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1.
Bioengineering (Basel) ; 11(2)2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38391673

RESUMO

Different studies suggest an impact of biofilms on carcinogenic lesion formation in varying human tissues. However, the mechanisms of cancer formation are difficult to examine in vivo as well as in vitro. Cell culture approaches, in most cases, are unable to keep a bacterial steady state without any overgrowth. In our approach, we aimed to develop an immunocompetent 3D tissue model which can mitigate bacterial outgrowth. We established a three-dimensional (3D) co-culture of human primary fibroblasts with pre-differentiated THP-1-derived macrophages on an SIS-muc scaffold which was derived by decellularisation of a porcine intestine. After establishment, we exposed the tissue models to define the biofilms of the Pseudomonas spec. and Staphylococcus spec. cultivated on implant mesh material. After 3 days of incubation, the cell culture medium in models with M0 and M2 pre-differentiated macrophages presented a noticeable turbidity, while models with M1 macrophages presented no noticeable bacterial growth. These results were validated by optical density measurements and a streak test. Immunohistology and immunofluorescent staining of the tissue presented a positive impact of the M1 macrophages on the structural integrity of the tissue model. Furthermore, multiplex ELISA highlighted the increased release of inflammatory cytokines for all the three model types, suggesting the immunocompetence of the developed model. Overall, in this proof-of-principle study, we were able to mitigate bacterial overgrowth and prepared a first step for the development of more complex 3D tissue models to understand the impact of biofilms on carcinogenic lesion formation.

2.
Histochem Cell Biol ; 159(5): 389-400, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36790468

RESUMO

An improved oxygen availability in air-liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-D-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Células Epiteliais , Animais , Suínos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células Epiteliais/metabolismo , Trifosfato de Adenosina , Lactatos/metabolismo , Glucose/metabolismo , RNA Mensageiro/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 43(2): 300-311, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36475703

RESUMO

BACKGROUND: DNA-PK (DNA-dependent protein kinase) is a stress-activated serine/threonine kinase that plays a central role in vascular smooth muscle cell proliferation and vascular proliferative disease processes such as neointimal formation. In this study, we link the activation of DNA-PK to the function of the transcription factor YB-1 (Y-box binding protein). METHODS: To identify YB-1 phosphorylation by DNA-PK, we generated different YB-1-expressing vectors. YB-1 nuclear translocation was investigated using immunoblotting and immunofluorescence staining. For YB-1 activity, luciferase assays were performed. RESULTS: We show by mutational analysis and kinase assay that the transcriptional regulator YB-1 is a substrate of DNA-PK. Blockade of DNA-PK by specific inhibitors revealed its critical involvement in YB-1phosphorylation as demonstrated by inhibition of an overexpressed YB-1 reporter construct. Using DNA-PK-deficient cells, we demonstrate that the shuttling of YB-1 from the cytoplasm to the nucleus is dependent on DNA-PK and that the N-terminal domain of YB-1 is phosphorylated at threonine 89. Point mutation of YB-1 at this residue abrogated the translocation of YB-1 into the nucleus. The phosphorylation of YB-1 by DNA-PK increased cellular DNA repair after exposure to ionizing radiation. Atherosclerotic tissue specimens were analyzed by immunohistochemistry. The DNA-PK subunits and YB-1 phosphorylated at T89 were found colocalized suggesting their in vivo interaction. In mice, the local application of the specific DNA-PK inhibitor NU7026 via thermosensitive Pluronic F-127 gel around dilated arteries significantly reduced the phosphorylation of YB-1. CONCLUSIONS: DNA-PK directly phosphorylates YB-1 and, this way, modulates YB-1 function. This interaction could be demonstrated in vivo, and colocalization in human atherosclerotic plaques suggests clinical relevance of our finding. Phosphorylation of YB-1 by DNA-PK may represent a novel mechanism governing atherosclerotic plaque progression.


Assuntos
Proteína Quinase Ativada por DNA , Proteínas Serina-Treonina Quinases , Animais , Humanos , Camundongos , DNA , Reparo do DNA , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo
4.
Int J Mol Sci ; 23(12)2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35743220

RESUMO

The NF-κB pathway is central pathway for inflammatory and immune responses, and IKKγ/NEMO is essential for NF-κB activation. In a previous report, we identified the role of glycogen synthase kinase-3ß (GSK-3ß) in NF-κB activation by regulating IKKγ/NEMO. Here, we show that NEMO phosphorylation by GSK-3ß leads to NEMO localization into multivesicular bodies (MVBs). Using the endosome marker Rab5, we observed localization into endosomes. Using siRNA, we identified the AAA-ATPase Vps4A, which is involved in recycling the ESCRT machinery by facilitating its dissociation from endosomal membranes, which is necessary for NEMO stability and NF-κB activation. Co-immunoprecipitation studies of NEMO and mutated NEMO demonstrated its direct interaction with Vps4A, which requires NEMO phosphorylation. The transfection of cells by a mutated and constitutively active form of Vps4A, Vps4A-E233Q, resulted in the formation of large vacuoles and strong augmentation in NEMO expression compared to GFP-Vps4-WT. In addition, the overexpression of the mutated form of Vps4A led to increased NF-κB activation. The treatment of cells with the pharmacologic V-ATPase inhibitor bafilomycin A led to a dramatic downregulation of NEMO and, in this way, inhibited NF-κB signal transduction. These results reveal an unexpected role for GSK-3ß and V-ATPase in NF-κB signaling activation.


Assuntos
Quinase I-kappa B , NF-kappa B , Adenosina Trifosfatases , Glicogênio Sintase Quinase 3 beta/genética , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Corpos Multivesiculares/metabolismo , NF-kappa B/metabolismo
5.
Bioconjug Chem ; 32(5): 897-903, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-33902282

RESUMO

The transfer of electrons across and along biological membranes drives the cellular energetics. In the context of artificial cells, it can be mimicked by minimal means, while using synthetic alternatives of the phospholipid bilayer and the electron-transducing proteins. Furthermore, the scaling up to biologically relevant and optically accessible dimensions may provide further insight and allow assessment of individual events but has been rarely attempted so far. Here, we visualized the mediated transmembrane oxidation of encapsulated NADH in giant unilamellar vesicles via confocal laser scanning and time-correlated single photon counting wide-field microscopy. To this end, we first augmented phospholipid membranes with an amphiphilic copolymer in order to check its influence on the oxidation kinetics spectrophotometrically. Then, we scaled up the compartments and followed the process microscopically.


Assuntos
Membrana Celular/metabolismo , NAD/metabolismo , Lipossomas Unilamelares/metabolismo , Oxirredução
6.
Exp Neurol ; 339: 113620, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33497646

RESUMO

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive, neurodegenerative disorder affecting upper and lower motor neurons. Approximately 10% of patients suffer from familial ALS (FALS) with mutations in different ubiquitously expressed genes including SOD1, C9ORF72, TARDBP, and FUS. There is compelling evidence for mitochondrial involvement in the pathogenic mechanisms of FALS and sporadic ALS (SALS), which is believed to be relevant for disease. Owing to the ubiquitous expression of relevant disease-associated genes, mitochondrial dysfunction is also detectable in peripheral patient tissue. We here report results of a detailed investigation of the functional impairment of mitochondrial oxidative phosphorylation (OXPHOS) in cultured skin fibroblasts from 23 SALS and 17 FALS patients, harboring pathogenic mutations in SOD1, C9ORF72, TARDBP and FUS. A considerable functional and structural mitochondrial impairment was detectable in fibroblasts from patients with SALS. Similarly, fibroblasts from patients with FALS, harboring pathogenic mutations in TARDBP, FUS and SOD1, showed mitochondrial defects, while fibroblasts from C9ORF72 associated FALS showed a very mild impairment detectable in mitochondrial ATP production rates only. While we could not detect alterations in the mtDNA copy number in the SALS or FALS fibroblast cultures, the impairment of OXPHOS in SALS fibroblasts and SOD1 or TARDBP FALS could be rescued by in vitro treatments with CoQ10 (5 µM for 3 weeks) or Trolox (300 µM for 5 days). This underlines the role of elevated oxidative stress as a potential cause for the observed functional effects on mitochondria, which might be relevant disease modifying factors.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Fibroblastos/metabolismo , Sequestradores de Radicais Livres/farmacologia , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Adulto , Idoso , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/patologia , Células Cultivadas , Feminino , Sequestradores de Radicais Livres/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Pele/efeitos dos fármacos , Pele/metabolismo , Adulto Jovem
7.
Sci Rep ; 10(1): 19714, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184358

RESUMO

The transition between synchronized and asynchronous behaviour of immobilized yeast cells of the strain Saccharomyces carlsbergensis was investigated by monitoring the autofluorescence of the coenzyme NADH. In populations of intermediate cell densities the individual cells remained oscillatory, whereas on the level of the cell population both a partially synchronized and an asynchronous state were accessible for experimental studies. In the partially synchronized state, the mean oscillatory frequency was larger than that of the cells in the asynchronous state. This suggests that synchronisation occurred due to entrainment by the cells that oscillated more rapidly. This is typical for synchronisation due to phase advancement. Furthermore, the synchronisation of the frequency of the glycolytic oscillations preceded the synchronisation of their phases. However, the cells did not synchronize completely, as the distribution of the oscillatory frequencies only narrowed but did not collapse to a unique frequency. Cells belonging to spatially denser clusters showed a slightly enhanced local synchronisation during the episode of partial synchronisation. Neither the clusters nor a transition from partially synchronized glycolytic oscillations to travelling glycolytic waves did substantially affect the degree of partial synchronisation. Chimera states, i.e., the coexistence of a synchronized and an asynchronous part of the population, could not be found.


Assuntos
Glucose/metabolismo , Glicólise , NAD/metabolismo , Saccharomyces cerevisiae/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais
8.
Cell Mol Life Sci ; 77(20): 4133-4142, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31932854

RESUMO

The transcription factors of the nuclear factor κB (NF-κB) family play a pivotal role in the cellular response to DNA damage. Genotoxic stress-induced activation of NF-κB differs from the classical canonical pathway by shuttling of the NF-κB Essential Modifier (IKKγ/NEMO) subunit through the nucleus. Here, we show that DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA double-strand break (DSB) repair, triggers the phosphorylation of NEMO by genotoxic stress, thereby enabling shuttling of NEMO through the nucleus with subsequent NF-κB activation. We identified serine 43 of NEMO as a DNA-PK phosphorylation site and point mutation of this serine to alanine led to a complete block of NF-κB activation by ionizing radiation (IR). Blockade of DNA-PK by a specific shRNA or by DNA-PKcs-deficient cells abrogated NEMO entry into the nucleus, as well. Accordingly, SUMOylation of NEMO, a prerequisite of nuclear NEMO, was abolished. Based on these observations, we propose a model in which NEMO phosphorylation by DNA-PK provides the first step in the nucleocytoplasmic trafficking of NEMO.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Alanina/metabolismo , Animais , Dano ao DNA/fisiologia , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Fosforilação/fisiologia , Serina/metabolismo , Transdução de Sinais/fisiologia
9.
Am J Transl Res ; 11(7): 4063-4076, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396318

RESUMO

Background: Monocytes (Mo) are the most important mediators in arteriogenesis. Previous results from our group demonstrated the great potential of allogenic Mo transplantation for improving collateral vessel growth, which appeared to be due to a considerable host vs. graft reaction. To prove this hypothesis and introduce this new method in clinical practice, we performed transplantation of human Mo (HuMo) in a mouse model. Methods and results: We ligated the femoral artery of BALB/c mice and transplanted Mo via the tail vein. Perfusion was measured by laser Doppler perfusion imaging (LDPI). We also performed clinical scoring based on behavior, wound healing, signs of inflammation and mobility of the ligated extremity. Finally, arteriogenesis and angiogenesis were examined histologically and by quantitative RT-PCR of the hind limb musculature. LDPI increased within one week after ligation when HuMo were transplanted and increased further up to day 21 (0.63±0.12 (n=12) in HuMo vs. 0.50±0.12 (n=17) in the control group (P<0.01)). A histological evaluation showed significantly more collateral arteries within the adductor muscles after HuMo transplantation. The promotion of collateral vessel growth after HuMo transplantation resulted in better clinical scores (0.33±0.26 (n=12) vs. 3.3 (n=9), SEM; P<0.01). Conclusions: Transplantation of HuMo improves collateral vessel growth and clinical outcomes in mice. These results verify our hypothesis that controlled triggering of the inflammatory mechanism resulted in collateral vessel growth by a local host vs. a graft reaction in the ischemic hind limbs and could represent a further step in the development of a clinical strategy for promoting arteriogenesis.

10.
Mol Neurobiol ; 56(10): 6833-6855, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30929164

RESUMO

Brain-derived neurotrophic factor (BDNF) is a secreted messenger molecule that is crucial for neuronal function and induction of synaptic plasticity. Although altered availability of BDNF underlies many neurological deficits and neurodegenerative disorders, secretion dynamics of endogenous BDNF are unexplored. We generated a BDNF-GFP knock-in (KiBE) mouse, in which GFP-labeled BDNF is expressed under the control of the unaltered endogenous mouse BDNF gene regulatory elements. This KiBE mouse model enables for the first time live cell imaging analysis of endogenous BDNF dynamics. We show that BDNF-GFP release and biological activity in vivo are unaffected by the GFP tag, since homozygous KiBE mice, which lack wild-type BDNF, are healthy and have a normal life expectancy. STED superresolution microscopy shows that 70% of BDNF-GFP vesicles in KiBE mouse neurites are localized in dendrites, being typically 200 nm away from synaptic release sites. Live cell imaging in hippocampal slices also reveals prominent targeting of endogenous BDNF-GFP vesicles to dendrites. Fusion pore opening and cargo release of dendritic BDNF vesicles start within 30 s after a strong depolarizing stimulus and continue for > 100 s thereafter, revealing an astonishingly delayed and prolonged release of endogenous BDNF.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dendritos/metabolismo , Exocitose , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Cromossomos de Mamíferos/genética , Marcação de Genes , Genoma , Hipocampo/metabolismo , Camundongos
11.
Blood Adv ; 3(6): 839-850, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30867143

RESUMO

Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)-dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Compartimento Celular , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Monócitos/citologia , Apresentação de Antígeno/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Humanos , Lisossomos/metabolismo , Monócitos/imunologia , Linfócitos T/imunologia
12.
Eur J Immunol ; 48(8): 1336-1349, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29749611

RESUMO

Mucosal-associated invariant T cells (MAIT) constitute the most abundant anti-bacterial CD8+ T-cell population in humans. MR1/TCR-activated MAIT cells were reported to organize cytotoxic and innate-like responses but knowledge about their molecular effector phenotype is still fragmentary. Here, we have examined the functional inventory of human MAIT cells (CD3+ Vα7.2+ CD161+ ) in comparison with those from conventional non-MAIT CD8+ T cells (cCD8+ ) and NK cells. Quantitative mass spectrometry characterized 5500 proteins of primary MAIT cells and identified 160 and 135 proteins that discriminate them from cCD8+ T cells and NK cells donor-independently. Most notably, MAIT cells showed a unique exocytosis machinery in parallel to a proinflammatory granzyme profile with high levels of the granzymes A, K, and M. Furthermore, 24 proteins were identified with highest abundances in MAIT cells, including CD26, CD98, and L-amino-oxidase (LAAO). Among those, expression of granzyme K and CD98 were validated as MAIT-specific with respect to non-MAIT CD8+ effector subsets and LAAO was found to be recruited together with granzymes, perforin, and CD107a at the immunological synapse of activated MAIT cells. In conclusion, this study complements knowledge on the molecular effector phenotype of MAIT cells and suggest novel immune regulatory functions as part of their cytotoxic responses.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Exocitose/fisiologia , Células Matadoras Naturais/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Proteoma/análise , Biomarcadores/análise , Células Cultivadas , Dipeptidil Peptidase 4/metabolismo , Proteína-1 Reguladora de Fusão/metabolismo , Granzimas/metabolismo , Humanos , L-Aminoácido Oxidase/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Espectrometria de Massas , Proteômica
13.
Biophys J ; 113(10): 2249-2260, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-28988699

RESUMO

The Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes.


Assuntos
Anquirinas/química , Anquirinas/metabolismo , Caveolina 1/química , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Mutação , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/genética , Difusão , Células HEK293 , Humanos , Modelos Moleculares , Oócitos/metabolismo , Ligação Proteica/genética , Domínios Proteicos , Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Xenopus laevis/metabolismo
14.
J Vis Exp ; (126)2017 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-28872119

RESUMO

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options. Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery. After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.


Assuntos
Microscopia Intravital/métodos , Monócitos/patologia , Doença Arterial Periférica/sangue , Doença Arterial Periférica/diagnóstico por imagem , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Neovascularização Fisiológica
15.
Sci Rep ; 7(1): 8358, 2017 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-28827723

RESUMO

The outcome of T cell activation is determined by mechanisms that balance Ca2+ influx and clearance. Here we report that murine CD4 T cells lacking Neuroplastin (Nptn -/-), an immunoglobulin superfamily protein, display elevated cytosolic Ca2+ and impaired post-stimulation Ca2+ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca2+ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn -/- T cells, suggesting an explanation for altered Ca2+ handling. Supporting this, Ca2+ extrusion was impaired while Ca2+ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn -/- T cells. Consistent with sustained Ca2+ levels, differentiation of Nptn -/- T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/fisiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/fisiologia , Linfócitos T/fisiologia , Animais , Sinalização do Cálcio , Diferenciação Celular , Núcleo Celular , Regulação da Expressão Gênica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia
16.
Cell Death Discov ; 3: 17001, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28250970

RESUMO

The intestinal porcine epithelial cell line IPEC-J2, cultured under the air-liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium-cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response.

17.
Sci Signal ; 10(462)2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-28096507

RESUMO

The enzymatic activity of the Src family tyrosine kinase p56Lck (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr394 and Tyr505 Phosphorylation of Tyr394 and the conformational opening of Lck are believed to activate the kinase, whereas Tyr505 phosphorylation is thought to generate a closed, inactive conformation of Lck. We investigated whether the conformation of Lck and its phosphorylation state act in concert to regulate the initiation of T cell receptor (TCR) signaling. With a sensitive biosensor, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the conformations of wild-type Lck and its phosphorylation-deficient mutants Y394F and Y505F and the double mutant Y394F/Y505F in unstimulated T cells and after TCR stimulation. With this approach, we separated the conformational changes of Lck from the phosphorylation state of its regulatory tyrosines. We showed that the conformational opening of Lck alone was insufficient to initiate signaling events in T cells. Rather, Lck additionally required phosphorylation of Tyr394 to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr394 occurred preferentially at the plasma membrane of Jurkat cells and primary human T cells. Our study supports the hypothesis that T cell activation through the TCR complex is accompanied by the de novo activation of Lck and that phosphorylation of Tyr394 plays a role in Lck function that goes beyond inducing an open conformation of the kinase.


Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Células Jurkat , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Microscopia Confocal , Microscopia de Fluorescência , Mutação , Fosforilação , Conformação Proteica , Linfócitos T/metabolismo , Tirosina/química , Tirosina/genética , Tirosina/metabolismo
18.
Sci Rep ; 6: 38553, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27929056

RESUMO

The NF-κB signaling pathway is central for the innate immune response and its deregulation is found in multiple disorders such as autoimmune, chronic inflammatory and metabolic diseases. IKKγ/NEMO is essential for NF-κB activation and NEMO dysfunction in humans has been linked to so-called progeria syndromes, which are characterized by advanced ageing due to age-dependent inflammatory diseases. It has been suggested that glycogen synthase kinase-3ß (GSK-3ß) participates in NF-κB regulation but the exact mechanism remained incompletely understood. In this study, we identified NEMO as a GSK-3ß substrate that is phosphorylated at serine 8, 17, 31 and 43 located within its N-terminal domain. The kinase forms a complex with wild-type NEMO while point mutations of NEMO at the specific serines abrogated GSK-3ß binding and subsequent phosphorylation of NEMO resulting in its destabilization. However, K63-linked polyubiquitination was augmented in mutated NEMO explaining an increased binding to IKKα and IKKß. Even IκBα was found degraded. Still, TNFα-stimulated NF-κB activation was impaired pointing towards an un-controlled signalling process. Our data suggest that GSK-3ß is critically important for ordered NF-κB signalling through modulation of NEMO phosphorylation.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , NF-kappa B/metabolismo , Animais , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Células MCF-7 , Camundongos , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Fosforilação , Ligação Proteica , Estabilidade Proteica
19.
Microsc Res Tech ; 79(6): 463-79, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27040755

RESUMO

Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM-CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM-CF operations elaborated by the workgroups of the German network of ALM-CFs, German Bio-Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM-CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463-479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC.


Assuntos
Instalações de Saúde , Laboratórios , Microscopia , Pesquisa Biomédica , Alemanha , Humanos
20.
PLoS One ; 10(7): e0132323, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26147118

RESUMO

The pig shows genetical and physiological resemblance to human, which predestines it as an experimental animal model especially for mucosal physiology. Therefore, the intestinal epithelial cell lines 1 and J2 (IPEC-1, IPEC-J2)--spontaneously immortalised cell lines from the porcine intestine--are important tools for studying intestinal function. A microarray (GeneChip Porcine Genome Array) was performed to compare the genome wide gene expression of IPECs. Different significantly up-regulated pathways were identified, like "lysosome", "pathways in cancer", "regulation of actin cytoskeleton" and "oxidative phosphorylation" in IPEC-J2 in comparison to IPEC-1. On the other hand, "spliceosome", "ribosome", "RNA-degradation" and "tight junction" are significantly down-regulated pathways in IPEC-J2 in comparison to IPEC-1. Examined pathways were followed up by functional analyses. ATP-, oxygen, glucose and lactate-measurement provide evidence for up-regulation of oxidative phosphorylation in IPEC-J2. These cells seem to be more active in their metabolism than IPEC-1 cells due to a significant higher ATP-content as well as a higher O2- and glucose-consumption. The down-regulated pathway "ribosome" was followed up by measurement of RNA- and protein content. In summary, IPEC-J2 is a morphologically and functionally more differentiated cell line in comparison to IPEC-1. In addition, IPEC-J2 cells are a preferential tool for in vitro studies with the focus on metabolism.


Assuntos
Células Epiteliais/citologia , Mucosa Intestinal/citologia , Intestinos/citologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Forma Celular/fisiologia , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Fosforilação , Suínos , Regulação para Cima
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