RESUMO
BACKGROUND: Parents who have a newborn with a congenital heart defect experience negative emotions, which may determine the emotional state of their children. METHODS: The study group included 154 parents of newborns and infants with cyanotic congenital heart disease, before cardiac surgery and after the procedure. HADS m and PSS-10 questionnaires were used to assess parental anxiety, depression, aggression, and the level of stress. RESULTS: High levels of depression, anxiety, total HADS and stress were diagnosed in a large group of parents, regardless of the stage of cardiac surgery treatment. A high level of stress was associated with a higher prevalence of emotional disturbance both in the total HADS (overall) and in all its individual domains. Anxiety and depression were more common in mothers. A high level of stress was a significant predictor of anxiety and depression in parents. CONCLUSIONS: A high level of stress was a significant predictor of anxiety and depression in parents of infants with congenital heart disease. The parents' psychological condition is one of many potential determinants over the course of their child's treatment and recovery.
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Potassium channels emerge as one of the crucial groups of proteins that shape the biology of cancer cells. Their involvement in processes like cell growth, migration, or electric signaling, seems obvious. However, the relationship between the function of K+ channels, glucose metabolism, and cancer glycome appears much more intriguing. Among the typical hallmarks of cancer, one can mention the switch to aerobic glycolysis as the most favorable mechanism for glucose metabolism and glycome alterations. This review outlines the interconnections between the expression and activity of potassium channels, carbohydrate metabolism, and altered glycosylation in cancer cells, which have not been broadly discussed in the literature hitherto. Moreover, we propose the potential mediators for the described relations (e.g., enzymes, microRNAs) and the novel promising directions (e.g., glycans-orinented drugs) for further research.
Assuntos
MicroRNAs , Neoplasias , Humanos , Canais de Potássio/metabolismo , Glicosilação , MicroRNAs/metabolismo , Glucose/metabolismo , GlicóliseRESUMO
MicroRNAs (miRNAs) have great therapeutic potential; however, their delivery still faces huge challenges, especially given the short half-life of naked miRNAs due to rapid hydrolysis or inactivation by abundant nucleases in the systemic circulation. Therefore, the search for reliable miRNA delivery systems is crucial. Nanogels are one of the more effective nanocarriers because they are biocompatible and have a high drug-loading capacity. In this study, acrylamide-based nanogels containing cationic groups and redox-sensitive crosslinkers were developed for cellular delivery of anti-miR21 (a-miR21). To achieve this, post-polymerization loading of a-miR21 oligonucleotides into nanogels was performed by utilizing the electrostatic interaction between positively charged nanogels and negatively charged oligonucleotides. Different molar ratios of the amine groups (N) on the cationic nanogel and phosphate groups (P) on the miRNA were investigated. An N/P ratio of 2 allowed high miRNA loading capacity (MLC, 6.7% w/w) and miRNA loading efficiency (MLE, 99.7% w/w). Successful miRNA loading was confirmed by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) measurements. miRNA-loaded nanogels (NG/miRNA) formed stable dispersions in biological media and showed an enhanced miRNA release profile in the presence of glutathione (GSH). Moreover, the addition of heparin to dissociate the miRNA from the cationic nanogels resulted in the complete release of miRNA. Lastly, a cell uptake study indicated that NG/miRNA could be easily taken up by cancer cells.
Assuntos
MicroRNAs , Nanogéis , MicroRNAs/genética , Polietilenoglicóis , Oxirredução , Acrilamidas , Portadores de FármacosRESUMO
The use of smart nanocarriers that can modulate therapeutic release aided by biological cues can prevent undesirable cytotoxicity caused by the premature release of cytotoxic drugs during nanocarrier circulation. In this report, degradable nanocarriers based on pH/reduction dual-responsive nanogels were synthesized to encapsulate doxorubicin hydrochloride (DOX) and specifically boost the release of DOX in conditions characteristic of the cancer microenvironment. Nanogels containing anionic monomer 2-carboxyethyl acrylate (CEA) and N,N'-bis(acryloyl)cystamine (CBA) as a degradable crosslinker have been successfully synthesized via photoinitiated free radical polymerization. The loading process was conducted after polymerization by taking advantage of the electrostatic interaction between the negatively charged nanogels and the positively charged DOX. In this case, a high drug loading capacity (DLC) of up to 27.89% was achieved. The entrapment of DOX into a nanogel network could prevent DOX from aggregating in biological media at DOX concentrations up to ~160 µg/mL. Anionic nanogels had an average hydrodynamic diameter (dH) of around 90 nm with a negative zeta (ζ) potential of around -25 mV, making them suitable for targeting cancer tissue via the enhanced permeation effect. DOX-loaded nanogels formed a stable dispersion in different biological media, including serum-enriched cell media. In the presence of glutathione (GSH) and reduced pH, drug release was enhanced, which proves dual responsivity. An in vitro study using the HCT 116 colon cancer cell line demonstrated the enhanced cytotoxic effect of the NG-CBA/DOX-1 nanogel compared to free DOX. Taken together, pH/reduction dual-responsive nanogels show promise as drug delivery systems for anticancer therapy.
