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Pesquisa : A10.082.500 [Categoria DeCS]
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  1 / 23518 MEDLINE  
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[PMID]:28463416
[Au] Autor:Kaja S; Payne AJ; Naumchuk Y; Koulen P
[Ad] Endereço:Departments of Ophthalmology and Molecular Pharmacology & Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois.
[Ti] Título:Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes.
[So] Source:Curr Protoc Toxicol;72:2.26.1-2.26.10, 2017 May 02.
[Is] ISSN:1934-9262
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Drug discovery heavily relies on cell viability studies to assess the potential toxicity of drug candidates. L-Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that catalyzes the concomitant interconversions of pyruvate to L-lactate and NADH to NAD during glycolysis, and the reverse reactions during the Cori cycle. In response to cellular damage, induced by endogenous cellular mechanisms or as a result of exogenously applied insults, LDH is released from the cytoplasm into the extracellular environment. Its stability in cell culture medium makes it a well-suited correlate for the presence of damage and toxicity in tissues and cells. We herein present protocols for a reproducible and validated LDH assay optimized for several cell types. In contrast to commercially available LDH assays, often associated with proprietary formulations and high cost, our protocols provide ample opportunities for experiment-specific optimization with low variability and cost. © 2017 by John Wiley & Sons, Inc.
[Mh] Termos MeSH primário: Astrócitos/enzimologia
Sobrevivência Celular
L-Lactato Desidrogenase/análise
Toxicologia/métodos
[Mh] Termos MeSH secundário: Animais
Técnicas de Cultura de Células
Linhagem Celular
Linhagem Celular Tumoral
Meios de Cultura/química
Citoplasma/enzimologia
Espaço Extracelular/enzimologia
Glicólise
Seres Humanos
Neurônios/efeitos dos fármacos
Fármacos Neuroprotetores/farmacologia
Nervo Óptico/citologia
Nervo Óptico/efeitos dos fármacos
Cultura Primária de Células
Ratos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Culture Media); 0 (Neuroprotective Agents); EC 1.1.1.27 (L-Lactate Dehydrogenase)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180308
[Lr] Data última revisão:
180308
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170503
[St] Status:MEDLINE
[do] DOI:10.1002/cptx.21


  2 / 23518 MEDLINE  
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[PMID]:29337067
[Au] Autor:Yassa ME; Mansour IA; Sewelam NI; Hamza H; Gaafar T
[Ad] Endereço:Department of Clinical and Chemical Pathology, Kasr Al-Ainy School of Medicine, Cairo University, Kasr Al-Ainy St., 11562, Cairo, Egypt. Electronic address: marianne.yassa@kasralainy.edu.eg.
[Ti] Título:The impact of growth factors on human induced pluripotent stem cells differentiation into cardiomyocytes.
[So] Source:Life Sci;196:38-47, 2018 Mar 01.
[Is] ISSN:1879-0631
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:Human induced pluripotent stem cells (hiPSCs) act as a promising therapeutic alternative for cardiovascular diseases. They yield a large number of functional cardiomyocytes (CMs) from autologous cell sources without ethical or immunological problems. However, significant limitations still remain in terms of line-to-line variability in CM yield and reproducibility. AIM: To efficiently enhance NP0040 hiPSCs differentiation into CMs. MAIN METHODS: Following a standard cardiac differentiation protocol using small molecules targeting the canonical Wnt signaling, growth factors (BMP4 and FGF2) and ascorbic acid were added further in order to increase the cardiac differentiation efficiency. All cultures were conducted in serum-free, feeder-free monolayer system followed by lactate purification. KEY FINDINGS: Using NP0040 hiPSCs, the CM yield resulting from modulation of the Wnt signaling pathway alone was inefficient compared to previous studies while the addition of BMP4, FGF2 and ascorbic acid resulted in enhanced cardiac differentiation outcome. The later resulted in a high yield (up to 92%) of cardiac troponin-T (cTnT) + CMs contracting spontaneously as organized sheets in 15 independent experiments. They were validated structurally and functionally using immunofluorescent staining for sarcomeric α-actinin, cTnT, MLC2v and Connexin 43. Reverse-transcriptase PCR revealed cardiac transcription factors and cardiac-specific genes expression. CMs were electrically connected to one another. Recorded action potential (AP) showed waves of relatively mature ventricular-like phenotype. SIGNIFICANCE: We demonstrated that hiPSC lines respond differently to a standard cardiac differentiation protocol and that a well-orchestrated interplay between Wnt, BMP4, FGF/MEK and Ascorbic acid MEK/ERK1/2 signaling pathways is beneficial in enhancing the differentiation outcome.
