共查询到20条相似文献,搜索用时 15 毫秒
1.
Neha Dhami Drupad K. Trivedi Royston Goodacre David Mainwaring David P. Humphreys 《Metabolomics : Official journal of the Metabolomic Society》2018,14(10):136
Introduction
Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.Objectives
The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.Methods
To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.Results
Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.Conclusion
Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.2.
N. Cesbron A.-L. Royer Y. Guitton A. Sydor B. Le Bizec G. Dervilly-Pinel 《Metabolomics : Official journal of the Metabolomic Society》2017,13(8):99
Introduction
Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.Objectives
In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.Methods
The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.Results
A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.Conclusion
The workflow generated repeatable and informative fingerprints for robust metabolome characterization.3.
Background
The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown.Methods
We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells.Results
Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division.Conclusions
Taken together, the data suggest a model in which mitochondria are transported by a microtubule-mediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis.4.
Polona Žigon Katjuša Mrak-Poljšak Katja Lakota Matic Terčelj Saša Čučnik Matija Tomsic Snezna Sodin-Semrl 《Metabolomics : Official journal of the Metabolomic Society》2016,12(5):92
Introduction
Human primary cells originating from different locations within the body could differ greatly in their metabolic phenotypes, influencing both how they act during physiological/pathological processes and how susceptible/resistant they are to a variety of disease risk factors. A novel way to monitor cellular metabolism is through cell energetics assays, so we explored this approach with human primary cell types, as models of sclerotic disorders.Objectives
In order to better understand pathophysiological processes at the cellular level, our goals were to measure metabolic pathway activities of endothelial cells and fibroblasts, and determine their metabolic phenotype profiles.Methods
Biolog Phenotype MicroArray? technology was used for the first time to characterize metabolic phenotypes of diverse primary cells. These colorimetric assays enable detection of utilization of 367 specific biochemical substrates by human endothelial cells from the coronary artery (HCAEC), umbilical vein (HUVEC) and normal, healthy lung fibroblasts (NHLF).Results
Adenosine, inosine, d-mannose and dextrin were strongly utilized by all three cell types, comparable to glucose. Substrates metabolized solely by HCAEC were mannan, pectin, gelatin and prevalently tricarballylic acid. HUVEC did not show any uniquely metabolized substrates whereas NHLF exhibited strong utilization of sugars and carboxylic acids along with amino acids and peptides.Conclusion
Taken together, we show for the first time that this simple energetics assay platform enables metabolic characterization of primary cells and that each of the three human cell types examined gives a unique and distinguishable profile.5.
Karimeh Haghani Pouyan Asadi Gholamreza Taheripak Ali Noori-Zadeh Shahram Darabi Salar Bakhtiyari 《生物学前沿》2018,13(6):406-417
Background
Diabetes mellitus (DM) is one of the most prevalent chronic diseases, and its prevalence continues to increase globally. The impact of mitochondrial dysfunction and lipid metabolism on diabetes mellitus and insulin resistance (IR) has been implicated in several previous reports; however, the results of studies are confusing despite four decades of study.Methods/Results
This review has evaluated updated understanding of the role of mitochondrial dysfunction and lipid metabolism on type 2 diabetes, and found that mitochondrial dysfunction and lipid metabolism disorder induce the dysregulation of liver and pancreatic beta cells, insulin resistance, and type 2 diabetes.Conclusion
Mitochondrial dysfunction and lipid metabolism induce metabolic dysregulation and finally increasing the possibility of diabetes.6.
Background
Friedreich ataxia is a neurological disease originating from an iron-sulfur cluster enzyme deficiency due to impaired iron handling in the mitochondrion, aconitase being particularly affected. As a mean to counteract disease progression, it has been suggested to chelate free mitochondrial iron. Recent years have witnessed a renewed interest in this strategy because of availability of deferiprone, a chelator preferentially targeting mitochondrial iron.Method
Control and Friedreich's ataxia patient cultured skin fibroblasts, frataxin-depleted neuroblastoma-derived cells (SK-N-AS) were studied for their response to iron chelation, with a particular attention paid to iron-sensitive aconitase activity.Results
We found that a direct consequence of chelating mitochondrial free iron in various cell systems is a concentration and time dependent loss of aconitase activity. Impairing aconitase activity was shown to precede decreased cell proliferation.Conclusion
We conclude that, if chelating excessive mitochondrial iron may be beneficial at some stage of the disease, great attention should be paid to not fully deplete mitochondrial iron store in order to avoid undesirable consequences.7.
