Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 13.745
Filtrar
1.
BMC Evol Biol ; 19(1): 146, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324143

RESUMO

BACKGROUND: Antioxidative enzymes contribute to a parasite's ability to counteract the host's intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite's life history? RESULTS: Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site. CONCLUSIONS: PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.


Assuntos
Antioxidantes/metabolismo , Ascorbato Peroxidases/metabolismo , Catalase/metabolismo , Parasitos/enzimologia , Fotossíntese , Sequência de Aminoácidos , Animais , Ascorbato Peroxidases/química , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/isolamento & purificação , Peróxido de Hidrogênio/metabolismo , Cinética , Modelos Moleculares , Parasitos/genética , Filogenia , Homologia Estrutural de Proteína , Frações Subcelulares/metabolismo
2.
Chem Commun (Camb) ; 55(44): 6197-6200, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31070615

RESUMO

Here we report a convenient, universal "DNA encoding loop program (DELP)" strategy that significantly enhanced the sensitivity of subcellular imaging of microRNA. The assay relies on the dynamic, ultrafast clustering of multiple plasmonic gold nanoparticles actuated by a DNA-programmed recycling process.


Assuntos
DNA/metabolismo , MicroRNAs/metabolismo , Frações Subcelulares/metabolismo , Animais , Linhagem Celular Tumoral , Ouro/química , Humanos , Nanopartículas Metálicas/química , Camundongos , Células RAW 264.7
3.
Anal Bioanal Chem ; 411(19): 4963-4971, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31093701

RESUMO

Cu isotope fractionation was investigated in the human neuroblastoma SH-SY5Y cell line, in a proliferating/tumor phase (undifferentiated cells), and in a differentiated state (neuron-like cells), induced using retinoic acid (RA). The SH-SY5Y cell line displays genetic aberrations due to its cancerous origin, but differentiation drives the cell line towards phenotypes suitable for the research of neurological diseases (e.g., Alzheimer's disease or Parkinson's disease). Cellular Cu distribution was first explored by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging and, subsequently, Cu isotopic analysis was performed at cellular and sub-cellular levels via multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS). The SH-SY5Y cells showed a re-distribution of intracellular Cu upon RA differentiation. Both undifferentiated and differentiated cells became systematically enriched in the light 63Cu isotope with increasing intracellular Cu content. Differentiated neuron-like SH-SY5Y cells showed a heavier Cu isotopic composition (+ 0.3‰) than did the undifferentiated proliferating cells when exposed to Cu for 24 h. However, after a longer exposure time (72 h), no difference was observed between both cellular phenotypes. Mitochondrial fractions were enriched in the light 63Cu isotope, compared to whole cells, for both undifferentiated and differentiated cells (no significant difference). The Cu isotopic composition of the remaining cell lysates was heavier than that of the whole cells and + 0.2‰ heavier in the differentiated cells than in the undifferentiated cells. These results indicate that neuronal differentiation affects the Cu isotope fractionation accompanying Cu uptake in the cells, but this effect does not seem to be associated with the mitochondrial Cu pathway. Cu isotope fractionation can be an interesting tool for studying Cu metabolism at a (sub)-cellular level in functional neurons. Graphical abstract.


Assuntos
Fracionamento Celular , Cobre/isolamento & purificação , Isótopos/isolamento & purificação , Neuroblastoma/metabolismo , Neurônios/metabolismo , Frações Subcelulares/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Mitocôndrias/metabolismo , Neuroblastoma/patologia , Neurônios/citologia
4.
Anal Bioanal Chem ; 411(20): 5223-5231, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31111180

RESUMO

The visualization of subcellular structures is critical for understanding their intracellular function. We prepared two triarylboron-based multinuclear Zn2+ complexes, TAB-1-3Zn2+ and TAB-2-2Zn2+, which can be used as fluorescent probes for nucleoside polyphosphate (NPP) and RNA because their multi Zn2+ center can selectively combine with the phosphate side chain of NPP or RNA, accompanied by a corresponding fluorescence change. Among them, TAB-2-2Zn2+ is more suitable than TAB-1-3Zn2+ for live cell imaging because of its excellent cell membrane permeability resulting from amphiphilicity. Since the various membrane structures in cells are also composed of phosphoric acid bilayers, TAB-2-2Zn2+ may also be used to image various membrane structures. Various colocalization experiments further confirmed that TAB-2-2Zn2+ can achieve clear simultaneous imaging of the cell membrane, the endoplasmic reticulum, and the nucleolus. Graphical abstract.