[Mh] Termos MeSH primário: Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia
Miócitos Cardíacos/efeitos dos fármacos
[Mh] Termos MeSH secundário: Potenciais de Ação/efeitos dos fármacos
Ácido Ascórbico/farmacologia
Proteína Morfogenética Óssea 4/metabolismo
Diferenciação Celular/efeitos dos fármacos
Espaço Extracelular/efeitos dos fármacos
Expressão Gênica/efeitos dos fármacos
Glucose/deficiência
Seres Humanos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos
Contração Miocárdica
Miócitos Cardíacos/metabolismo
Troponina T/metabolismo
Vitaminas/farmacologia
Via de Sinalização Wnt/efeitos dos fármacos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bone Morphogenetic Protein 4); 0 (Intercellular Signaling Peptides and Proteins); 0 (Troponin T); 0 (Vitamins); IY9XDZ35W2 (Glucose); PQ6CK8PD0R (Ascorbic Acid)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180117
[St] Status:MEDLINE


  3 / 23518 MEDLINE  
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[PMID]:28458358
[Au] Autor:Sasaki T; Nishimura Y; Ikegaya Y
[Ad] Endereço:Graduate School of Pharmaceutical Sciences, The University of Tokyo.
[Ti] Título:Simultaneous Recordings of Central and Peripheral Bioelectrical Signals in a Freely Moving Rodent.
[So] Source:Biol Pharm Bull;40(5):711-715, 2017.
[Is] ISSN:1347-5215
[Cp] País de publicação:Japan
[La] Idioma:eng
[Ab] Resumo:Understanding physiological interactions between the central and peripheral nervous systems requires an experimental strategy to simultaneously monitor activity patterns of the brain and peripheral organs. In this study, we developed a novel method to record extracellular field potential signals from a wide range of brain regions together with electrocardiograms, electromyograms, and breathing signals from a freely moving rodent. This method collects all recorded signals into a single device mounted on an animal's head, allowing the reduction of experimental costs and the simplification of data processing. The methodological concept is applicable to a number of biological research issues of how the brain-body association is altered in response to various environmental changes, emotional challenges, and acute and chronic dysfunction of internal organs.
[Mh] Termos MeSH primário: Sistema Nervoso Central/fisiologia
Eletrofisiologia/métodos
Sistema Nervoso Periférico/fisiologia
[Mh] Termos MeSH secundário: Animais
Comportamento Animal/fisiologia
Eletrocardiografia
Eletrodos Implantados
Eletromiografia
Fenômenos Eletrofisiológicos
Eletrofisiologia/instrumentação
Emoções
Espaço Extracelular/fisiologia
Masculino
Ratos
Ratos Wistar
Respiração
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180212
[Lr] Data última revisão:
180212
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170502
[St] Status:MEDLINE
[do] DOI:10.1248/bpb.b17-00070


  4 / 23518 MEDLINE  
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[PMID]:29283227
[Au] Autor:Kolesnikov SS; Bystrova MF
[Ti] Título:Cyclic AMP: Second Messenger as the First Messenger.
[So] Source:Usp Fiziol Nauk;47(3):3-16, 2016 Jul-Sep.
