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1.
Invest Ophthalmol Vis Sci ; 55(3): 1393-401, 2014 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-24458145

RESUMO

PURPOSE: Resuscitation of newborns is one of the most frequent procedures in neonatal medicine. The use of supplementary oxygen during resuscitation of the asphyxiated newborn has been shown to be detrimental to vulnerable tissues. We wanted to assess transcriptional changes in ocular tissue after the acute use of oxygen in the delivery room in a hypoxia-reoxygenation model of the newborn mouse. METHODS: C57BL/6 mice (n = 57), postnatal day 7, were randomized to receive either 120 minutes of hypoxia, at 8% O2, followed by 30 minutes of reoxygenation with 21, 40, 60, or 100% O2 or to normoxia followed by 30 minutes of 21% or 100% O2. Whole ocular homogenates were analyzed by Affymetrix 750k expression array, and RT-PCR was performed for validation. Bayesian analysis of variance for microarray data (BAMarray) was used to identify single significant genes, and Gene Set Enrichment Analysis (GSEA) was applied to reveal significant pathway systems. RESULTS: In total, ∼ 92% of the gene expression changes were altered in response to reoxygenation with 60% or 100% O2 compared to expression at the lower percentages of 21% and 40%. After 100% O2 treatment, genes involved in inflammation (Ccl12), angiogenesis (Igfr1, Stat3), and metabolism (Hk2) were upregulated. Pathway analyses after hypoxia-reoxygenation revealed significant alterations of six pathways which included apoptosis, TGF-beta signaling, oxidative phosphorylation, voltage-gated calcium channel complex, mitochondrion, and regulation of RAS protein signal transduction. CONCLUSIONS: Hypoxia-reoxygenation can induce immediate transcriptional responses in ocular tissue involving inflammation, angiogenesis, energy failure, and Ras signaling.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hiperóxia/genética , Hipóxia/genética , Proteínas Quimioatraentes de Monócitos/genética , Estresse Oxidativo/genética , RNA/genética , Fator de Transcrição STAT3/genética , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Hiperóxia/metabolismo , Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Proteínas Quimioatraentes de Monócitos/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/biossíntese , Transdução de Sinais
2.
Pediatr Res ; 75(4): 517-26, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24375083

RESUMO

BACKGROUND: Supplemental oxygen used during resuscitation can be detrimental to the newborn brain. The aim was to determine how different oxygen therapies affect gene transcription in a hypoxia-reoxygenation model. METHODS: C57BL/6 mice (n = 56), postnatal day 7, were randomized either to 120 min of hypoxia 8% O2 followed by 30 min of reoxygenation with 21, 40, 60, or 100% O2, or to normoxia followed by 30 min of 21 or 100% O2. Affymetrix 750k expression array was applied with RT-PCR used for validation. Histopathology and immunohistochemistry 3 d after hypoxia-reoxygenation compared groups reoxygenated with 21 or 100% O2 with normoxic controls (n = 22). RESULTS: In total, ~81% of the gene expression changes were altered in response to reoxygenation with 60 or 100% O2 and constituted many inflammatory-responsive genes (i.e., C5ar2, Stat3, and Ccl12). Oxidative phosphorylation was downregulated after 60 or 100% O2. Iba1(+) cells were significantly increased in the striatum and hippocampal CA1 after both 21 and 100% O2. CONCLUSION: In the present model, hypoxia-reoxygenation induces microglial accumulation in subregions of the brain. The transcriptional changes dominating after applying hyperoxic reoxygenation regimes include upregulating genes related to inflammatory responses and suppressing the oxidative phosphorylation pathway.


Assuntos
Encéfalo/metabolismo , Perfilação da Expressão Gênica , Hiperóxia/metabolismo , Hipóxia/metabolismo , Inflamação/metabolismo , Transcriptoma , Animais , Animais Recém-Nascidos , Análise por Conglomerados , Metabolismo Energético/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos
3.
Pediatr Res ; 74(5): 536-44, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23999071