Background
Mitochondrial impairment has been implicated in the pathogenesis of Huntington’s disease (HD). However, how mutant huntingtin impairs mitochondrial function and thus contributes to HD has not been fully elucidated. In this study, we used striatal cells expressing wild type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin protein, and cortical neurons expressing the exon 1 of the huntingtin protein with physiological or pathological polyglutamine domains, to examine the interrelationship among specific mitochondrial functions.Results
Depolarization induced by KCl resulted in similar changes in calcium levels without compromising mitochondrial function, both in wild type and mutant cells. However, treatment of mutant cells with thapsigargin (a SERCA antagonist that raises cytosolic calcium levels), resulted in a pronounced decrease in mitochondrial calcium uptake, increased production of reactive oxygen species (ROS), mitochondrial depolarization and fragmentation, and cell viability loss. The mitochondrial dysfunction in mutant cells was also observed in cortical neurons expressing exon 1 of the huntingtin protein with 104 Gln residues (Q104-GFP) when they were exposed to calcium stress. In addition, calcium overload induced opening of the mitochondrial permeability transition pore (mPTP) in mutant striatal cells. The mitochondrial impairment observed in mutant cells and cortical neurons expressing Q104-GFP was prevented by pre-treatment with cyclosporine A (CsA) but not by FK506 (an inhibitor of calcineurin), indicating a potential role for mPTP opening in the mitochondrial dysfunction induced by calcium stress in mutant huntingtin cells.Conclusions
Expression of mutant huntingtin alters mitochondrial and cell viability through mPTP opening in striatal cells and cortical neurons.8.
Bing-Qian Su Ying-Qian Han Shuang-Shuang Fan Sheng-Li Ming Bo Wan Wei-Fei Lu Bei-Bei Chu Guo-Yu Yang Jiang Wang 《Biotechnology letters》2018,40(4):641-648
Objective
The purpose of the article is to evaluate the changes in lipid metabolism in bovine mammary-gland epithelial MAC-T cells after PKM2 knockdown.Results
MAC-T cells stably expressing low levels of PKM2 were established with lentivirus-mediated small hairpin RNA. Although the knockdown of PKM2 had no effect on MAC-T cell growth, the reduced expression of PKM2 attenuated the mRNA and protein expression of key enzymes involved in sterol synthesis through the SREBP pathway.Conclusions
The downregulation of PKM2 significantly influenced lipid synthesis in bovine mammary-gland epithelial MAC-T cells. These findings extend our understanding of the crosstalk between glycolysis and lipid metabolism in bovine mammary-gland epithelial cells.9.
Arunachalam Kalaiarasi Renu Sankar Chidambaram Anusha Kandasamy Saravanan Kalyanasundaram Aarthy Selvaraj Karthic Theodore lemuel Mathuram Vilwanathan Ravikumar 《Biotechnology letters》2018,40(2):249-256
Objectives
Copper oxide nanoparticles (CuO NPs) promoting anticancer activity may be due to the regulation of various classes of histone deacetylases (HDACs).Results
Green-synthesized CuO NPs significantly arrested total HDAC level and also suppressed class I, II and IV HDACs mRNA expression in A549 cells. A549 cells treated with CuO NPs downregulated oncogenes and upregulated tumor suppressor protein expression. CuO NPs positively regulated both mitochondrial and death receptor-mediated apoptosis caspase cascade pathway in A549 cells.Conclusion
Green-synthesized CuO NPs inhibited HDAC and therefore shown apoptosis mediated anticancer activity in A549 lung cancer cell line.10.