[Is] ISSN:0301-1798
[Cp] País de publicação:Russia (Federation)
[La] Idioma:rus
[Ab] Resumo:Cell-to-cell communications and autocrine/paracrine regulations are mediated by an extracellular signaling network involving secretion of a variety of different factors, hormones, neurotransmitters, and other signaling molecules that are recognized in an extracellular medium by multiple molecular receptors operating in the plasma membrane of cells. Most of plasma membrane receptors belong to the superfamily of heptahelical receptors, many of which are coupled by G-proteins to adenylate cyclase responsible for cAMP production in the cell cytoplasm. The canonical role of cAMP in cell physiology is to serve as a second messenger and universal regulator of intracellular processes. Meanwhile, increasing body of evidence leaves little doubts that stimulated cells can release cAMP into intercellular space, where it may serve as signaling molecule in cell-to-cell communications and autocrine regulations. This review considers the basic concept on mechanisms of intracellular and extracellular signaling with cAMP as the second and first messenger.
[Mh] Termos MeSH primário: Adenilil Ciclases/metabolismo
AMP Cíclico/metabolismo
Células Eucarióticas/metabolismo
Receptores de Superfície Celular/metabolismo
Sistemas do Segundo Mensageiro/genética
[Mh] Termos MeSH secundário: Adenilil Ciclases/genética
Animais
Comunicação Celular
Membrana Celular/metabolismo
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo
Células Eucarióticas/citologia
Espaço Extracelular/metabolismo
Regulação da Expressão Gênica
Hormônios/genética
Hormônios/metabolismo
Seres Humanos
Neurotransmissores/genética
Neurotransmissores/metabolismo
Receptores de Superfície Celular/genética
Transdução de Sinais
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (Hormones); 0 (Neurotransmitter Agents); 0 (Receptors, Cell Surface); E0399OZS9N (Cyclic AMP); EC 3.1.4.35 (Cyclic Nucleotide Phosphodiesterases, Type 6); EC 4.6.1.1 (Adenylyl Cyclases)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180116
[Lr] Data última revisão:
180116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171229
[St] Status:MEDLINE


  5 / 23518 MEDLINE  
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[PMID]:29261777
[Au] Autor:Noll F; Behnke J; Leiting S; Troidl K; Alves GT; Müller-Redetzky H; Preissner KT; Fischer S
[Ad] Endereço:Institute of Biochemistry, Medical School, Justus-Liebig-University, Giessen, Germany.
[Ti] Título:Self-extracellular RNA acts in synergy with exogenous danger signals to promote inflammation.
[So] Source:PLoS One;12(12):e0190002, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Self-extracellular RNA (eRNA), released from stressed or injured cells upon various pathological situations such as ischemia-reperfusion-injury, has been shown to act as an alarmin by inducing procoagulatory and proinflammatory responses. In particular, M1-polarization of macrophages by eRNA resulted in the expression and release of a variety of cytokines, including tumor necrosis factor (TNF)-α or interleukin-6 (IL-6). The present study now investigates in which way self-eRNA may influence the response of macrophages towards various Toll-like receptor (TLR)-agonists. Isolated agonists of TLR2 (Pam2CSK4), TLR3 (PolyIC), TLR4 (LPS), or TLR7 (R848) induced the release of TNF-α in a concentration-dependent manner in murine macrophages, differentiated from bone marrow-derived stem cells by mouse colony stimulating factor. Here, the presence of eRNA shifted the dose-response curve for Pam2CSK4 (Pam) considerably to the left, indicating that eRNA synergistically enhanced the cytokine liberation from macrophages even at very low Pam-levels. The synergistic activation of TLR2 by eRNA/Pam was duplicated by other TLR2-agonists such as FSL-1 or Pam3CSK4. In contrast, for TLR4-agonists such as LPS a synergistic effect of eRNA was much weaker, and was not existent for TLR3-, or TLR7-agonists. The synergistic eRNA/Pam action was dependent on the NFκB-signaling pathway as well as on p38MAP- and MEK1/ERK-kinases and was prevented by predigestion of eRNA with RNase1 or by antibodies against TLR2. Thus, the presence of self-eRNA as alarming molecule sensitizes innate immune responses towards pathogen-associated molecular patterns (PAMPs) in a synergistic way and may thereby contribute to the differentiated outcome of inflammatory responses.