Assuntos
Compostos de Boro/química , Membrana Celular/metabolismo , Nucléolo Celular/metabolismo , Complexos de Coordenação/química , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Compostos de Zinco/química , Células 3T3 , Animais , Células HeLa , Células Hep G2 , Humanos , Camundongos , Fótons , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Frações Subcelulares/metabolismo
5.
Mol Biotechnol ; 61(6): 432-441, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963480

RESUMO

D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM D-fructose, and 20 mM Mg2+ when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.


Assuntos
Trifosfato de Adenosina/metabolismo , Carboidratos Epimerases/metabolismo , Frutose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biotransformação , Carboidratos Epimerases/genética , Cátions Bivalentes , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose/biossíntese , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Magnésio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polifosfatos/metabolismo , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismo
6.
Methods Mol Biol ; 1933: 173-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945184

RESUMO

Plant lncRNAs are expected to play important roles in various plant processes including response to abiotic stress, although the functions of most plant lncRNAs are still unknown. To investigate these potential functions, integrated approaches that employ genetic, transgenic, and molecular biology methods are required. Here, we describe general methods to study the function of lncRNAs in plant response to drought and salt stresses. First, the expression pattern and subcellular localization of lncRNA are analyzed by GUS staining and RNA fluorescence in situ hybridization (FISH). Then the responses of lncRNA mutant and overexpressing transgenic plants to drought and salt stress treatments are characterized, and their sensitivities to ABA are also assayed. To understand the molecular mechanism of lncRNAs' function in stress response, transcriptome sequencing (RNA-seq) and real-time quantitative PCR are performed to analyze altered expression of stress-related genes. Finally, proline content and ROS content are measured to reveal the accumulation of osmolytes and second messengers in these plants in response to drought and salt stress treatments.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , RNA Longo não Codificante/genética , RNA de Plantas/genética , Tolerância ao Sal , Estresse Fisiológico , Frações Subcelulares/metabolismo , Metilação de DNA , Regulação da Expressão Gênica de Plantas
7.
Cell Mol Biol (Noisy-le-grand) ; 65(3): 25-31, 2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30942153

RESUMO

Flowering is a very important developmental stage in the plant life cycle. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) has been shown to participate in epigenetic silencing of flowering genes. Here, for the first time, we isolated and characterized six CmLHP1 homolog genes from the important day-neutral ornamental Chrysanthemum morifolium cultivar 'Jin budiao'. These homolog genes were most likely generated by whole-genome duplication. Bioinformatic analysis showed that chrysanthemum LHP1 homologs present low similarity to other plant LHP1-like genes. However, three nuclear localization signals and two domains were highly conserved among them. The secondary structures of the CmLHP1 homologs mainly include α-helices and random coils, indicating that the proteins are mixed proteins. Phylogenetic tree analysis indicated that the six CmLHP1 genes constituted a small clade and had the closest relationship with LsLHP1 (Lactuca sativa LHP1). Quantitative RT-PCR analysis showed that the CmLHP1 homologs were expressed in different tissues during the developmental period of chrysanthemum, but they were highly expressed in the buds, especially during the key S1 stage of the inflorescence. Furthermore, the expression patterns of CmLHP1 homologs showed divergence under different photoperiods. Both CmLHP1b and CmLHP1e exhibited photoperiod sensitivity in leaves. Intriguingly, CmLHP1c was insensitive to photoperiod in both the shoot apexes and the leaves. Subcellular localization revealed that the six CmLHP1 proteins were located in the nucleus. These results reveal that CmLHP1 homolog genes could be strong candidates as important regulators of flowering time in chrysanthemum.


