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Runx1 is a transcription factor that plays a key role in determining the proliferative and differential state of multiple cell types, during both development and adulthood. Here, we report how Runx1 is specifically upregulated at the injury site during zebrafish heart regeneration, and that absence of runx1 results in increased myocardial survival and proliferation, and overall heart regeneration, accompanied by decreased fibrosis. Using single cell sequencing, we found that the wild-type injury site consists of Runx1-positive endocardial cells and thrombocytes that induce expression of smooth muscle and collagen genes. Both these populations cannot be identified in runx1 mutant wounds that contain less collagen and fibrin. The reduction in fibrin in the mutant is further explained by reduced myofibroblast formation and upregulation of components of the fibrin degradation pathway, including plasminogen receptor annexin 2A as well as downregulation of plasminogen activator inhibitor serpine1 in myocardium and endocardium, resulting in increased levels of plasminogen. Our findings suggest that Runx1 controls the regenerative response of multiple cardiac cell types and that targeting Runx1 is a novel therapeutic strategy for inducing endogenous heart repair.
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Cicatriz/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Coração/fisiopatologia , Miocárdio/patologia , Regeneração , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Anexina A2/metabolismo , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Endocárdio/patologia , Regulação da Expressão Gênica no Desenvolvimento , Músculo Liso/metabolismo , Mutação/genética , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Cadeias Pesadas de Miosina/metabolismo , Regulação para Cima/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
There is currently no widely adopted standard for the optical characterization of fluorescence microscopes. We used laser written fluorescence to generate two- and three-dimensional patterns to deliver a quick and quantitative measure of imaging performance. We report on the use of two laser written patterns to measure the lateral resolution, illumination uniformity, lens distortion and color plane alignment using confocal and structured illumination fluorescence microscopes.
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Anthocyanins are water soluble pigments which have been proved to exhibit health benefits. Several studies have investigated their effects on several types of cancer, but little attention has been given to melanoma. The phytochemical content of nine different berry samples was assessed by liquid chromatography followed by electrospray ionization mass spectrometry (LC-ESI+-MS). Twenty-six anthocyanins were identified, after a previous C18 Sep-pak clean-up procedure. Chokeberry and red grape anthocyanins rich extracts (C-ARE and RG-ARE) were selected to be tested on normal and melanoma cell lines, due to their different chemical pattern. C-ARE composition consists of cyanidin aglycone glycosylated with different sugars; while RG-ARE contains glucosylated derivatives of five different aglycones. Both C-ARE and RG-ARE anthocyanins reduced proliferation, increased oxidative stress biomarkers and diminished mitochondrial membrane potential in melanoma cells, having no negative influence on normal cells. A synergistic response may be attributed to the five different aglycones present in RG-ARE, which proved to exert greater effects on melanoma cells than the mixture of cyanidin derivatives with different sugars (C-ARE). In conclusion, C-ARE and RG-ARE anthocyanins may inhibit melanoma cell proliferation and increase the level of oxidative stress, with opposite effect on normal cells. Therefore, anthocyanins might be recommended as active ingredients for cosmetic and nutraceutical industry. Graphical Abstract á .