Background
In HIV-1 infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with progression of infection toward AIDS. HIV-1 Tat protein, by interacting with TLR4-MD2 at the membrane level, induces IL-10 production by primary human monocytes and macrophages. In the present study we evaluated the effect of the TLR4 antagonist Eritoran tetrasodium (E5564) on HIV-1 Tat-induced IL-10 production.Findings
Here, we confirm that the recombinant HIV-1 Tat protein and the GST-Tat 1–45 fusion protein efficiently stimulate IL-10 production by primary monocytes and macrophages and that this stimulation is inhibited by blocking anti-TLR4 mAbs. We show that a similar inhibition is observed by preincubating the cells with the TLR4 antagonist E5564.Conclusion
This study provides compelling data showing for the first time that the TLR4 antagonist E5564 inhibits the immunosuppressive cytokine IL-10 production by primary human monocytes and macrophages incubated in the presence of HIV-1 Tat protein.11.
Angela Lombardi Bruno Trimarco Guido Iaccarino Gaetano Santulli 《Cell communication and signaling : CCS》2017,15(1):47
Background
One of the most common side effects of the immunosuppressive drug tacrolimus (FK506) is the increased risk of new-onset diabetes mellitus. However, the molecular mechanisms underlying this association have not been fully clarified.Methods
We studied the effects of the therapeutic dose of tacrolimus on mitochondrial fitness in beta-cells.Results
We demonstrate that tacrolimus impairs glucose-stimulated insulin secretion (GSIS) in beta-cells through a previously unidentified mechanism. Indeed, tacrolimus causes a decrease in mitochondrial Ca2+ uptake, accompanied by altered mitochondrial respiration and reduced ATP production, eventually leading to impaired GSIS.Conclusion
Our observations individuate a new fundamental mechanism responsible for the augmented incidence of diabetes following tacrolimus treatment. Indeed, this drug alters Ca2+ fluxes in mitochondria, thereby compromising metabolism-secretion coupling in beta-cells.12.
Background
Weaning stress affects the small intestine of piglets. MiR-146b is differentially expressed in suckling and weaned piglets. In this study, we evaluated the effects of miR-146b on cell viability, proliferation, and apoptosis in IPEC-J2 cells.Results
Transfection with miR-146b mimics successfully increased miR-146b levels by 1000× (P?<?0.001). The over-expression of miR-146b significantly promoted the apoptosis (P?<?0.01) of IPEC-J2 cells, with no significant effects on cell viability or proliferation. MiR-146b suppressed the luciferase activity of the miR-TLR4-wt by 57% compared with the negative control, while mutation of the miR-146b binding site significantly blocked the suppressive effect (P?<?0.05). Western blot results showed that TLR4 levels decreased in IPEC-J2 cells transfected with miR-146b mimics (P?<?0.05).Conclusions
The over-expression of miR-146b promotes IPEC-J2 cell apoptosis. TLR4 is a direct target of miR-146b in IPEC-J2 cells.Reviewers
This article was reviewed by Eugene Berezikov and Jan B Hoek.13.
Daqiang Pan Caroline Lindau Simon Lagies Nils Wiedemann Bernd Kammerer 《Metabolomics : Official journal of the Metabolomic Society》2018,14(5):59
Introduction
Subcellular compartmentalization enables eukaryotic cells to carry out different reactions at the same time, resulting in different metabolite pools in the subcellular compartments. Thus, mutations affecting the mitochondrial energy metabolism could cause different metabolic alterations in mitochondria compared to the cytoplasm. Given that the metabolite pool in the cytosol is larger than that of other subcellular compartments, metabolic profiling of total cells could miss these compartment-specific metabolic alterations.Objectives
To reveal compartment-specific metabolic differences, mitochondria and the cytoplasmic fraction of baker’s yeast Saccharomyces cerevisiae were isolated and subjected to metabolic profiling.Methods
Mitochondria were isolated through differential centrifugation and were analyzed together with the remaining cytoplasm by gas chromatography–mass spectrometry (GC–MS) based metabolic profiling.Results
Seventy-two metabolites were identified, of which eight were found exclusively in mitochondria and sixteen exclusively in the cytoplasm. Based on the metabolic signature of mitochondria and of the cytoplasm, mutants of the succinate dehydrogenase (respiratory chain complex II) and of the FOF1-ATP-synthase (complex V) can be discriminated in both compartments by principal component analysis from wild-type and each other. These mitochondrial oxidative phosphorylation machinery mutants altered not only citric acid cycle related metabolites but also amino acids, fatty acids, purine and pyrimidine intermediates and others.Conclusion
By applying metabolomics to isolated mitochondria and the corresponding cytoplasm, compartment-specific metabolic signatures can be identified. This subcellular metabolomics analysis is a powerful tool to study the molecular mechanism of compartment-specific metabolic homeostasis in response to mutations affecting the mitochondrial metabolism.14.