Assuntos
Proteínas Cromossômicas não Histona/genética , Chrysanthemum/genética , Clonagem Molecular/métodos , Regulação da Expressão Gênica de Plantas , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas Cromossômicas não Histona/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Frações Subcelulares/metabolismo
8.
Ecotoxicol Environ Saf ; 176: 162-169, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-30927637

RESUMO

Bacteria have been applied for the bioremediation of cadmium-contaminated environment. Less is known about the bioaccumulation of high concentration of Cd over time under the oligotrophic environment. Burkholderia cepacia GYP1, which was isolated from multiple heavy metal contaminated farmland, was studied for its bioaccumulation mechanism of Cd under oligotrophic condition. GYP1 possessed highly accumulation capacity for cadmium reaching 116 mg Cd/g biomass (dry weight). ATR-FTIR, electron microscopy, flow cytometry along with subcellular fraction demonstrated that the uptake and distribution of cadmium varied with the increased amount of cadmium of GYP1 cell during the 7-day treatment time: the accumulation of cadmium was mainly on the outer membrane at the beginning (within 1 day), and the intracellular cadmium kept increased and held stable after 2 days, after that, the increased amount of cadmium mainly located extracellularly, related to the secreted EPS. Further mechanism analysis of bioaccumulation of Cd by GYP1 based on iTRAQ-based proteomics showed that Cd(II) could trigger the up-regulation of the Cd2+/Zn2+-exporting ATPase, type VI protein secretion systems, and glutathione-S-transferase that are related to cadmium response, which may contribute to maintain the intracellular cadmium homeostasis. In summary, the immobilization of Cd(II) by B. cepacia GYP1 contains three steps:(1) fast immobilization of Cd(II) on the cell surface coordinated with functional groups, (2) transport of Cd(II) to cells and accumulation in cytoplasm, and (3) efflux of intracellular Cd(II) depended on energy and the entrapped or adsorbed of extracellular Cd(II) by EPS. Our study provided the understanding of the cadmium accumulation process of B. cepacia GYP1 under oligotrophic condition, which would be helpful in bioremediation of natural cadmium contaminated environment.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia cepacia/metabolismo , Cádmio/metabolismo , Poluentes Ambientais/metabolismo , Proteoma/metabolismo , Biodegradação Ambiental , Transporte Biológico , Biomassa , Nutrientes/deficiência , Frações Subcelulares/metabolismo
9.
Int J Mol Sci ; 20(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889841

RESUMO

Human cells, when exposed to both real and simulated microgravity (s-µg), form 3D tissue constructs mirroring in vivo architectures (e.g., cartilage, intima constructs, cancer spheroids and others). In this study, we exposed human foetal osteoblast (hFOB 1.19) cells to a Random Positioning Machine (RPM) for 7 days and 14 days, with the purpose of investigating the effects of s-µg on biological processes and to engineer 3D bone constructs. RPM exposure of the hFOB 1.19 cells induces alterations in the cytoskeleton, cell adhesion, extra cellular matrix (ECM) and the 3D multicellular spheroid (MCS) formation. In addition, after 7 days, it influences the morphological appearance of these cells, as it forces adherent cells to detach from the surface and assemble into 3D structures. The RPM-exposed hFOB 1.19 cells exhibited a differential gene expression of the following genes: transforming growth factor beta 1 (TGFB1, bone morphogenic protein 2 (BMP2), SRY-Box 9 (SOX9), actin beta (ACTB), beta tubulin (TUBB), vimentin (VIM), laminin subunit alpha 1 (LAMA1), collagen type 1 alpha 1 (COL1A1), phosphoprotein 1 (SPP1) and fibronectin 1 (FN1). RPM exposure also induced a significantly altered release of the cytokines and bone biomarkers sclerostin (SOST), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), interleukin 1 beta (IL-1ß) and tumour necrosis factor 1 alpha (TNF-1α). After the two-week RPM exposure, the spheroids presented a bone-specific morphology. In conclusion, culturing cells in s-µg under gravitational unloading represents a novel technology for tissue-engineering of bone constructs and it can be used for investigating the mechanisms behind spaceflight-related bone loss as well as bone diseases such as osteonecrosis or bone injuries.