Assuntos
Antineoplásicos , Cistamina , Antígeno Carcinoembrionário/metabolismo , Doxorrubicina/farmacologia , Portadores de Fármacos/metabolismo , Liberação Controlada de Fármacos , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Nanogéis , Polietilenoglicóis , PolietilenoiminaRESUMO
For several decades, natural products have been widely researched and their native scaffolds are the basis for the design and synthesis of new potential therapeutic agents. Betulin is an interesting biologically attractive natural parent molecule with a high safety profile and can easily undergo a variety of structural modifications. Herein, we describe the synthesis of new molecular hybrids of betulin via covalent linkage with an alkyltriphenylphosphonium moiety. The proposed strategy enables the preparation of semi-synthetic derivatives (28-TPP⊕ BN and 3,28-bisTPP⊕ BN) from betulin through simple transformations in high yields. The obtained results showed that the presence of a lipophilic cation improved the solubility of the tested analogs compared to betulin, and increased their cytotoxicity. Among the triphenylphosphonium derivatives tested, analogs 7a (IC50 of 5.56 µM) and 7b (IC50 of 5.77 µM) demonstrated the highest cytotoxicity against the colorectal carcinoma cell line (HCT 116). TPP⊕-conjugates with betulin showed antimicrobial properties against Gram-positive reference Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228 bacteria, at a 200 µM concentration in water. Hence, the conjugation of betulin's parent backbone with a triphenylphosphonium moiety promotes transport through the hydrophobic barriers of the mitochondrial membrane, making it a promising strategy to improve the bioavailability of natural substances.
Assuntos
Anti-Infecciosos , Antineoplásicos , Triterpenos , Antibacterianos/química , Anti-Infecciosos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Triterpenos/químicaRESUMO
Trehalose has been widely studied as a treatment for a variety of human disorders due to its ability to stimulate autophagy. Trehalose, however, is poorly adsorbed and is hydrolyzed in the intestinal mucosa, and oral delivery requires relatively high doses to induce autophagy. The parenteral injection of trehalose-releasing nanogels proposed in this study offers an alternative mode of delivery. This study aimed to develop stable colloidal dispersions of trehalose-rich nanogels that could sustainably release trehalose under physiologically relevant conditions. The nanogel design was based on the covalent incorporation of 6-O-acryloyl-trehalose within a polymer network. A series of nine trehalose-rich nanogels with highly conjugated trehalose (up to 59 % w/w) were synthesized and shown to sustainably release trehalose at a rate that is not dose dependent. The nanogels were optimized to keep colloidal stability in serum-enriched cell culture media. The stable nanogels were not cytotoxic to primary HUVECs. Two selected nanogels with opposite surface charges were subjected to extended in vitro characterization that included a cellular uptake study and a hemocompatibility assay. Both nanogels were efficiently taken up by HUVECs during a short incubation. They also proved not to be hemolytic to human RBCs in concentrations up to 2.0 mg/mL. Finally, an in vivo autophagy stimulation study employing transgenic zebrafish and Drosophila larvae demonstrated that prolonged exposure to a cationic trehalose-releasing nanogel can induce autophagic activity in in vivo systems without any detectable toxicity.