RESUMO

BACKGROUND: The use of oxygen in acute treatment of asphyxiated term newborns is associated with increased mortality. It is unclear how hyperoxic reoxygenation after hypoxia affects transcriptional changes in the newborn lung. METHODS: On postnatal day 7, C57BL/6 mice (n = 62) were randomized to 120-min hypoxia (fraction of inspired oxygen (FiO2) 0.08) or normoxia. The hypoxia group was further randomized to reoxygenation for 30 min with FiO2 0.21, 0.40, 0.60, or 1.00, and the normoxia group to FiO2 0.21 or 1.00. Transcriptome profiling was performed on homogenized lung tissue using the Affymetrix 750k expression array, and validation was carried out by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: The hypoxia-reoxygenation model induced hypoxia-inducible factor 1 (HIF-1) targets like Vegfc, Adm, and Aqp1. In total, ~70% of the significantly differentially expressed genes were detected in the two high hyperoxic groups (FiO2 0.60 and 1.00). Reoxygenation with 100% oxygen after hypoxia uniquely upregulated Gadd45g, Dusp1, Peg3, and Tgm2. Pathway analysis identified mammalian target of rapamycin (mTOR) signaling pathway, DNA repair, c-jun N-terminal kinase (JNK)-pathway regulation, and cell cycle after hyperoxic reoxygenation was applied. CONCLUSION: Acute hypoxia induces HIF-1 targets independent of the reoxygenation regime applied. Hyperoxic reoxygenation affects pathways regulating cell growth and survival. DNA-damage-responsive genes are restricted to reoxygenation with 100% oxygen.


Assuntos
Animais Recém-Nascidos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipóxia/terapia , Pulmão/metabolismo , Oxigênio/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos/genética , Análise por Conglomerados , Primers do DNA/genética , Reparo do DNA/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Modelos Lineares , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Oxigênio/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo
4.
PLoS One ; 5(12): e14261, 2010 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-21151608

RESUMO

BACKGROUND: Perinatal hypoxia-ischemia is a major cause of mortality and cerebral morbidity, and using oxygen during newborn resuscitation may further harm the brain. The aim was to examine how supplementary oxygen used for newborn resuscitation would influence early brain tissue injury, cell death and repair processes and the regulation of genes related to apoptosis, neurodegeneration and neuroprotection. METHODS AND FINDINGS: Anesthetized newborn piglets were subjected to global hypoxia and then randomly assigned to resuscitation with 21%, 40% or 100% O(2) for 30 min and followed for 9 h. An additional group received 100% O(2) for 30 min without preceding hypoxia. The left hemisphere was used for histopathology and immunohistochemistry and the right hemisphere was used for in situ zymography in the corpus striatum; gene expression and the activity of various relevant biofactors were measured in the frontal cortex. There was an increase in the net matrix metalloproteinase gelatinolytic activity in the corpus striatum from piglets resuscitated with 100% oxygen vs. 21%. Hematoxylin-eosin (HE) staining revealed no significant changes. Nine hours after oxygen-assisted resuscitation, caspase-3 expression and activity was increased by 30-40% in the 100% O(2) group (n = 9/10) vs. the 21% O(2) group (n = 10; p<0.04), whereas brain-derived neurotrophic factor (BDNF) activity was decreased by 65% p<0.03. CONCLUSIONS: The use of 100% oxygen for resuscitation resulted in increased potentially harmful proteolytic activities and attenuated BDNF activity when compared with 21%. Although there were no significant changes in short term cell loss, hyperoxia seems to cause an early imbalance between neuroprotective and neurotoxic mechanisms that might compromise the final pathological outcome.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Caspase 3/biossíntese , Metaloproteinases da Matriz/biossíntese , Oxigênio/metabolismo , Ressuscitação/métodos , Animais , Animais Recém-Nascidos , Apoptose , Morte Celular , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Hipocampo/metabolismo , Oxigênio/química , Oxigênio/uso terapêutico , Suínos , Fatores de Tempo
5.
Anticancer Res ; 30(10): 3879-87, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21036698

RESUMO

5-Fluorouracil (5-FU) is frequently used in cancer treatment. Previous studies with 5-FU suggest that proapoptotic protein BAX and tumor suppressor protein TP53 are central factors in this process. As the leukemic T cell line Jurkat E6 has mutations in both these genes, we investigated a possible activation of alternative death pathways following 5-FU treatment. Here we show that 5-FU triggers apoptosis in Jurkat cells in a dose-dependent manner. Death responses were only moderately attenuated in the presence of a general caspase inhibitor. However, flow cytometric analysis showed activation of caspase 3 and a slight increase in ROS generation in a time- and dose-dependent manner. Furthermore, we observed 5-FU induced PARP cleavage and notably, reduced expression of antiapoptotic MCL-1L associated with the appearance of proapoptotic MCL-1S. Our results demonstrate the activation of alternative death pathways following treatment with 5-FU, despite mutations in the TP53 and BAX genes.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Fluoruracila/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Morte Celular/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Proteína de Sequência 1 de Leucemia de Células Mieloides , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Linfócitos T/enzimologia , Linfócitos T/patologia
6.
Pediatr Res ; 67(3): 250-6, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20010314