Assuntos
Osso e Ossos/fisiologia , Feto/citologia , Osteoblastos/citologia , Engenharia Tecidual/métodos , Proteína Morfogenética Óssea 2/metabolismo , Forma Celular , Células Cultivadas , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Organoides/citologia , Osteoblastos/metabolismo , Osteogênese , Ligação Proteica , Transdução de Sinais , Solubilidade , Frações Subcelulares/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Simulação de Ausência de Peso
10.
Int J Mol Sci ; 20(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889878

RESUMO

Diacylglycerol kinase (DGK) is an enzyme that plays a pivotal role in abiotic and biotic stress responses in plants by transforming the diacylglycerol into phosphatidic acid. However, there is no report on the characterization of soybean DGK genes in spite of the availability of the soybean genome sequence. In this study, we performed genome-wide analysis and expression profiling of the DGK gene family in the soybean genome. We identified 12 DGK genes (namely GmDGK1-12) which all contained conserved catalytic domains with protein lengths and molecular weights ranging from 436 to 727 amino acids (aa) and 48.62 to 80.93 kDa, respectively. Phylogenetic analyses grouped GmDGK genes into three clusters-cluster I, cluster II, and cluster III-which had three, four, and five genes, respectively. The qRT-PCR analysis revealed significant GmDGK gene expression levels in both leaves and roots coping with polyethylene glycol (PEG), salt, alkali, and salt/alkali treatments. This work provides the first characterization of the DGK gene family in soybean and suggests their importance in soybean response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of this gene family.


Assuntos
Diacilglicerol Quinase/genética , Perfilação da Expressão Gênica , Genômica , Família Multigênica , Soja/enzimologia , Soja/genética , Estresse Fisiológico/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Cromossomos de Plantas/genética , Diacilglicerol Quinase/química , Diacilglicerol Quinase/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Especificidade de Órgãos/genética , Filogenia , Regiões Promotoras Genéticas/genética , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo
11.
Anal Bioanal Chem ; 411(19): 4481-4508, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30927013

RESUMO

G protein-coupled receptors (GPCRs), G proteins, and their signaling associates are major signal transducers that control the majority of cellular signaling and regulate key biological functions including immune, neurological, cardiovascular, and metabolic processes. These pathways are targeted by over one-third of drugs on the market; however, the current understanding of their function is limited and primarily derived from cell-destructive approaches providing an ensemble of static, multi-cell information about the status and composition of molecules. Spatiotemporal behavior of molecules involved is crucial to understanding in vivo cell behaviors both in health and disease, and the advent of genetically encoded fluorescence proteins and small fluorophore-based biosensors has facilitated the mapping of dynamic signaling in cells with subcellular acuity. Since we and others have developed optogenetic methods to regulate GPCR-G protein signaling in single cells and subcellular regions using dedicated wavelengths, the desire to develop and adopt optogenetically amenable assays to measure signaling has motivated us to take a broader look at the available optical tools and approaches compatible with measuring single-cell and subcellular GPCR-G protein signaling. Here we review such key optical approaches enabling the examination of GPCR, G protein, secondary messenger, and downstream molecules such as kinase and lipid signaling in living cells. The methods reviewed employ both fluorescence and bioluminescence detection. We not only further elaborate the underlying principles of these sensors but also discuss the experimental criteria and limitations to be considered during their use in single-cell and subcellular signal mapping.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Análise de Célula Única , Frações Subcelulares/metabolismo , Fluorescência , Expressão Gênica/fisiologia , Humanos , Ligação Proteica , Receptores Acoplados a Proteínas-G/fisiologia
12.
Nat Commun ; 10(1): 1351, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30903027

RESUMO

The inability to inspect metabolic activities within subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Here, we describe a spatial-fluxomics approach for inferring metabolic fluxes in mitochondria and cytosol under physiological conditions, combining isotope tracing, rapid subcellular fractionation, LC-MS-based metabolomics, computational deconvolution, and metabolic network modeling. Applied to study reductive glutamine metabolism in cancer cells, shown to mediate fatty acid biosynthesis under hypoxia and defective mitochondria, we find a previously unappreciated role of reductive IDH1 as the sole net contributor of carbons to fatty acid biosynthesis under standard normoxic conditions in HeLa cells. In murine cells with defective SDH, we find that reductive biosynthesis of citrate in mitochondria is followed by a reversed CS activity, suggesting a new route for supporting pyrimidine biosynthesis. We expect this spatial-fluxomics approach to be a highly useful tool for elucidating the role of metabolic dysfunction in human disease.