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Antocianinas/farmacologia , Melanoma/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Antocianinas/análise , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Humanos , Malondialdeído/metabolismo , Melanoma/patologia , Camundongos , Extratos Vegetais/química , Prunus/química , Vitis/químicaRESUMO
RATIONALE AND OBJECTIVE: Acute myocardial infarction (AMI) results in the recruitment of leukocytes to injured myocardium. Additionally, myocardium remote to the infarct zone also becomes inflamed and is associated with adverse left ventricular remodelling. Renal ischaemic syndromes have been associated with remote organ inflammation and impaired function. Here, we tested the hypothesis that AMI results in remote organ (renal) inflammation. METHODS: Mice were subjected to either AMI, sham procedure or no procedure and the inflammatory response in peripheral blood, injured and remote myocardium, and kidneys was studied at 24 h. RESULTS: AMI resulted in increased circulating neutrophils (P < 0.001) and monocytes (P < 0.001). mRNA for inflammatory mediators significantly increased in infarcted myocardium and in remote myocardium. VCAM-1 mRNA was increased in both infarcted and remote myocardium. VCAM-1 protein was also increased in the kidneys of AMI mice (P < 0.05) and immunofluorescence revealed localisation of VCAM-1 to glomeruli, associated with leukocyte infiltration and increased local inflammatory mRNA expression. CONCLUSIONS: We conclude that in addition to local inflammation, AMI results in remote organ inflammation evidenced by (1) increased expression of mRNA for inflammatory cytokines, (2) marked upregulation of VCAM-1 in renal glomeruli, and (3) the recruitment and infiltration of leukocytes in the kidney.
Assuntos
Glomerulonefrite/etiologia , Glomérulos Renais/imunologia , Infarto do Miocárdio/complicações , Miocardite/etiologia , Miocárdio/imunologia , Animais , Citocinas/genética , Feminino , Glomerulonefrite/imunologia , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/imunologia , Miocardite/imunologia , RNA Mensageiro/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
OBJECTIVE: Nicotinic acid (NA) treatment has been associated with benefits in atherosclerosis that are usually attributed to effects on plasma lipoproteins. The NA receptor GPR109A is expressed in monocytes and macrophages, suggesting a possible additional role for NA in modulating function of these immune cells. We hypothesize that NA has the potential to act directly on monocytes to alter mediators of inflammation that may contribute to its antiatherogenic effects in vivo. METHODS AND RESULTS: In human monocytes activated by Toll-like receptor (TLR)-4 agonist lipopolysaccharide, NA reduced secretion of proinflammatory mediators: TNF-α (by 49.2±4.5%); interleukin-6 (by 56.2±2.8%), and monocyte chemoattractant protein-1 (by 43.2±3.1%) (P<0.01). In TLR2 agonist, heat-killed Listeria monocytogenes-activated human monocytes, NA reduced secretion of TNF-α (by 48.6±7.1%), interleukin-6 (by 60.9±1.6%), and monocyte chemoattractant protein-1 (by 59.3±5.3%) (P<0.01; n=7). Knockdown of GPR109A by siRNA resulted in a loss of this anti-inflammatory effect in THP-1 monocytes. However, inhibition of prostaglandin D2 receptor by MK0524 or COX2 by NS398 did not alter the anti-inflammatory effects of NA observed in activated human monocytes. Preincubation of THP-1 monocytes with NA 0.1 mmol/L reduced phosphorylated IKKß by 42±2% (P<0.001) IKB-α by 54±14% (P<0.01). Accumulation of nuclear p65 NF-κB in response to lipopolysaccharide treatment was also profoundly inhibited, by 89±1.3% (n=4; P<0.01). NA potently inhibited monocyte adhesion to activated HUVEC, and VCAM, mediated by the integrin, very late antigen 4. Monocyte chemotaxis was also significantly reduced (by 45.7±1.2%; P<0.001). CONCLUSION: NA displays a range of effects that are lipoprotein-independent and potentially antiatherogenic. These effects are mediated by GPR109A and are independent of prostaglandin pathways. They suggest a rationale for treatment with NA that is not dependent on levels of plasma cholesterol and possible applications beyond the treatment of dyslipidemia.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/metabolismo , Monócitos/efeitos dos fármacos , Niacina/farmacologia , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Inflamação/genética , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Integrina alfa4beta1/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/imunologia , Monócitos/metabolismo , Fosforilação , Pirazinas/farmacologia , Interferência de RNA , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
OBJECTIVE: Endothelial cell activation is an important mediator of monocyte recruitment to sites of vascular inflammation. We hypothesized that high-affinity dual-ligand microparticles of iron oxide (MPIO), targeted to P-selectin and vascular cell adhesion molecule-1 (PV-MPIO), would identify activated endothelial cells during atherosclerosis progression. METHODS AND RESULTS: In vivo magnetic resonance imaging in apolipoprotein E-deficient mice showed rapid binding of PV-MPIO to the aortic root, which was maximal 30 minutes post-MPIO injection and maintained at 60 minutes. Minimal binding was observed for control IgG-MPIO. Intensely low magnetic resonance signal areas, corresponding to PV-MPIO binding, were detected early (14 weeks), during foam cell formation. Contrast effects increased at 20 weeks during fibrofatty lesion development (P<0.05), but reduced by 30 weeks (P<0.01). Across all lesion severities, magnetic resonance imaging contrast effects correlated with lesion macrophage area quantified by immunohistochemistry (R=0.53; P<0.01). Near-infrared fluorescently labeled PV-MPIO were shown, by flow cytometry, to bind only activated endothelial cells and not to macrophages. Using en face immunofluorescence, we further demonstrate selective PV-MPIO accumulation at atherosclerosis-susceptible sites, with minimal binding to atherosclerosis-spared regions. CONCLUSIONS: This high-affinity leukocyte-mimetic magnetic resonance imaging agent reveals endothelial activation. PV-MPIO demonstrate exceptionally rapid in vivo steady state accumulation, providing conspicuous magnetic resonance contrast effects that can be objectively quantified. In atherosclerosis progression, PV-MPIO tracked closely with the burden and distribution of plaque macrophages, not merely plaque size. On a biocompatible platform, this approach has potential for quantitative magnetic resonance imaging of inflammatory disease activity.
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Aorta/patologia , Doenças da Aorta/diagnóstico , Aterosclerose/diagnóstico , Materiais Biomiméticos , Meios de Contraste , Endotélio Vascular/patologia , Leucócitos/patologia , Macrófagos/patologia , Imageamento por Ressonância Magnética , Animais , Anticorpos , Afinidade de Anticorpos , Aorta/imunologia , Aorta/metabolismo , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Sítios de Ligação de Anticorpos , Meios de Contraste/farmacocinética , Modelos Animais de Doenças , Progressão da Doença , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Feminino , Compostos Férricos , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Leucócitos/imunologia , Leucócitos/metabolismo , Ligantes , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Selectina-P/imunologia , Selectina-P/metabolismo , Tamanho da Partícula , Espectroscopia de Luz Próxima ao Infravermelho , Fatores de Tempo , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Differential scanning calorimetry (DSC) and X-ray powder diffraction (PXRD) have been used to determine the phase behavior of the binary mixtures of undecanoic acid (A) and undecylamine (B) in the bulk. In addition, we report DSC data that indicates very similar behavior for the solid monolayers of these materials adsorbed on the surface of graphite. The two species are found to form a series of stoichiometric complexes of the type AB, A(2)B, and A(3)B on the acid rich side of the phase diagram. Interestingly, no similar series of complexes is evident on the amine rich side. As a result of this complexation, the solid monolayers of the binary mixtures exhibit a very pronounced enhancement in stability relative to the pure adsorbates.
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The title compound, C(10)H(24)N(+)·C(8)H(15)O(2) (-), forms a layered structure in which inter-molecular N(+)-Hâ¯O hydrogen bonds connect anions and cations, forming a two-dimensional network parallel to (010). The n-alkyl chains of the decyl-ammonium cations pack according to an ortho-rhom-bic 'subcell' with approximate dimensions 5.1 × 7.3â Å, and they are significantly distorted from planarity.
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Commercial fluorescence microscope stands and fully automated XYZt fluorescence imaging systems are generally beyond the limited budgets available for teaching and outreach. We have addressed this problem by developing "Microscopi", an accessible, affordable, DIY automated imaging system that is built from 3D printed and commodity off-the-shelf hardware, including electro-mechanical, computer and optical components. Our design features automated sample navigation and image capture with a simple web-based graphical user interface, accessible with a tablet or other mobile device. The light path can easily be switched between different imaging modalities. The open source Python-based control software allows the hardware to be driven as an integrated imaging system. Furthermore, the microscope is fully customisable, which also enhances its value as a learning tool. Here, we describe the basic design and demonstrate imaging performance for a range of easily sourced specimens.
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INTRODUCTION: Exogenous ketones potentially provide an alternative, energetically advantageous fuel to power exercising skeletal muscle. However, there is limited evidence regarding their relative contribution to energy expenditure during exercise. Furthermore, the effect of blood ketone concentration and exercise intensity on exogenous ketone oxidation rates is unknown. METHODS: Six athletes completed cycling ergometer exercise on three occasions within a single-blind, random-order controlled, crossover design study. Exercise duration was 60 min, consisting of 20-min intervals at 25%, 50%, and 75% maximal power output (WMax). Participants consumed (i) bitter flavored water (control), (ii) a low-dose ß-hydroxybutyrate (ßHB) ketone monoester (KME; 252 mg·kg BW-1, "low ketosis"), or (iii) a high-dose ßHB KME (752 mg·kg BW-1, "high ketosis"). The KME contained a 13C isotope label, allowing for the determination of whole-body exogenous ßHB oxidation rates through sampled respiratory gases. RESULTS: Despite an approximate doubling of blood ßHB concentrations between low- and high-ketosis conditions (~2 mM vs ~4.4 mM), exogenous ßHB oxidation rates were similar at rest and throughout exercise. The contribution of exogenous ßHB oxidation to energy expenditure peaked during the 25% WMax exercise intensity but was relatively low (4.46% ± 2.71%). Delta efficiency during cycling exercise was significantly greater in the low-ketosis (25.9% ± 2.1%) versus control condition (24.1% ± 1.9%; P = 0.027). CONCLUSIONS: Regardless of exercise intensity, exogenous ßHB oxidation contributes minimally to energy expenditure and is not increased by elevating circulating concentrations greater than ~2 mM. Despite low exogenous ßHB oxidation rates, exercise efficiency was significantly improved when blood ßHB concentration was raised to ~2 mM.
Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Atletas , Metabolismo Energético/fisiologia , Exercício Físico/fisiologia , Cetonas/sangue , Músculo Esquelético/metabolismo , Ácido 3-Hidroxibutírico/administração & dosagem , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/urina , Estudos Cross-Over , Teste de Esforço , Feminino , Glicogênio/metabolismo , Humanos , Cetonas/administração & dosagem , Cetose/metabolismo , Masculino , Fibras Musculares Esqueléticas/metabolismo , Oxirredução , Esforço Físico , Método Simples-Cego , Fatores de Tempo , Adulto JovemRESUMO
The Na(+)-K(+)-ATPase (NKA) can affect intracellular Ca(2+) concentration regulation via coupling to the Na(+)-Ca(2+) exchanger and may be important in myogenic tone. We previously reported that in mice carrying a transgene for the NKA alpha(2)-isoform in smooth muscle (alpha(2sm+)), the alpha(2)-isoform protein as well as the alpha(1)-isoform (not contained in the transgene) increased to similar degrees (2-7-fold). Aortas from alpha(2sm+) mice relaxed faster from a KCl-induced contraction, hypothesized to be related to more rapid Ca(2+) clearance. To elucidate the mechanisms underlying this faster relaxation, we therefore measured the expression and distribution of proteins involved in Ca(2+) clearance. Na(+)-Ca(2+) exchanger, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and plasma membrane Ca(2+)-ATPase (PMCA) proteins were all elevated up to approximately fivefold, whereas actin, myosin light chain, and calponin proteins were not changed in smooth muscle from alpha(2sm+) mice. Interestingly, the corresponding Ca(2+) clearance mRNA levels were unchanged. Immunocytochemical data indicate that the Ca(2+) clearance proteins are distributed similarly in wild-type and alpha(2sm+) aorta cells. In studies measuring relaxation half-times from a KCl-induced contraction in the presence of pharmacological inhibitors of SERCA and PMCA, we estimated that together these proteins were responsible for approximately 60-70% of relaxation in aorta. Moreover, the percent contribution of SERCA and PMCA to relaxation rates in alpha(2sm+) aorta was not significantly different from that in wild-type aorta. The coordinate expressions of NKA and Ca(2+) clearance proteins without change in the relative contributions of each individual protein to smooth muscle function suggest that NKA may be but one component of a larger functional Ca(2+) clearance system.
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Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transporte Biológico , Pressão Sanguínea , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , RNA Mensageiro/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Fatores de Tempo , Vasoconstrição , VasodilataçãoRESUMO
BACKGROUND: Human artificial chromosomes (HAC) are small functional extrachromosomal elements, which segregate correctly during each cell division. In human cells, they are mitotically stable, however when the HAC are transferred to murine cells they show an increased and variable rate of loss. In some cell lines the HAC are lost over a short period of time, while in others the HAC become stable without acquiring murine DNA. RESULTS: In this study, we linked the loss rate to the position of the HAC in the murine cell nucleus with respect to the chromocenters. HAC that associated preferentially with the chromocenter displayed a lower loss rate compared to the HAC that are less frequently associated. The chromocenter acts as a hub for the deposition of heterochromatic markers, controlling centromeric and pericentromeric DNA replication timing and chromosome segregation. The HAC which localized more frequently outside the chromocenters bound variable amounts of histone H3 tri-methylated at lysine 9, and the high level of intraclonal variability was associated with an increase in HAC segregation errors and delayed DNA replication timing. CONCLUSION: This is a novel result indicating that HAC segregation is closely linked to the position in the murine nucleus and gives important insight for HAC gene expression studies in murine cells and establishing murine models of human genetic disease.
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Cromatina/química , Instabilidade Cromossômica , Cromossomos Artificiais Humanos/genética , Cromossomos Artificiais Humanos/metabolismo , Animais , Linhagem Celular , Núcleo Celular/genética , Centrômero/fisiologia , Cromatina/metabolismo , Técnicas de Transferência de Genes , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Metáfase , Camundongos , MitoseRESUMO
X-linked recessive hypoparathyroidism, due to parathyroid agenesis, has been mapped to a 906-kb region on Xq27 that contains 3 genes (ATP11C, U7snRNA, and SOX3), and analyses have not revealed mutations. We therefore characterized this region by combined analysis of single nucleotide polymorphisms and sequence-tagged sites. This identified a 23- to 25-kb deletion, which did not contain genes. However, DNA fiber-FISH and pulsed-field gel electrophoresis revealed an approximately 340-kb insertion that replaced the deleted fragment. Use of flow-sorted X chromosome-specific libraries and DNA sequence analyses revealed that the telomeric and centromeric breakpoints on X were, respectively, approximately 67 kb downstream of SOX3 and within a repetitive sequence. Use of a monochromosomal somatic cell hybrid panel and metaphase-FISH mapping demonstrated that the insertion originated from 2p25 and contained a segment of the SNTG2 gene that lacked an open reading frame. However, the deletion-insertion [del(X)(q27.1) inv ins (X;2)(q27.1;p25.3)], which represents a novel abnormality causing hypoparathyroidism, could result in a position effect on SOX3 expression. Indeed, SOX3 expression was demonstrated, by in situ hybridization, in the developing parathyroid tissue of mouse embryos between 10.5 and 15.5 days post coitum. Thus, our results indicate a likely new role for SOX3 in the embryonic development of the parathyroid glands.