Assuntos
Excipientes , Trealose , Animais , Autofagia , Drosophila , Humanos , Nanogéis , Polímeros , Trealose/administração & dosagem , Peixe-ZebraRESUMO
The gonadal steroids, including androgens, estrogens and progestogens, are involved in the control of body fat distribution in humans. Nevertheless, not only the size and localization of the fat depots depend on the sex steroids levels, but they can also highly affect the functioning of adipose tissue. Namely, the gonadocorticoids can directly influence insulin signaling, lipid metabolism, fatty acid uptake and adipokine production. They may also alter energy balance and glucose homeostasis in adipocytes in an indirect way, e.g., by changing the expression level of aquaglyceroporins. This work presents the recent advances in understanding the molecular mechanism of how the gonadal steroids influence the functioning of adipose tissue leading to a set of detrimental metabolic consequences. Special attention is given here to highlighting the sexual dimorphism of adipocyte functioning in terms of health and disease. Particularly, we discuss the molecular background of metabolic disturbances occurring in consequence of hormonal imbalance which is characteristic of some common endocrinopathies such as the polycystic ovary syndrome. From this perspective, we highlight the potential drug targets and the active substances which can be used in personalized sex-specific management of metabolic diseases, in accord with the patient's hormonal status.
Assuntos
Tecido Adiposo/fisiologia , Doenças Metabólicas/metabolismo , Esteroides/metabolismo , Adipócitos/metabolismo , Animais , Aquaporinas/metabolismo , Distribuição da Gordura Corporal , Feminino , Hormônios Esteroides Gonadais/fisiologia , Humanos , Resistência à Insulina/fisiologia , Lipogênese/fisiologia , Masculino , MicroRNAs/metabolismo , Síndrome do Ovário Policístico/metabolismo , Fatores Sexuais , Esteroides/fisiologiaRESUMO
Pentacyclic lupane-type triterpenoids, such as betulin and its synthetic derivatives, display a broad spectrum of biological activity. However, one of the major drawbacks of these compounds as potential therapeutic agents is their high hydrophobicity and low bioavailability. On the other hand, the presence of easily transformable functional groups in the parent structure makes betulin have a high synthetic potential and the ability to form different derivatives. In this context, research on the synthesis of new betulin derivatives as conjugates of naturally occurring triterpenoid with a monosaccharide via a linker containing a heteroaromatic 1,2,3-triazole ring was presented. It has been shown that copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition reaction (CuAAC) provides an easy and effective way to synthesize new molecular hybrids based on natural products. The chemical structures of the obtained betulin glycoconjugates were confirmed by spectroscopic analysis. Cytotoxicity of the obtained compounds was evaluated on a human breast adenocarcinoma cell line (MCF-7) and colorectal carcinoma cell line (HCT 116). The obtained results show that despite the fact that the obtained betulin glycoconjugates do not show interesting antitumor activity, the idea of adding a sugar unit to the betulin backbone may, after some modifications, turn out to be correct and allow for the targeted transport of betulin glycoconjugates into the tumor cells.
Assuntos
Alcinos/farmacologia , Antineoplásicos/farmacologia , Azidas/farmacologia , Cobre/química , Glicoconjugados/farmacologia , Triterpenos/farmacologia , Alcinos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Azidas/química , Catálise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reação de Cicloadição , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glicoconjugados/síntese química , Glicoconjugados/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Triterpenos/síntese química , Triterpenos/químicaRESUMO
Perturbations of glycosaminoglycan metabolism lead to mucopolysaccharidoses (MPS)-lysosomal storage diseases. One type of MPS (type VI) is associated with a deficiency of arylsulfatase B (ARSB), for which we previously established a cellular model using pulmonary artery endothelial cells with a silenced ARSB gene. Here, we explored the effects of silencing the ARSB gene on the growth of human pulmonary artery smooth muscle cells in the presence of different concentrations of dermatan sulfate (DS). The viability of pulmonary artery smooth muscle cells with a silenced ARSB gene was stimulated by the dermatan sulfate. In contrast, the growth of pulmonary artery endothelial cells was not affected. As shown by microarray analysis, the expression of the arylsulfatase G (ARSG) in pulmonary artery smooth muscle cells increased after silencing the arylsulfatase B gene, but the expression of genes encoding other enzymes involved in the degradation of dermatan sulfate did not. The active site of arylsulfatase G closely resembles that of arylsulfatase B, as shown by molecular modeling. Together, these results lead us to propose that arylsulfatase G can take part in DS degradation; therefore, it can affect the functioning of the cells with a silenced arylsulfatase B gene.
Assuntos
Dermatan Sulfato/metabolismo , Miócitos de Músculo Liso/enzimologia , N-Acetilgalactosamina-4-Sulfatase/fisiologia , Sequência de Aminoácidos , Arilsulfatases/biossíntese , Arilsulfatases/química , Arilsulfatases/genética , Domínio Catalítico , Dermatan Sulfato/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Inativação Gênica , Humanos , Modelos Moleculares , Mucopolissacaridose VI/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , N-Acetilgalactosamina-4-Sulfatase/química , Especificidade de Órgãos , Ligação Proteica , Conformação Proteica , Artéria Pulmonar/citologia , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Análise Serial de Tecidos , Regulação para CimaRESUMO
BACKGROUND: Rapid changes in the expression of many messenger RNA (mRNA) species follow exposure of cells to ionizing radiation. One of the hypothetical mechanisms of this response may include microRNA (miRNA) regulation, since the amounts of miRNAs in cells also vary upon irradiation. To address this possibility, we designed experiments using cancer-derived cell lines transfected with luciferase reporter gene containing sequences targeted by different miRNA species in its 3'- untranslated region. We focus on the early time-course response (1 h past irradiation) to eliminate secondary mRNA expression waves. RESULTS: Experiments revealed that the irradiation-induced changes in the mRNA expression depend on the miRNAs which interact with mRNA. To identify the strongest interactions, we propose a mathematical model which predicts the mRNA fold expression changes, caused by perturbation of microRNA-mRNA interactions. Model was applied to experimental data including various cell lines, irradiation doses and observation times, both ours and literature-based. Comparison of modelled and experimental mRNA expression levels given miRNA level changes allows estimating how many and which miRNAs play a significant role in transcriptome response to stress conditions in different cell types. As an example, in the human melanoma cell line the comparison suggests that, globally, a major part of the irradiation-induced changes of mRNA expression can be explained by perturbed miRNA-mRNA interactions. A subset of about 30 out of a few hundred miRNAs expressed in these cells appears to account for the changes. These miRNAs play crucial roles in regulatory mechanisms observed after irradiation. In addition, these miRNAs have a higher average content of GC and a higher number of targeted transcripts, and many have been reported to play a role in the development of cancer. CONCLUSIONS: Our proposed mathematical modeling approach may be used to identify miRNAs which participate in responses of cells to ionizing radiation, and other stress factors such as extremes of temperature, exposure to toxins, and drugs.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Modelos Biológicos , Neoplasias/genética , RNA Mensageiro/metabolismo , Radiação Ionizante , Estresse Fisiológico , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Neoplasias/metabolismo , Neoplasias/fisiopatologiaRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy originating from T-cell precursors. The genetic landscape of T-ALL has been largely characterized by next-generation sequencing. Yet, the transcriptome of miRNAs (miRNome) of T-ALL has been less extensively studied. Using small RNA sequencing, we characterized the miRNome of 34 pediatric T-ALL samples, including the expression of isomiRs and the identification of candidate novel miRNAs (not previously annotated in miRBase). For the first time, we show that immunophenotypic subtypes of T-ALL present different miRNA expression profiles. To extend miRNome characteristics in T-ALL (to 82â¯T-ALL cases), we combined our small RNA-seq results with data available in Gene Expression Omnibus. We report on miRNAs most abundantly expressed in pediatric T-ALL and miRNAs differentially expressed in T-ALL versus normal mature T-lymphocytes and thymocytes, representing candidate oncogenic and tumor suppressor miRNAs. Using eight target prediction algorithms and pathway enrichment analysis, we identified differentially expressed miRNAs and their predicted targets implicated in processes (defined in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) of potential importance in pathogenesis of T-ALL, including interleukin-6-mediated signaling, mTOR signaling, and regulation of apoptosis. We finally focused on hsa-mir-106a-363 cluster and functionally validated direct interactions of hsa-miR-20b-5p and hsa-miR-363-3p with 3' untranslated regions of their predicted targets (PTEN, SOS1, LATS2), overrepresented in regulation of apoptosis. hsa-mir-106a-363 is a paralogue of prototypic oncogenic hsa-mir-17-92 cluster with yet unestablished role in the pathogenesis of T-ALL. Our study provides a firm basis and data resource for functional analyses on the role of miRNA-mRNA interactions in T-ALL.