Assuntos
Compartimento Celular , Glutamina/metabolismo , Análise do Fluxo Metabólico , Neoplasias/metabolismo , Animais , Isótopos de Carbono , Hipóxia Celular , Citrato (si)-Sintase/metabolismo , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Citosol/metabolismo , Células HeLa , Humanos , Isocitrato Desidrogenase/metabolismo , Metaboloma , Camundongos , Mitocôndrias/metabolismo , Frações Subcelulares/metabolismo , Succinato Desidrogenase/metabolismo
13.
Oxid Med Cell Longev ; 2019: 4565238, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30918579

RESUMO

A surgical connection between portal and inferior cava veins was performed to generate an experimental model of high circulating ammonium and hepatic hypofunctioning. After 13 weeks of portacaval anastomosis (PCA), hyperammonemia and shrinkage in the liver were observed. Low glycemic levels accompanied by elevated levels of serum alanine aminotransferase were recorded. However, the activity of serum aspartate aminotransferase was reduced, without change in circulating urea. Histological and ultrastructural observations revealed ongoing vascularization and alterations in the hepatocyte nucleus (reduced diameter with indentations), fewer mitochondria, and numerous ribosomes in the endoplasmic reticulum. High activity of hepatic caspase-3 suggested apoptosis. PCA promoted a marked reduction in lipid peroxidation determined by TBARs in liver homogenate but specially in the mitochondrial and microsomal fractions. The reduced lipoperoxidative activity was also detected in assays supplemented with Fe2+. Only discreet changes were observed in conjugated dienes. Fluorescent probes showed significant attenuation in mitochondrial membrane potential, reactive oxygen species (ROS), and calcium content. Rats with PCA also showed reduced food intake and decreased energy expenditure through indirect calorimetry by measuring oxygen consumption with an open-flow respirometric system. We conclude that experimental PCA promotes an angiogenic state in the liver to confront the altered blood flow by reducing the prooxidant reactions associated with lower metabolic rate, along with significant reduction of mitochondrial content, but without a clear hepatic dysfunction.


Assuntos
Peroxidação de Lipídeos , Fígado/metabolismo , Fígado/cirurgia , Derivação Portocava Cirúrgica , Anastomose Cirúrgica , Animais , Membrana Celular/metabolismo , Metabolismo Energético , Comportamento Alimentar , Corantes Fluorescentes/metabolismo , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Fígado/patologia , Fígado/ultraestrutura , Masculino , Mitocôndrias/metabolismo , Oxidantes/metabolismo , Ratos Wistar , Frações Subcelulares/metabolismo
14.
Methods Mol Biol ; 1941: 201-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707436

RESUMO

Subcellular fractionation methods permit the isolation, purification, and/or enrichment of specific cellular compartments from complex tissue samples. Enrichment of multiple subcellular compartments from the same tissue sample permits comparisons of the spatial distribution of target proteins between specific intracellular compartments and, in some cases, can provide information about spatiotemporal processing of key cellular components. Here we describe a method to generate subcellular fractions enriched for heavy membranes and nuclei, rough and smooth endoplasmic reticulum membranes, light membranes and cytosol, synapses, and other intermediate cellular membranes from postmortem human brain tissue. These subcellular fractions can be used in a variety of downstream applications to assess the localization, relative abundance, and stoichiometry of glutamate receptor subunits along the forward trafficking pathway.