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Sequência de Bases/genética , Cromossomos Humanos Par 2/genética , Cromossomos Humanos X/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Hipoparatireoidismo/genética , Mutagênese Insercional/genética , Deleção de Sequência/genética , Animais , Inversão Cromossômica/genética , Análise Mutacional de DNA/métodos , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Recessivos/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Hipoparatireoidismo/patologia , Hibridização in Situ Fluorescente/métodos , Masculino , Camundongos , Fases de Leitura Aberta/genética , Glândulas Paratireoides/embriologia , Glândulas Paratireoides/patologia , Linhagem , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genéticaRESUMO
Increased production of reactive oxygen species and loss of endothelial NO bioactivity are key features of vascular disease states such as diabetes mellitus. Tetrahydrobiopterin (BH4) is a required cofactor for eNOS activity; pharmacologic studies suggest that BH4 may mediate some of the adverse effects of diabetes on eNOS function. We have now investigated the importance and mechanisms of BH4 availability in vivo using a novel transgenic mouse model with endothelial-targeted overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate-cyclohydrolase I (GTPCH). Transgenic (GCH-Tg) mice demonstrated selective augmentation of endothelial BH4 levels. In WT mice, induction of diabetes with streptozotocin (STZ) increased vascular oxidative stress, resulting in oxidative loss of BH4, forming BH2 and biopterin. Endothelial cell superoxide production in diabetes was increased, and NO-mediated endothelium-dependent vasodilatation was impaired. In diabetic GCH-Tg mice, superoxide production from the endothelium was markedly reduced compared with that of WT mice, endothelial BH4 levels were maintained despite some oxidative loss of BH4, and NO-mediated vasodilatation was preserved. These findings indicate that BH4 is an important mediator of eNOS regulation in diabetes and is a rational therapeutic target to restore NO-mediated endothelial function in diabetes and other vascular disease states.
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Biopterinas/análogos & derivados , Biopterinas/fisiologia , Diabetes Mellitus Experimental/metabolismo , Endotélio Vascular/fisiologia , GTP Cicloidrolase/fisiologia , Óxido Nítrico/fisiologia , Animais , Diabetes Mellitus Experimental/terapia , GTP Cicloidrolase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Estresse Oxidativo , RNA Mensageiro/análise , Estreptozocina , Superóxidos/metabolismoRESUMO
Ligand-conjugated microparticles of iron oxide (MPIO) have the potential to provide high sensitivity contrast for molecular magnetic resonance imaging (MRI). However, the accumulation and persistence of non-biodegradable micron-sized particles in liver and spleen precludes their clinical use and limits the translational potential of MPIO-based contrast agents. Here we show that ligand-targeted MPIO derived from multiple iron oxide nanoparticles may be coupled covalently through peptide linkers that are designed to be cleaved by intracellular macrophage proteases. The synthesized particles possess potential characteristics for targeted MRI contrast agents, including high relaxivity, unappreciable sedimentation, clearance from circulation and no overt toxicity. Importantly, we demonstrate that these particles are rapidly degraded both in vitro and in vivo, and that the targeted probes can be used for detection of inflammation in vivo using MRI. This approach provides a platform for molecular MRI contrast agents that is potentially more suitable for translation to humans.