RESUMO

The optimal oxygen concentration for newborn resuscitation is still discussed. Oxygen administration during reoxygenation may induce short- and long-term pathologic changes via oxidative stress and has been associated to later childhood cancer. The aim was to study changes in oxidative stress-associated markers in liver and lung tissue of newborn pigs after acute hypoxia followed by reoxygenation for 30 min with 21, 40, or 100% oxygen compared with room air or to ventilation with 100% oxygen without preceding hypoxia. Nine hours after resuscitation, we found a dose-dependent increase in the matrix metalloproteinase gelatinase activity in liver tissue related to percentage oxygen supply by resuscitation (100% versus 21%; p = 0.002) pointing at more extensive tissue damage. Receiving 100% oxygen for 30 min without preceding hypoxia decreased the expression of VEGFR2 and TGFBR3 mRNA in liver tissue, but not in lung tissue. MMP-, VEGF-, and TGFbeta-superfamily are vital for the development, growth, and functional integrity of most tissues and our data rise concern about both short- and long-term consequences of even a brief hyperoxic exposure.


Assuntos
Hipóxia/terapia , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Oxigenoterapia , Respiração Artificial , Ressuscitação/métodos , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipóxia/enzimologia , Hipóxia/genética , Hipóxia/patologia , Fígado/enzimologia , Fígado/patologia , Pulmão/enzimologia , Pulmão/patologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Estresse Oxidativo/efeitos dos fármacos , Oxigenoterapia/efeitos adversos , Proteoglicanas/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Respiração Artificial/efeitos adversos , Suínos , Fatores de Tempo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
7.
Brain Res ; 1217: 37-49, 2008 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-18502402

RESUMO

Cytokines are released in response to brain injury and inflammation. By binding to receptors, they can cause, exacerbate or inhibit cellular injury and repair. We studied RNA expression of cytokine receptors and members of inflammatory pathways in human NT2-N neurons during 3 h of hypoxia and glucose deprivation followed by 21 h of reoxygenation, and the impact of acidosis. Right after acidotic hypoxia, RNA of IL-10RA and CXCR4 were significantly increased relative to acidotic control, while Bcl-2 and Bcl-xL were significantly decreased. After 21 h of neutral reoxygenation after neutral hypoxia, there was a significant increase in RNA of CXCR1 (relative quantification (RQ)=4.1, p<0.05), CXCR2 (3.6, p<0.05), CCR2 (3.8, p<0.05), Hsp70 (2.4, p<0.05), HIF-1alpha (1.5, p<0.001), TRAF6 (1.3, p<0.05) and TNFR1 (1.6, p<0.05). After 21 h of acidotic reoxygenation after acidotic hypoxia, we found a significant increase in RNA of IL-1R1, IL-10RA, CXCR4 and Hsp70 compared to control, and a significant decrease in FAS and TRAF6. There was a significant increase in Bax expression and a significant decrease in Bcl-2 and Bcl-xL expression in three out of four pH groups after 21 h of reoxygenation. Acidotic, relative to neutral, hypoxia and reoxygenation also influenced the expression of various genes. We conclude that inflammatory receptors and pathways are activated during hypoxia and reoxygenation in NT2-N neurons, and that this activation is pH dependent. This supports the concept that inflammatory pathways play a role in cerebral hypoxic-ischemic damage, and that they may represent important pharmacological targets.


Assuntos
Acidose/metabolismo , Hipóxia Celular/fisiologia , Inflamação/metabolismo , Neurônios/metabolismo , Receptores de Citocinas/biossíntese , Células Cultivadas , Expressão Gênica , Humanos , Traumatismo por Reperfusão/metabolismo
8.
Neurochem Int ; 48(6-7): 579-85, 2006 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16517018

RESUMO

The cerebellar granule cells have been extensively used for studies on metabolism, neurotransmission and neurotoxicology, since they can easily be grown in cultures. However, knowledge about the development of different proteins essential for synaptic transmission in these cells is lacking. This study has characterized the developmental profiles of the vesicular glutamate transporters (VGLUTs) and the synaptic vesicle proteins synapsins and synaptophysin in cerebellar granule cells and in co-cultures containing both granule cells and astrocytes. The protein levels of VGLUT2 decreased by approximately 70% from days 2 to 7 in vitro, whereas the levels of VGLUT1 increased by approximately 95%. Protein levels of synapsin I, synapsin IIIa and synaptophysin showed a developmental pattern similar to VGLUT1 while synapsin II and VGLUT3 were absent. The mRNA expressions of VGLUT1 and VGLUT2 were in accordance with the protein levels. The results indicate both that cerebellar granule cells are mature at approximately 7 days in vitro, and that the up-regulation of VGLUT1 and down-regulation of VGLUT2 in cerebellar granule cells are both independent of surrounding astrocytes and neuronal input. The results of this study are discussed in relation to general developmental profiles of VGLUTs in other brain regions.


Assuntos
Cerebelo/metabolismo , Neurônios/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/biossíntese , Proteína Vesicular 2 de Transporte de Glutamato/biossíntese , Proteínas Vesiculares de Transporte de Glutamato/biossíntese , Animais , Astrócitos/citologia , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Técnicas de Cocultura , Regulação da Expressão Gênica no Desenvolvimento , Ratos , Ratos Wistar , Sinapsinas/biossíntese , Sinaptofisina/biossíntese
9.
J Neurochem ; 96(5): 1458-66, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16478532

RESUMO

Studies of synapsin-deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild-type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double-labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.


Assuntos
Neurotransmissores/metabolismo , Sinapsinas/fisiologia , Proteínas Vesiculares de Transporte de Neurotransmissores/metabolismo , Animais , Imunofluorescência/métodos , Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal/métodos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Frações Subcelulares/metabolismo , Sinapsinas/deficiência , Sinaptossomos/metabolismo , Proteínas Vesiculares de Transporte de Neurotransmissores/classificação
10.
Immunol Lett ; 95(1): 25-30, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15325794

RESUMO

Fungal infections by molds like Aspergillus fumigatus are an increasing health problem which can be fatal in immuno-compromised patients. In healthy individuals, these infections are easily eliminated by the innate and acquired immune system. Complement factor 3 (C3) has a key place within the complement cascade and C3 RNA expression can therefore be used to monitor an impending immune response. Employing a liver cell line (HepG2) as a model system, we have examined their responses to A. fumigatus or beta-glucan, a major component of the fungal wall. C3 RNA expression was increased after stimulation with both LPS and A. fumigatus as well as after incubation with beta-glucan, although with different kinetics. C3 protein release into the supernatant followed an inverse bell-shaped curve when cells were incubated with A. fumigatus or beta-glucan while during LPS stimulation, the release was more stable. HepG2 cells also express Toll-like receptors (TLRs) and both for TLR2 and TLR4, an expression increase was found. These data demonstrate that liver cells are able to react specifically to a fungal pathogen without the help of Kupffer cells.


Assuntos
Aspergillus fumigatus/fisiologia , Complemento C3/metabolismo , Hepatócitos/imunologia , Hepatócitos/microbiologia , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Antígenos de Fungos/imunologia , Antígenos de Fungos/farmacologia , Aspergillus fumigatus/imunologia , Linhagem Celular , Complemento C3/genética , Expressão Gênica , Humanos , RNA/metabolismo , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Regulação para Cima , beta-Glucanas/farmacologia
11.
Anticancer Res ; 23(2B): 1229-34, 2003 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12820376

RESUMO

BACKGROUND: The intestinal mitosis-inhibiting peptide pyroglu-His-GlyOH (pEHG), inhibits normal intestinal epithelial cells and the human colon adenocarcinoma cell line HT-29 and increases the expression of c-fos (1). In this study, we investigated the mechanisms of the growth-inhibiting effects of pEHG. MATERIALS AND METHODS: cDNA expression array was hybridized with cDNA from HT-29 cells exposed to pEHG or control. The results were confirmed with Northern blot or real-time PCR. RESULTS: pEHG(1 nM) provoked a significant increase in the expression of the early growth response protein 1 (egr-1) after an incubation of 20 minutes, while c-fos was confirmed up-regulated by the same treatment. We further studied the expression of fosB, c-jun and junB, in the AP-1 complex. fosB was up-regulated 20-fold, but only minor effects on jun variants were observed. CONCLUSION: pEHG stimulates the gene expression of some immediate-early transcription factors involved in cell proliferation.


Assuntos
Carcinoma/patologia , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Precoces/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Proteínas Imediatamente Precoces/biossíntese , Proteínas de Neoplasias/biossíntese , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-fos , Proteínas de Bactérias/biossíntese , DNA Complementar/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Perfilação da Expressão Gênica , Genes fos/efeitos dos fármacos , Genes jun/efeitos dos fármacos , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mitose/efeitos dos fármacos , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-jun/biossíntese , Ácido Pirrolidonocarboxílico/análogos & derivados , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
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