with or without AS treatment, indicating that the effect of the AS on SABP was independent of the thyroid status. Because the encephalic renin-angiotensin system seems to be crucial in the development and/or maintenance of hypertension in SHR, we investigated the effect of antisense inhibition of TRH on that system and found that TRH antisense treatment significantly diminished the elevated diencephalic angiotensin II (Ang II) content in the SHR without any effect in control animals, suggesting that the Ang II system is involved in the TRH cardiovascular effects. To summarize, the central TRH system seems to be involved in the etiopathogenesis of hypertension in this model of essential hypertension.
Assuntos
DNA Antissenso/farmacologia , Hipertensão/etiologia , Hormônio Liberador de Tireotropina/fisiologia , Angiotensina II/metabolismo , Animais , Pressão Sanguínea , Ventrículos Cerebrais/metabolismo , DNA Antissenso/administração & dosagem , Modelos Animais de Doenças , Hipertensão/genética , Hipertensão/metabolismo , Masculino , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacologia , Radioimunoensaio , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Sistema Renina-Angiotensina/fisiologia , Hormônios Tireóideos/sangue , Hormônio Liberador de Tireotropina/análise , Hormônio Liberador de Tireotropina/biossíntese , Hormônio Liberador de Tireotropina/genéticaRESUMO
Gene therapy for hypertension is needed for the next generation of antihypertensive drugs. Current drugs, although effective, have poor compliance, are expensive and short-lasting (hours or one day). Gene therapy offers a way to produce long-lasting antihypertensive effects (weeks, months or years). We are currently using two strategies: a) antisense oligodeoxynucleotides (AS-ODN) and b) antisense DNA delivered in viral vectors to inhibit genes associated with vasoconstrictive properties. It is not necessary to know all the genes involved in hypertension, since many years of experience with drugs show which genes need to be controlled. AS-ODN are short, single-stranded DNA that can be injected in naked form or in liposomes. AS-ODN, targeted to angiotensin type 1 receptors (AT1-R), angiotensinogen (AGT), angiotensin converting enzyme, and ss1-adrenergic receptors effectively reduce hypertension in rat models (SHR, 2K-1C) and cold-induced hypertension. A single dose is effective up to one month when delivered with liposomes. No side effects or toxic effects have been detected, and repeated injections can be given. For the vector, adeno-associated virus (AAV) is used with a construct to include a CMV promoter, antisense DNA to AGT or AT1-R and a reporter gene. Results in SHR demonstrate reduction and slowing of development of hypertension, with a single dose administration. Left ventricular hypertrophy is also reduced by AAV-AGT-AS treatment. Double transgenic mice (human renin plus human AGT) with high angiotensin II causing high blood pressure, treated with AAV-AT1-R-AS, show a normalization of blood pressure for over six months with a single injection of vector. We conclude that ODNs will probably be developed first because they can be treated like drugs for the treatment of hypertension with long-term effects. Viral vector delivery needs more engineering to be certain of its safety, but one day may be used for a very prolonged control of blood pressure.