Assuntos
Regulação Leucêmica da Expressão Gênica , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Interferência de RNA , RNA Mensageiro/genética , Transcriptoma , Apoptose/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Genes Reporter , Imunofenotipagem , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnósticoRESUMO
A series of thermoresponsive glycomicrogels with trehalose in the cross-links or with trehalose in the cross-links and as pending moieties was synthesized. These materials were obtained by surfactant-free precipitation copolymerization of N-isopropylacrylamide and various amounts of trehalose monomers. The resultant particles showed a spherical shape and a submicrometer hydrodynamic size with a narrow size distribution. At 25 °C, glycomicrogels in solutions with physiological ionic strength formed stable colloids, which further gelled upon heating to physiological temperature forming a macroscopic hydrogel with an interconnected porous structure. These extremely soft matrices with dynamic storage modulus in the range of 9-70 Pa were examined in 3D culture systems for HeLa cell culture in comparison to traditional 2D mode. They showed relatively low syneresis over time, especially when glycomicrogels with a high content of hydrophilic trehalose were used as building blocks. An incorporated pending trehalose composed of two α,α'-1,1'-linked d-glucose moieties was used with the intention of providing multivalent interactions with glucose transporters (GLUTs) expressed on the cell surface. A better cell viability was observed when a soft hydrogel with the highest content of trehalose and the lowest syneresis was used as a matrix compared to a 2D control assay.
Assuntos
Técnicas de Cultura de Células/métodos , Géis/química , Temperatura , Trealose/química , Células HeLa , Humanos , Células Tumorais CultivadasRESUMO
This study described the synthesis and in vitro evaluation of eight new derivatives of uridine as antifungal agents and inhibitors of chitin synthase. Dimeric uridinyl derivatives synthesized by us did not exhibit significant activity. One of the studied monomeric derivative, 5'-(N-succinyl)-5'-amino-5'-deoxyuridine methyl ester (compound 7) showed activities against several fungal strains (MIC range 0.06-1.00 mg/mL) and inhibited chitin synthase from Saccharomyces cerevisiae (IC50=0.8mM). Moreover compound 7 exhibited synergistic interaction with caspofungin against Candida albicans (FIC index=0.28).
Assuntos
Antifúngicos/química , Quitina Sintase/antagonistas & inibidores , Inibidores Enzimáticos/química , Uridina/análogos & derivados , Antifúngicos/síntese química , Antifúngicos/farmacologia , Aspergillus niger/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Quitina Sintase/metabolismo , Dimerização , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Testes de Sensibilidade Microbiana , Rhizopus/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Uridina/síntese química , Uridina/farmacologiaRESUMO
Radiation-induced bystander effect, appearing as different biological changes in cells that are not directly exposed to ionizing radiation but are under the influence of molecular signals secreted by irradiated neighbors, have recently attracted considerable interest due to their possible implication for radiotherapy. However, various cells present diverse radiosensitivity and bystander responses that depend, inter alia, on genetic status including TP53, the gene controlling the cell cycle, DNA repair and apoptosis. Here we compared the ionizing radiation and bystander responses of human colorectal carcinoma HCT116 cells with wild type or knockout TP53 using a transwell co-culture system. The viability of exposed to X-rays (0-8 Gy) and bystander cells of both lines showed a roughly comparable decline with increasing dose. The frequency of micronuclei was also comparable at lower doses but at higher increased considerably, especially in bystander TP53-/- cells. Moreover, the TP53-/- cells showed a significantly elevated frequency of apoptosis, while TP53+/+ counterparts expressed high level of senescence. The cross-matched experiments where irradiated cells of one line were co-cultured with non-irradiated cells of opposite line show that both cell lines were also able to induce bystander effects in their counterparts, however different endpoints revealed with different strength. Potential mediators of bystander effects, IL-6 and IL-8, were also generated differently in both lines. The knockout cells secreted IL-6 at lower doses whereas wild type cells only at higher doses. Secretion of IL-8 by TP53-/- control cells was many times lower than that by TP53+/+ but increased significantly after irradiation. Transcription of the NFκBIA was induced in irradiated TP53+/+ mainly, but in bystanders a higher level was observed in TP53-/- cells, suggesting that TP53 is required for induction of NFκB pathway after irradiation but another mechanism of activation must operate in bystander cells.
Assuntos
Adenocarcinoma/genética , Apoptose/efeitos da radiação , Neoplasias Colorretais/genética , Genes p53 , Adenocarcinoma/patologia , Apoptose/genética , Efeito Espectador/efeitos da radiação , Linhagem Celular Tumoral/efeitos da radiação , Senescência Celular/efeitos da radiação , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Proteínas I-kappa B/biossíntese , Proteínas I-kappa B/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Interleucina-8/biossíntese , Interleucina-8/genética , Testes para Micronúcleos , Inibidor de NF-kappaB alfa , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase (ARSB). Pulmonary hypertension (PH) occurs in MPS VI patients and is a marker of bad prognosis. Malfunction of endothelium, which regulates vascular tonus and stimulates angiogenesis, can contribute to the occurrence of PH in MPS VI. AIM: The aim of the study was to establish a human MPS VI cellular model of pulmonary artery endothelial cells (HPAECs) and evaluate how it affects factors that may trigger PH such as proliferation, apoptosis, expression of endothelial nitric oxide synthase (eNOS), natriuretic peptide type C (NPPC), and vascular endothelial growth factor A (VEGFA). RESULTS: Increasing concentrations of dermatan sulfate (DS) reduce the viability of the cells in both ARSB deficiency and controls, but hardly influence apoptosis. The expression of eNOS in HPAECs is reduced up to two thirds in the presence of DS. NPPC shows a biphasic expression reaction with an increase at 50 µg/mL DS and reduction at 0 and 100 µg/mL DS. The expression of VEGFA decreases with increasing DS concentrations and absence of elastin, and increases with increasing DS in the presence of elastin. CONCLUSION: Our data suggest that MPS VI endothelium presents a prohypertensive phenotype due to the reduction of endothelium's proliferation ability and expression of vasorelaxing factors.
RESUMO
Interactions of living organisms with their environment mainly involve modulation of gene expression by stimulation or silencing. One of the epigenetic mechanisms that regulate translation and the levels of transcripts is RNA interference, in which microRNAs (miRNAs) address RNA-induced silencing complexes (RISCs) to degrade specific mRNAs or to silence their translation. In this mechanism, double stranded RNA structure is crucial for miRNA biogenesis and the action of RISCs. RNA molecules can be modified structurally by reactive oxygen and nitrogen species (ROS and RNS) that are produced in cells of all aerobic organisms and may be induced by environmental factors. Here we describe experiments in which changes of ROS and transcript levels are induced by X-irradiation and measured using flow cytometer and the fluorescent dye 2',7'-dichlorofluorescein diacetate and microarray methods in cultured human K562, Me45 and HCT116 cells. Analysis of the nucleotide sequences of mRNAs which are up- or down-regulated after irradiation shows significant differences in the distributions of miRNA-targeted motives between these two groups. Immediately after irradiation most miRNAs behave as "up-regulators", showing more targets in up-than in down-regulated transcripts, and this changes about 12h later when we also observe changes in ROS and miRNA levels. Our results suggest that the changes in the transcriptome could result from changes in RNA interference and that these effects could be related to the changed ROS levels in irradiated cells. We propose that such modulation of gene expression at the mRNA level may be implicated more generally in cellular responses to stresses where ROS levels change.
Assuntos
Redes Reguladoras de Genes/efeitos da radiação , MicroRNAs/metabolismo , Interferência de RNA , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Citometria de Fluxo , Regulação da Expressão Gênica , Células HCT116 , Humanos , Células K562 , MicroRNAs/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Regulação para Cima , Raios XRESUMO
The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G. mellonella was determined, using electrospray mass spectrometry. The MS analysis was performed on tryptic digests of JHBP. Our results show that the disulfide bridges link Cys10 and Cys17, and Cys151 and Cys195. Of the two potential N-glycosylation sites in JHBP, Asn4, and Asn94, only Asn94 is glycosylated. This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris.
Assuntos
Proteínas de Transporte/química , Cistina/metabolismo , Proteínas de Insetos , Animais , Proteínas de Transporte/sangue , Proteínas de Transporte/metabolismo , Glicosilação , Larva/química , Larva/metabolismo , Lepidópteros/química , Lepidópteros/metabolismo , Isoformas de Proteínas , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.