Assuntos
Biomarcadores/metabolismo , Encéfalo/metabolismo , Fracionamento Celular/métodos , Núcleo Celular/metabolismo , Organelas/metabolismo , Receptores de Glutamato/metabolismo , Frações Subcelulares/metabolismo , Humanos
15.
Genet Epidemiol ; 43(3): 330-341, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30614068

RESUMO

Single-cell microscopy image analysis has proved invaluable in protein subcellular localization for inferring gene/protein function. Fluorescent-tagged proteins across cellular compartments are tracked and imaged in response to genetic or environmental perturbations. With a large number of images generated by high-content microscopy while manual labeling is both labor-intensive and error-prone, machine learning offers a viable alternative for automatic labeling of subcellular localizations. Contrarily, in recent years applications of deep learning methods to large datasets in natural images and other domains have become quite successful. An appeal of deep learning methods is that they can learn salient features from complicated data with little data preprocessing. For such purposes, we applied several representative types of deep convolutional neural networks (CNNs) and two popular ensemble methods, random forests and gradient boosting, to predict protein subcellular localization with a moderately large cell image data set. We show a consistently better predictive performance of CNNs over the two ensemble methods. We also demonstrate the use of CNNs for feature extraction. In the end, we share our computer code and pretrained models to facilitate CNN's applications in genetics and computational biology.


Assuntos
Aprendizado Profundo , Processamento de Imagem Assistida por Computador , Microscopia/métodos , Redes Neurais (Computação) , Proteínas de Saccharomyces cerevisiae/metabolismo , Algoritmos , Modelos Genéticos , Saccharomyces cerevisiae/metabolismo , Tamanho da Amostra , Frações Subcelulares/metabolismo
16.
Int J Mol Sci ; 20(3)2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678326

RESUMO

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.


Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/metabolismo , Monoiodotirosina/metabolismo , Fatores de Terminação de Peptídeos/deficiência , Códon de Terminação/genética , Códon de Terminação/metabolismo , Monoiodotirosina/genética , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Frações Subcelulares/metabolismo
17.
Mol Cell ; 73(1): 166-182.e7, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30609389

RESUMO

Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.


Assuntos
Cromatografia Líquida , Espectrometria de Massas , Proteínas/metabolismo , Proteoma , Proteômica/métodos , Frações Subcelulares/metabolismo , Biomarcadores/metabolismo , Fracionamento Celular , Biologia Computacional , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Humanos , Focalização Isoelétrica , Células MCF-7 , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , Proteínas/antagonistas & inibidores , Proteínas/classificação , Proteínas/genética , Reprodutibilidade dos Testes , Frações Subcelulares/classificação , Frações Subcelulares/efeitos dos fármacos
18.
Mol Biol Cell ; 30(2): 173-180, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30640588

RESUMO

Crowding of the subcellular environment by macromolecules is thought to promote protein aggregation and phase separation. A challenge is how to parameterize the degree of crowding of the cell interior or artificial solutions that is relevant to these reactions. Here I review colloid osmotic pressure as a crowding metric. This pressure is generated by solutions of macromolecules in contact with pores that are permeable to water and ions but not macromolecules. It generates depletion forces that push macromolecules together in crowded solutions and thus promotes aggregation and phase separation. I discuss measurements of colloid osmotic pressure inside cells using the nucleus, the cytoplasmic gel, and fluorescence resonant energy transfer (FRET) biosensors as osmometers, which return a range of values from 1 to 20 kPa. I argue for a low value, 1-2 kPa, in frog eggs and perhaps more generally. This value is close to the linear range on concentration-pressure curves and is thus not crowded from an osmotic perspective. I discuss the implications of a low crowding pressure inside cells for phase separation biology, buffer design, and proteome evolution. I also discuss a pressure-tension model for nuclear shape, where colloid osmotic pressure generated by nuclear protein import inflates the nucleus.