[Mh] Termos MeSH primário: Espaço Extracelular/metabolismo
Inflamação/metabolismo
RNA/metabolismo
Transdução de Sinais
[Mh] Termos MeSH secundário: Animais
Citocinas/metabolismo
Diglicerídeos/farmacologia
Lipopeptídeos/farmacologia
Macrófagos/efeitos dos fármacos
Macrófagos/metabolismo
Camundongos Endogâmicos C57BL
Oligopeptídeos/farmacologia
Padrões Moleculares Associados a Patógenos/metabolismo
Transdução de Sinais/efeitos dos fármacos
Fatores de Tempo
Receptores Toll-Like/antagonistas & inibidores
Receptores Toll-Like/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Cytokines); 0 (Diglycerides); 0 (FSL-1 lipoprotein, synthetic); 0 (Lipopeptides); 0 (Oligopeptides); 0 (Pam2CSK4 lipopeptide); 0 (Pathogen-Associated Molecular Pattern Molecules); 0 (Toll-Like Receptors); 63231-63-0 (RNA)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180108
[Lr] Data última revisão:
180108
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171221
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0190002


  6 / 23518 MEDLINE  
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[PMID]:29211992
[Au] Autor:Scheppach C; Robinson HPC
[Ad] Endereço:Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom; Institute of Physics, University of Freiburg, Freiburg im Breisgau, Germany. Electronic address: christian.scheppach@physik.uni-freiburg.de.
[Ti] Título:Fluctuation Analysis in Nonstationary Conditions: Single Ca Channel Current in Pyramidal Neurons.
[So] Source:Biophys J;113(11):2383-2395, 2017 Dec 05.
[Is] ISSN:1542-0086
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Fluctuation analysis is a method that allows measurement of the single-channel current of ion channels even when it is too small to be resolved directly with the patch-clamp technique. This is the case for voltage-gated calcium channels. They are present in all mammalian central neurons, controlling presynaptic release of transmitter, postsynaptic signaling, and synaptic integration. The amplitudes of their single-channel currents in a physiological concentration of extracellular calcium, however, are small and not well determined. But measurement of this quantity is essential for estimating numbers of functional voltage-gated calcium channels in the membrane and the size of channel-associated calcium signaling domains, and for understanding the stochastic nature of calcium signaling. Here, we recorded the voltage-gated calcium channel current in nucleated patches from layer 5 pyramidal neurons in rat neocortex, in physiological external calcium (1-2 mM). The ensemble-averaging of current responses required for conventional fluctuation analysis proved impractical because of the rapid rundown of calcium channel currents. We therefore developed a more robust method, using mean current fitting of individual current responses and band-pass filtering. Furthermore, voltage-ramp stimulation proved useful. We validated the accuracy of the method by analyzing simulated data. At an external calcium concentration of 1 mM, and a membrane potential of -20 mV, we found that the average single-channel current amplitude was ∼0.04 pA, increasing to 0.065 pA at 2 mM external calcium, and 0.12 pA at 5 mM. The relaxation time constant of the fluctuations was in the range 0.2-0.8 ms. The results are relevant to understanding the stochastic properties of dendritic Ca spikes in neocortical layer 5 pyramidal neurons. With the reported method, single-channel current amplitude of native voltage-gated calcium channels can be resolved accurately despite conditions of unstable rundown.
[Mh] Termos MeSH primário: Canais de Cálcio/metabolismo
Células Piramidais/metabolismo
[Mh] Termos MeSH secundário: Animais
Cálcio/farmacologia
Relação Dose-Resposta a Droga
Espaço Extracelular/efeitos dos fármacos
Espaço Extracelular/metabolismo
Modelos Neurológicos
Células Piramidais/citologia
Células Piramidais/efeitos dos fármacos
Ratos
Ratos Wistar
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Calcium Channels); SY7Q814VUP (Calcium)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180103
[Lr] Data última revisão:
180103
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171207
[St] Status:MEDLINE


  7 / 23518 MEDLINE  
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[PMID]:29216450
[Au] Autor:Mateo Y; Johnson KA; Covey DP; Atwood BK; Wang HL; Zhang S; Gildish I; Cachope R; Bellocchio L; Guzmán M; Morales M; Cheer JF; Lovinger DM
[Ad] Endereço:Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, US National Institutes of Health, Rockville, MD, USA.
[Ti] Título:Endocannabinoid Actions on Cortical Terminals Orchestrate Local Modulation of Dopamine Release in the Nucleus Accumbens.
[So] Source:Neuron;96(5):1112-1126.e5, 2017 Dec 06.
[Is] ISSN:1097-4199
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Dopamine (DA) transmission mediates numerous aspects of behavior. Although DA release is strongly linked to firing of DA neurons, recent developments indicate the importance of presynaptic modulation at striatal dopaminergic terminals. The endocannabinoid (eCB) system regulates DA release and is a canonical gatekeeper of goal-directed behavior. Here we report that extracellular DA increases induced by selective optogenetic activation of cholinergic neurons in the nucleus accumbens (NAc) are inhibited by CB1 agonists and eCBs. This modulation requires CB1 receptors on cortical glutamatergic afferents. Dopamine increases driven by optogenetic activation of prefrontal cortex (PFC) terminals in the NAc are similarly modulated by activation of these CB1 receptors. We further demonstrate that this same population of CB1 receptors modulates optical self-stimulation sustained by activation of PFC afferents in the NAc. These results establish local eCB actions on PFC terminals within the NAc that inhibit mesolimbic DA release and constrain reward-driven behavior.
[Mh] Termos MeSH primário: Dopamina/metabolismo
Endocanabinoides/farmacologia
Núcleo Accumbens/efeitos dos fármacos
Núcleo Accumbens/metabolismo
Córtex Pré-Frontal/efeitos dos fármacos
Terminações Pré-Sinápticas/efeitos dos fármacos
Terminações Pré-Sinápticas/metabolismo
[Mh] Termos MeSH secundário: Animais
Fenômenos Eletrofisiológicos/efeitos dos fármacos
Espaço Extracelular/efeitos dos fármacos
Espaço Extracelular/metabolismo
Glutamatos/fisiologia
Masculino
Camundongos
Camundongos Endogâmicos C57BL
Núcleo Accumbens/citologia
Córtex Pré-Frontal/citologia
Receptor CB1 de Canabinoide/agonistas
Recompensa
Autoestimulação
Transmissão Sináptica/efeitos dos fármacos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Endocannabinoids); 0 (Glutamates); 0 (Receptor, Cannabinoid, CB1); VTD58H1Z2X (Dopamine)
[Em] Mês de entrada:1712
[Cu] Atualização por classe:171220
[Lr] Data última revisão:
171220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171208
[St] Status:MEDLINE


  8 / 23518 MEDLINE  
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Texto completo SciELO Brasil
[PMID]:28746471
[Au] Autor:Wanandi SI; Yustisia I; Neolaka GMG; Jusman SWA
[Ad] Endereço:Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
[Ti] Título:Impact of extracellular alkalinization on the survival of human CD24-/CD44+ breast cancer stem cells associated with cellular metabolic shifts.
[So] Source:Braz J Med Biol Res;50(8):e6538, 2017 Jul 20.
[Is] ISSN:1414-431X
[Cp] País de publicação:Brazil
[La] Idioma:eng
[Ab] Resumo:Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.
[Mh] Termos MeSH primário: Neoplasias da Mama/metabolismo
Antígeno CD24/metabolismo
Receptores de Hialuronatos/metabolismo
Células-Tronco Neoplásicas/metabolismo
[Mh] Termos MeSH secundário: Apoptose
Proliferação Celular
Sobrevivência Celular
Espaço Extracelular
Feminino
Regulação Neoplásica da Expressão Gênica
Seres Humanos
Reação em Cadeia da Polimerase Via Transcriptase Reversa
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (CD24 Antigen); 0 (CD24 protein, human); 0 (CD44 protein, human); 0 (Hyaluronan Receptors)
[Em] Mês de entrada:1712
[Cu] Atualização por classe:171204
[Lr] Data última revisão:
171204
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170727
[St] Status:MEDLINE


  9 / 23518 MEDLINE  
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[PMID]:29045498
[Au] Autor:Elamin AA; Steinicke S; Oehlmann W; Braun Y; Wanas H; Shuralev EA; Huck C; Maringer M; Rohde M; Singh M
[Ad] Endereço:LIONEX Diagnostics and Therapeutics GmbH, Braunschweig, Germany.
[Ti] Título:Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.
[So] Source:PLoS One;12(10):e0186801, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection.
[Mh] Termos MeSH primário: Antibacterianos/farmacologia
Vias Biossintéticas/efeitos dos fármacos
Parede Celular/metabolismo
Técnicas de Silenciamento de Genes
Pseudomonas aeruginosa/citologia
Pseudomonas aeruginosa/genética
[Mh] Termos MeSH secundário: Animais
Parede Celular/efeitos dos fármacos
Parede Celular/genética
Contagem de Colônia Microbiana
DNA Bacteriano/genética
Espaço Extracelular/química
Feminino
Genes Bacterianos
Vetores Genéticos/metabolismo
Alface/microbiologia
Lipopolissacarídeos/biossíntese
Pulmão/microbiologia
Pulmão/patologia
Macrófagos/microbiologia
Camundongos
Modelos Biológicos
Mutação/genética
Peptidoglicano/biossíntese
Doenças das Plantas/microbiologia
Infecções por Pseudomonas/microbiologia
Pseudomonas aeruginosa/crescimento & desenvolvimento
Pseudomonas aeruginosa/patogenicidade
Doenças Respiratórias/microbiologia
Doenças Respiratórias/patologia
Virulência/efeitos dos fármacos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Anti-Bacterial Agents); 0 (DNA, Bacterial); 0 (Lipopolysaccharides); 0 (Peptidoglycan)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171031
[Lr] Data última revisão:
171031
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171019
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0186801


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[PMID]:29045415
[Au] Autor:Teng T; Liang L; Chen K; Xi B; Xie J; Xu P
[Ad] Endereço:Wuxi Fisheries College, Nanjing Agricultural University, Wuxi, Jiangsu, China.
[Ti] Título:Isolation, identification and phenotypic and molecular characterization of pathogenic Vibrio vulnificus isolated from Litopenaeus vannamei.
[So] Source:PLoS One;12(10):e0186135, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The morphology and the drug sensitivity of the strain GYX2014-1 isolated from the hepatic pancreatic tissue of moribund Litopenaeus vannamei were evaluated by conventional culture characteristics, physical and chemical characteristics, and molecular biology methods. Detection of extracellulase and hemolysin activity shows that the isolated GYX2014-1 has protease, lipase, gelatinase activity, but none of amylase, or lecithinase activity. The 16S rRNA gene (GenBank accession number: KT781675) was analyzed, and a phylogenetic tree analysis showed that the isolated pathogen was most closely related to V. vulnificus (GenBank accession number: NR 118570)-a match of more than 99%. The phenotypic traits and molecular biology of isolated bacteria, determined their identity as Vibrio vulnificus (V. vulnificus). In addition, artificially infected L. vannamei with Vibrio vulnificus appeared with the same disease symptoms as those of naturally infected shrimp. Drug sensitivity tests showed that V. vulnificus is highly sensitive to fosfomycin, cefradine and sinomin, and was resistant to penicillin, amikacin and kanamycin. This experiment is the first to separate V. vulnificus from L. vannamei, and the findings of this study can be used as a reference for disease control and health management.
[Mh] Termos MeSH primário: Penaeidae/virologia
Vibrio vulnificus/genética
Vibrio vulnificus/isolamento & purificação
[Mh] Termos MeSH secundário: Animais
Sequência de Bases
Contagem de Colônia Microbiana
Espaço Extracelular/enzimologia
Testes de Sensibilidade Microbiana
Fenótipo
Filogenia
RNA Ribossômico 16S/genética
Vibrioses/microbiologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (RNA, Ribosomal, 16S)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171031
[Lr] Data última revisão:
171031
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171019
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0186135



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