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Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Peptídeo Hidrolases/metabolismo , Animais , Anticorpos/metabolismo , Meios de Contraste/química , Compostos Férricos/química , Humanos , Nanopartículas de Magnetita/ultraestrutura , Masculino , Camundongos , Tamanho da Partícula , Células RAW 264.7 , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Monogenic human disorders have been used as paradigms for complex genetic disease and as tools for establishing important insights into mechanisms of gene regulation and transcriptional control. Maturity-onset diabetes of the young (MODY) is a monogenic dominantly inherited form of diabetes that is characterized by defective insulin secretion from the pancreatic beta-cells. A wide variety of mutation types in five different genes have been identified that result in this condition. There have been no reports of a chromosome deletion or translocation resulting in MODY. We report a pedigree where MODY cosegregates with a balanced translocation [karyotype 46, XX t(3;20) (p21.2;q12)]. The chromosome 20 break point, 20q12, is within the region of one of the known MODY genes, hepatocyte nuclear factor-4alpha (HNF4A). Fluorescence in situ hybridization analysis demonstrated that the break point does not disrupt the coding region of this gene, but it lies at least 6 kb upstream of the conventional promoter (P1). We propose that this mutation disrupts the spatial relationship between the recently described alternate distal pancreatic promoter (P2) and HNF4A. This is the first case of MODY due to a balanced translocation, and it provides evidence to confirm the crucial role of an upstream regulator of HNF4A gene expression in the beta-cell.
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Cromossomos Humanos Par 20 , Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 2/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Translocação Genética , Adolescente , Adulto , Fatores Etários , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Mapeamento Cromossômico , Feminino , Fator 4 Nuclear de Hepatócito , Humanos , Insulina/sangue , Cariotipagem , Linhagem , Reação em Cadeia da Polimerase , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
We have identified a novel gene, upstream of the cytokine gene cluster region in 5q23-31, residing within one of the most common deleted segments associated with MDS. The novel gene exhibits significant alternative splicing generating at least six splice variants encoding four putative proline-rich protein isoforms, one of which is Golgi-associated. The gene is ubiquitously expressed and conserved among species with the C. elegans homologue being the most interesting, since it resides within an operon with two other genes, phospholipase D and dishevelled, a member of the Wnt pathway, suggesting a functional association. In addition, the novel gene and other key regulatory genes of the region, such IL3, Ril, AF5q31 and TCF-1, were found to be deleted in an atypical CML case, thus underscoring the significance of this subregion in the leukemogenesis process.
Assuntos
Processamento Alternativo , Proteínas de Transporte/genética , Cromossomos Humanos Par 5 , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia , Sequência de Bases , Deleção Cromossômica , Expressão Gênica , Humanos , Proteínas dos Microfilamentos , Dados de Sequência MolecularRESUMO
Angiogenesis is an essential component of tumour growth and, consequently, an important target both therapeutically and diagnostically. The cell adhesion molecule α(v)ß(3) integrin is a specific marker of angiogenic vessels and the most prevalent vascular integrin that binds the amino acid sequence arginine-glycine-aspartic acid (RGD). Previous studies using RGD-targeted nanoparticles (20-50 nm diameter) of iron oxide (NPIO) for magnetic resonance imaging (MRI) of tumour angiogenesis, have identified a number of limitations, including non-specific extravasation, long blood half-life (reducing specific contrast) and low targeting valency. The aim of this study, therefore, was to determine whether conjugation of a cyclic RGD variant [c(RGDyK)], with enhanced affinity for α(v)ß(3), to microparticles of iron oxide (MPIO) would provide a more sensitive contrast agent for imaging of angiogenic tumour vessels. Cyclic RGD [c(RGDyK)] and RAD [c(RADyK)] based peptides were coupled to 2.8 µm MPIO, and binding efficacy tested both in vitro and in vivo. Significantly greater specific binding of c(RGDyK)-MPIO to S-nitroso-n-acetylpenicillamine (SNAP)-stimulated human umbilical vein endothelial cells in vitro than PBS-treated cells was demonstrated under both static (14-fold increase; P < 0.001) and flow (44-fold increase; P < 0.001) conditions. Subsequently, mice bearing subcutaneous colorectal (MC38) or melanoma (B16F10) derived tumours underwent in vivo MRI pre- and post-intravenous administration of c(RGDyK)-MPIO or c(RADyK)-MPIO. A significantly greater volume of MPIO-induced hypointensities were found in c(RGDyK)-MPIO injected compared to c(RADyK)-MPIO injected mice, in both tumour models (P < 0.05). Similarly, administration of c(RGDyK)-MPIO induced a greater reduction in mean tumour T(2)* relaxation times than the control agent in both tumour models (melanoma P < 0.001; colorectal P < 0.0001). Correspondingly, MPIO density per tumour volume assessed immunohistochemically was significantly greater for c(RGDyK)-MPIO than c(RADyK)-MPIO injected animals, in both melanoma (P < 0.05) and colorectal (P < 0.0005) tumours. In both cases, binding of c(RGDyK)-MPIO co-localised with α(v)ß(3) expression. Comparison of RGD-targeted and dynamic contrast enhanced (DCE) MRI assessment of tumour perfusion indicated sensitivity to different vascular features. This study demonstrates specific binding of c(RGDyK)-MPIO to α(v)ß(3) expressing neo-vessels, with marked and quantifiable contrast and rapid clearance of unbound particles from the blood circulation compared to NPIO. Combination of this molecular MRI approach with conventional DCE MRI will enable integrated molecular, anatomical and perfusion tumour imaging.
Assuntos
Compostos Férricos/administração & dosagem , Compostos Férricos/análise , Imageamento por Ressonância Magnética/métodos , Neoplasias/diagnóstico , Neovascularização Patológica/diagnóstico por imagem , Oligopeptídeos/administração & dosagem , Oligopeptídeos/análise , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Humanos , Camundongos Endogâmicos C57BL , Microesferas , Neoplasias/terapia , Ligação Proteica , RadiografiaRESUMO
Targeting particles to sites of inflammation is of considerable interest for applications relating to molecular imaging and drug delivery. We and others have described micron-sized particles of iron oxide (MPIO) that can be directed using specific ligands (e.g. antibodies, peptides and oligosaccharides) to bind to mediators of vascular inflammation in vivo. Since leukocyte binding to these molecules can induce changes in the target cell, an outstanding question has been whether the binding of imaging particles to these mediators induces biologically significant changes in the endothelial cells, potentially initiating or propagating inflammation. Here, we address these questions by looking for changes in endothelial cells following binding of contrast agent. Specifically, we have quantified calcium flux, rearrangement of the actin cytoskeleton, production of reactive oxygen species (ROS), apoptosis and potential secondary changes, such as changes in gene and protein expression follow binding events to primary endothelial cells in vitro. Although leukocytes induced changes to endothelial cell function, we did not see any significant changes to endothelial calcium flux, cytoskeletal organisation, production of ROS or induction of apoptosis in response to antibody-MPIO binding. Furthermore, there were no changes to gene expression monitored via real-time RT-PCR or presentation of protein on the cell surface measured using flow cytometry. Our experiments demonstrate that whilst antibody-targeted microparticles mimic the binding capability of leukocytes to inflamed endothelium, they do not trigger the same cellular responses and do not appear to initiate or compound inflammation. These properties are desirable for targeted therapeutic and diagnostic agents.
Assuntos
Meios de Contraste/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Compostos Férricos/metabolismo , Material Particulado/metabolismo , Actinas/metabolismo , Cálcio/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Human artificial chromosomes (HAC) are a valuable tool in the analysis of complex chromatin structures such as the human centromere because of their small size and relative simplicity compared with normal human chromosomes. This report includes a comprehensive study of the centromere and chromatin composition of HAC, expressing human genes, generated in human cells and transferred to murine cells. The analysis involved chromatin immuno-precipitation and immuno-FISH on metaphase chromosomes and chromatin fibres. In both the cell types, the HAC consisted of alphoid and non-alphoid DNA and were mainly euchromatic in composition, although a pericentromeric heterochromatic region was present on all the HAC. Fibre-FISH and chromatin immuno-precipitation data indicated that the position of the centromere differed between HAC in human cells and in murine cells. Our work highlights the importance and utilisation of HAC for understanding the epigenetic aspects of chromosome biology.