Purpose of Review
This review summarizes the fungal diagnostic measures currently available for use in paediatric patients at high risk for developing invasive fungal disease (IFD) and those suspected of having an IFD. The clinical utility of each test is described based on reported performances of individual tests in specific paediatric populations.Recent Findings
Available studies in the paediatric population are scarce and are characterized by a huge heterogeneity in underlying diseases (e.g. different risk for IFD), different study objectives and management strategies (screening versus diagnostic) used.Summary
A final valuation of paediatric studies on fungal diagnostic tools is limited. While the galactomannan and fungal PCR assays are useful to exclude the presence of IFD, it is unclear if mannan, mannan antibodies and β-D-glucan are of benefit due to a lack of studies or validation of the cut-off, respectively. Well-designed multicentre paediatric studies are urgently needed to improve the outcome of IFD.15.
Lifan Zhuang Jianhua Tian Xinzhi Zhang Hong Wang Chenghui Huang 《Cellular & molecular biology letters》2018,23(1):43
Background
Lnc-DC is a specific group of long non-coding (Lnc) RNAs in dendritic cells (DCs). Its function has been previously studied, and includes roles in dendritic cell differentiation and the progression of some diseases. In this study, we observed the critical role of Lnc-DC in regulating the differentiation, growth, and apoptosis of dendritic cells.Methods
We first isolated peripheral blood mononuclear cells to culture and induce into DCs, which were then co-cultured with hepatitis B virus (HBV)-secreting HepG2.2.15 cells for the detection of changes in Lnc-DC. The expression levels of TLR9, p-STAT3, and SOCS3 were tested with qPCR and western blot. MTT assays were used to analyze the cell proliferation, cell cycle, and apoptosis. We used ELISA to test the expression of TNF-α, IL-1β, IL-6, IL-12p40, and IFN-γ.Results
Co-culture with HBV-secreting HepG2.2.15 cells increased the level of Lnc-DC and activated TLR9/STAT3 signaling. The HBV DNA level (IU/ml) was positively correlated with levels of Lnc-DC and TLR9, further demonstrating that Lnc-DC was associated with the immune response of HBV. Lnc-DC was shown to regulate TLR9/STAT3 signaling in dendritic cells. More interestingly, the regulation of Lnc-DC controlled the immune response by reducing the concentration of secreted TNF-α, IL-6, IL-12, and IFN-γ, as well as increasing the IL-1β concentration in dendritic cells.Conclusion
Lnc-DC is important in regulating the growth, apoptosis, and immune response of dendritic cells mediated by TLR9/STAT3 signaling, and was also activated by HBV. This study provides a previously unidentified mechanism underlying the immune response in dendritic cells.16.
RAC3 influences the chemoresistance of colon cancer cells through autophagy and apoptosis inhibition
María Fernanda Rubio María Cecilia Lira Francisco Damián Rosa Adrían Dario Sambresqui María Cecilia Salazar Güemes Mónica Alejandra Costas 《Cancer cell international》2017,17(1):111
Background
RAC3 coactivator overexpression has been implicated in tumorigenesis, contributing to inhibition of apoptosis and autophagy. Both mechanisms are involved in resistance to treatment with chemotherapeutic agents. The aim of this study was to investigate its role in chemoresistance of colorectal cancer.Methods
The sensitivity to 5-fluorouracil and oxaliplatin in colon cancer cells HT-29, HCT 116 and Lovo cell lines, expressing high or low natural levels of RAC3, was investigated using viability assays.Results
In HCT 116 cells, we found that although 5-fluorouracil was a poor inducer of apoptosis, autophagy was strongly induced, while oxaliplatin has shown a similar ability to induce both of them. However, in HCT 116 cells expressing a short hairpin RNA for RAC3, we found an increased sensitivity to both drugs if it is compared with control cells. 5-Fluorouracil and oxaliplatin treatment lead to an enhanced caspase 3-dependent apoptosis and produce an increase of autophagy. In addition, both process have shown to be trigged faster than in control cells, starting earlier after stimulation.Conclusions
Our results suggest that RAC3 expression levels influence the sensitivity to chemotherapeutic drugs. Therefore, the knowledge of RAC3 expression levels in tumoral samples could be an important contribution to design new improved therapeutic strategies in the future.17.
Katie?R?Hagen Xiangbin?Zeng Mi-Young?Lee Shannon?Tucker Kahn Mary?Kathryn?Harrison Pitner Sandra?S?Zaky Yuan?Liu Ruth?M?O’Regan Xingming?Deng Harold?I?Saavedra
Background
The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery.Methods
To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks.Results
Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis.Conclusion
Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.18.
Background
For many years, yeast cell walls (YCW) and mannan oligosaccharides (MOS) have been used as alternatives to antibiotics and health feed additives to enhance the growth performance and health of food animals. In the present study, the inhibitory effects of YCWand MOS on the adhesion of enteropathogenic bacteria to intestinal epithelial cells were tested.Methods
YCW and MOS were extracted from Saccharomyces cerevisiae (XM 0315), and the morphology of YCW and MOS bound to pathogenic bacteria was observed by scanning electron microscopy (SEM). Real-time fluorescent quantitative PCR was used to quantitatively analyze the effects of YCW and MOS on the adhesion of Escherichia coli (CVCC3367) and Salmonella pullorum (CVCC520) to Caco-2 cells.Results
The results showed that YCW inhibited E. coli and S. pullorum binding to Caco-2 cells by 95% and 74%, respectively, whereas MOS prevented E. coli and S. pullorum binding by 67% and 50%, respectively.Conclusions
These data suggest that YCW has a stronger ability than MOS to inhibit pathogenic bacteria from adhering to Caco-2 cells in vitro.19.
Samantha M. Carlisle Patrick J. Trainor Xinmin Yin Mark A. Doll Marcus W. Stepp J. Christopher States Xiang Zhang David W. Hein 《Metabolomics : Official journal of the Metabolomic Society》2016,12(7):111
Introduction
Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. Expression of NAT1 is elevated in several cancers including breast cancer. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear.Objective
This study explored polar metabolome differences between MDA-MB-231 breast cancer cells expressing varying levels of NAT1 activity using an untargeted approach.Methods
Three MDA-MB-231 breast adenocarcinoma cell lines that stably express wild-type, increased, and decreased levels of human NAT1 were investigated for differences in polar metabolic profile using a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) system.Results
Increased levels of human NAT1 in the transformed cell lines resulted in a statistically significant decreased abundance of the metabolite palmitoleic acid (q = 0.0006), when compared to normal and decreased levels of human NAT1. The fatty acid synthesis pathway utilizes acetyl coenzyme A (acetyl-CoA) in the first two reactions of the pathway and eventually leads to the synthesis of palmitoleic acid.Conclusion
These data suggest a link between increased levels of NAT1 activity and decreased flux of acetyl-CoA through this portion of the fatty acid synthesis pathway.20.
Meng Wang Peiwei Zhang Yufei Zhu Xiangyin Kong Zhenguo Zhang Landian Hu 《BMC medical genomics》2017,10(1):55