Assuntos
Coloides/química , Substâncias Macromoleculares/metabolismo , Osmose , Animais , Hidrodinâmica , Modelos Biológicos , Frações Subcelulares/metabolismo
19.
BMC Biol ; 17(1): 5, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678683

RESUMO

BACKGROUND: RNA localization involves cis-motifs that are recognized by RNA-binding proteins (RBP), which then mediate localization to specific sub-cellular compartments. RNA localization is critical for many different cell functions, e.g., in neuronal dendrites, localization is a critical step for long-lasting synaptic potentiation. However, there is little consensus regarding which RNAs are localized and the role of alternative isoforms in localization. A comprehensive catalog of localized RNA can help dissect RBP/RNA interactions and localization motifs. Here, we utilize a single cell sub-cellular RNA sequencing approach to profile differentially localized RNAs from individual cells across multiple single cells to help identify a consistent set of localized RNA in mouse neurons. RESULTS: Using independent RNA sequencing from soma and dendrites of the same neuron, we deeply profiled the sub-cellular transcriptomes to assess the extent and variability of dendritic RNA localization in individual hippocampal neurons, including an assessment of differential localization of alternative 3'UTR isoforms. We identified 2225 dendritic RNAs, including 298 cases of 3'UTR isoform-specific localization. We extensively analyzed the localized RNAs for potential localization motifs, finding that B1 and B2 SINE elements are up to 5.7 times more abundant in localized RNA 3'UTRs than non-localized, and also functionally characterized the localized RNAs using protein structure analysis. CONCLUSION: We integrate our list of localized RNAs with the literature to provide a comprehensive list of known dendritically localized RNAs as a resource. This catalog of transcripts, including differentially localized isoforms and computationally hypothesized localization motifs, will help investigators further dissect the genome-scale mechanism of RNA localization.


Assuntos
Dendritos/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Isoformas de RNA/genética , RNA Mensageiro/genética , Regiões 3' não Traduzidas , Animais , Camundongos , Isoformas de RNA/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Frações Subcelulares/metabolismo , Transcriptoma
20.
Plant Biol (Stuttg) ; 21(4): 634-642, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30664832

RESUMO

Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator due to its high Pb tolerance and accumulation ability. However, the mechanisms that support Pb accumulation and tolerance in P. crinitum are not yet clearly understood. An indoor hydroponic experiment was conducted by cultivating P. crinitum seedlings exposed to intermittent Pb stress for 60 days, divided into four stages (T1, T2, T3 and T4), with a 15-day duration per stage. The following concentrations of Pb were used: 0, 500, 0, 500 mg·l-1 and 0, 1000, 0, 1000 mg·l-1 ). Antioxidant enzyme activity, Pb concentration and subcellular distribution of Pb were measured at each of the above stages. The results showed that superoxide dismutase (SOD) activity in shoots, and SOD, peroxidase (POD) and malondialdehyde (MDA) activity in shoots and roots significantly increased from T1 (no Pb stress) to T2 (Pb stress) in both 500 mg·l-1 and 1000 mg·l-1 treatments; however, no significant difference was noted between stages T3 (no Pb stress) and T4 (Pb stress). There was no obvious effect of Pb stress on catalase (CAT) activity in shoots and roots among different stages. The Pb concentration in shoots was up to 5090.90 mg·kg-1 and 7573.57 mg·kg-1 , and the bioconcentration factor (BFC) was 10.18 and 7.57 for the 500 mg·l-1 and 1000 mg·l-1 treatments, respectively, which confirmed the Pb hyperaccumulator characteristics of P. crinitum. For plants under Pb stress, most of the Pb was fixed in the cell walls, with a smaller amount in leaves and root vacuoles. Both SOD and POD scavenging of reactive oxygen radicals and fixing and compartmentalisation of Pb in the cell wall might play important roles in detoxification of P. crinitum seedlings in response to Pb stress. There was no phased response of P. crinitum to intermittent Pb stress and the physiological response to Pb stress may be contiguous.


Assuntos
Catalase/efeitos dos fármacos , Chumbo/metabolismo , Peroxidase/efeitos dos fármacos , Proteínas de Plantas/efeitos dos fármacos , Poaceae/efeitos dos fármacos , Plântula/efeitos dos fármacos , Superóxido Dismutase/efeitos dos fármacos , Catalase/metabolismo , Relação Dose-Resposta a Droga , Chumbo/toxicidade , Malondialdeído/metabolismo , Peroxidase/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Poaceae/enzimologia , Poaceae/crescimento & desenvolvimento , Poaceae/metabolismo , Plântula/enzimologia , Plântula/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Frações Subcelulares/metabolismo , Superóxido Dismutase/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA