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1.
The ability of lysolipids to enter into a membrane bi-layer and disturb the membrane structure was used to study the behavior of K562 erythroleukemic cells, K562 wild type (K562wt) as well as the multidrug resistant cells K562adr. Both types of cells, when analyzed by proton NMR spectroscopy exhibit the high resolution signals assigned to so-called "mobile lipid" signals, which, in most cases, are located outside the lipid bi-layer as lipid droplets. In order to perform these studies, the K562wt and K562adr cells were treated for 48h with lysophosphatidylcholine oleoyl (LPC18), lysophosphatidylcholine palmitoyl (LPC16) and L-alpha-lysophosphatidyslerine (LPS). After evaluating toxicity of lysolipids, proton NMR of whole treated cells was used to analyze the mobile lipid content. Nile red staining and fluorescence microscopy were used to detect the presence of intracellular lipid droplets. Membrane lipid asymmetry perturbation was estimated by annexin V staining with use of flow cytometry. Using fluorescence spectroscopy the functioning of P-glycoprotein (P-gp) responsible for multidrug resistance was also evaluated after the treatment with lysolipids. Lysolipids were found to be more toxic for K562wt than for K562adr cells. LPS and LPC16 produced an increased of a mobile lipid NMR signal and amount of lipid droplets in K562wt cells only. LPC18, with the lowest toxicity, has shown more intense effects on NMR spectra with a large increase of lipid NMR signal without changes in lipid droplet staining. The functioning of the P-gp pump and membrane asymmetry were not modified by any of the lysolipids used. 相似文献
2.
Background
The hERG potassium channel can modulate the proliferation of the chronic myelogenous leukemic K562 cells, and its role in the erythroid differentiation of K562 cells still remains unclear.Principal Findings
The hERG potassium channel blockage by a new 36-residue scorpion toxin BmKKx2, a potent hERG channel blocker with IC50 of 6.7±1.7 nM, enhanced the erythroid differentiation of K562 cells. The mean values of GPA (CD235a) fluorescence intensity in the group of K562 cells pretreated by the toxin for 24 h and followed by cytosine arabinoside (Ara-C) treatment for 72 h were about 2-fold stronger than those of K562 cells induced by Ara-C alone. Such unique role of hERG potassium channel was also supported by the evidence that the effect of the toxin BmKKx2 on cell differentiation was nullified in hERG-deficient cell lines. During the K562 cell differentiation, BmKKx2 could also suppress the expression of hERG channels at both mRNA and protein levels. Besides the function of differentiation enhancement, BmKKx2 was also found to promote the differentiation-dependent apoptosis during the differentiation process of K562 cells. In addition, the blockage of hERG potassium channel by toxin BmKKx2 was able to decrease the intracellular Ca2+ concentration during the K562 cell differentiation, providing an insight into the mechanism of hERG potassium channel regulating this cellular process.Conclusions/Significance
Our results revealed scorpion toxin BmKKx2 could enhance the erythroid differentiation of leukemic K562 cells via inhibiting hERG potassium channel currents. These findings would not only accelerate the functional research of hERG channel in different leukemic cells, but also present the prospects of natural scorpion toxins as anti-leukemic drugs. 相似文献3.
Xiaoqin Wang Weidong Xu Subhra Mohapatra Xiaoyuan Kong Xu Li Richard F Lockey Shyam S Mohapatra 《Genetic vaccines and therapy》2008,6(1):1-9
Background
For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants.Methods
To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls.Results
Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed.Conclusion
Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors. 相似文献4.
High-resolution proton NMR spectra of intact tumour cells generally exhibit intense signals due to isotropically mobile lipids (MLs) of still uncertain nature and origin. NMR studies performed on intact wild-type and caveolin-1-infected haematopoietic K562 cells showed that, under our experimental conditions, part of the ML signals are due to lipid complexes resistant to extraction in Triton X-100 at 4 degrees C. This evidence suggests that a portion of NMR-visible lipid structures are compatible with Triton-resistant membrane rafts and therefore biophysically distinct from NMR-visible Triton-soluble lipid bodies. Similarly to lipid rafts and caveolae, the organization of the Triton-insoluble ML domains could be compromised by treatment with beta-octylglucoside or methyl-beta-cyclodextrin. Exposure to exogenous sphingomyelinase caused an increase in ML NMR visibility, indicating the possible involvement of ceramides in ML formation. The mobility of these lipids was found to be temperature sensitive, suggesting a transition in cells going from 4 degrees C to 25-37 degrees C. These new results are here discussed in the light of possible contributions of plasma membrane microdomains to NMR-visible ML signals. 相似文献
5.
Yongni Shao Hui Fang Hong Zhou Qi Wang Yiming Zhu Yong He 《Biotechnology for biofuels》2017,10(1):300
In this study, confocal Raman microspectroscopy was used to detect lipids in microalgae rapidly and non-destructively. Microalgae cells were cultured under nitrogen deficiency. The accumulation of lipids in Scenedesmus obliquus was observed by Nile red staining, and the total amount of lipids accumulated in the cells was measured by gravimetric method. The signals from different microalgae cells were collected by confocal Raman microspectroscopy to establish a prediction model of intracellular lipid content, and surface scanning signals for drawing pseudo color images of lipids distribution. The images can show the location of pyrenoid and lipid accumulation in cells. Analyze Raman spectrum data and build PCA-LDA model using four different bands (full bands, pigments, lipids, and mixed features). Models of full bands or pigment characteristic bands were capable of identifying S. obliquus cells under different nitrogen stress culture time. The prediction accuracy of model of lipid characteristic bands is relatively low. The correlation between the fatty acid content measured by the gravimetric method and the integral Raman intensity of the oil characteristic peak (1445 cm?1) measured by Raman spectroscopy was analyzed. There was significant correlation (R 2 = 0.83), which means that Raman spectroscopy is applicable to semi-quantitative detection of microalgal lipid content. 相似文献
6.
Ying Liu Baoquan Song Yimeng Wei Fang Chen Ying Chi Huifang Fan Na Liu Zongjin Li Zhongchao Han Fengxia Ma 《Cytotherapy》2018,20(2):181-188
Background aims
Imatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM.Methods
hUC-MSC-Exo were isolated and identified. K562 cells were then treated or not with IM (1?µmol/L) in combination with hUC-MSC-Exo (50?µg/mL). Cell viability and apoptosis were determined by cell counting kit 8 (CCK-8) and annexin V/propidium iodide (PI) double staining, respectively. Apoptotic proteins, caspase and their cleaved forms were detected by Western blot.Results
It was shown that hUC-MSC-Exo alone had no effect on cell viability and apoptosis of K562 cells. However, hUC-MSC-Exo promoted IM-induced cell viability inhibition and apoptosis. Moreover, hUC-MSC-Exo enhanced the increased Bax expression and the decreased Bcl-2 expression that were induced by IM. Compared with IM alone, caspase-9 and caspase-3 were further activated by combination of hUC-MSC-Exo with IM. Finally, the effects of hUC-MSC-Exo on K562 cells could be reversed by pretreatment of K562 cells with caspase inhibitor Z-VAD-FMK (30?µmol/L)Discussion
These results indicate that hUC-MSC-Exo enhanced the sensitivity of K562 cells to IM via activation of caspase signaling pathway. Therefore, combining IM with hUC-MSC-Exo could be a promising approach to improve the efficacy of CML treatment. 相似文献7.
McCallum L Lu W Price S Lazar N Perbal B Irvine AE 《Journal of cell communication and signaling》2012,6(1):27-35
CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia
(CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role
for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 106 copies per 50 ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty
vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within
the subG0 phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1 μM and 5 μM) showed an increase in events within the subG0 phase of cell cycle over 96 h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells
treated with recombinant human Ccn3 (10 nM) in combination with Imatinib (5 μM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control)
which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase
in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib
induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies. 相似文献
8.
Yueh-Hsin Ping Chia-ying Chu Hong Cao Jean-Marc Jacque Mario Stevenson Tariq M Rana 《Retrovirology》2004,1(1):1-13
Background
Dendritic cell (DC) transmission of human immunodeficiency virus (HIV) to CD4+ T cells occurs across a point of cell-cell contact referred to as the infectious synapse. The relationship between the infectious synapse and the classically defined immunological synapse is not currently understood. We have recently demonstrated that human B cells expressing exogenous DC-SIGN, DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin, efficiently transmit captured HIV type 1 (HIV-1) to CD4+ T cells. K562, another human cell line of hematopoietic origin that has been extensively used in functional analyses of DC-SIGN and related molecules, lacks the principal molecules involved in the formation of immunological synaptic junctions, namely major histocompatibility complex (MHC) class II molecules and leukocyte function-associated antigen-1 (LFA-1). We thus examined whether K562 erythroleukemic cells could recapitulate efficient DC-SIGN-mediated HIV-1 transmission (DMHT).Results
Here we demonstrate that DMHT requires cell-cell contact. Despite similar expression of functional DC-SIGN, K562/DC-SIGN cells were inefficient in the transmission of HIV-1 to CD4+ T cells when compared with Raji/DC-SIGN cells. Expression of MHC class II molecules or LFA-1 on K562/DC-SIGN cells was insufficient to rescue HIV-1 transmission efficiency. Strikingly, we observed that co-culture of K562 cells with Raji/DC-SIGN cells impaired DMHT to CD4+ T cells. The K562 cell inhibition of transmission was not directly exerted on the CD4+ T cell targets and required contact between K562 and Raji/DC-SIGN cells.Conclusions
DMHT is cell type dependent and requires cell-cell contact. We also find that the cellular milieu can negatively regulate DC-SIGN transmission of HIV-1 in trans. 相似文献9.
Background
This study exploits the speed benefits of echo-planar spectroscopic imaging (EPSI) to acquire lipid spectra of skeletal muscle. The main purpose was to develop a high-resolution EPSI technique for clinical MR scanner, to visualise the bulk magnetic susceptibility (BMS) shifts of extra-myocellular lipid (EMCL) spectral lines, and to investigate the feasibility of this method for the assessment of intra-myocellular (IMCL) lipids.Methods
The study group consisted of six healthy volunteers. A two dimensional EPSI sequence with point-resolved spectroscopy (PRESS) spatial localization was implemented on a 3T clinical MR scanner. Measurements were performed by means of 64×64 spatial matrix and nominal voxel size 3×3×15 mm3. The total net measurement time was 3 min 12 sec for non-water-suppressed (1 acquisition) and 12 min 48 sec for water-suppressed scans (4 acquisitions).Results
Spectra of the human calf had a very good signal-to-noise ratio and linewidths sufficient to differentiate IMCL resonances from EMCL. The use of a large spatial matrix reduces inter-voxel signal contamination of the strong EMCL signals. Small voxels enabled visualisation of the methylene EMCL spectral line splitting and their BMS shifts up to 0.5 ppm relative to the correspondent IMCL line. The mean soleus muscle IMCL content of our six volunteers was 0.30±0.10 vol% (range 0.18–0.46) or 3.6±1.2 mmol/kg wet weight (range: 2.1–5.4).Conclusion
This study demonstrates that high-spatial resolution PRESS EPSI of the muscle lipids is feasible on standard clinical scanners. 相似文献10.
The relative intensities of the CH stretching vibrations are used to study the interaction of lecithin liposomes with valinomycin, a mobile carrier for alkali ions. In the case of dipalmitoyl lecithin liposomes, the lipid phase transition is not significantly affected by valinomycin. However, in dimyristoylphosphatidylcholine liposomes, the phase transition is broadened by the addition of 1 mol% valinomycin even at low K+ concentrations. This indicates that the carrier interacts with the hydrophobic core of the bilayer. In addition, these experiments showed that the lipid phase transitions which are reflected by the methylene groups and the terminal methyl groups are nearly equivalent. Therefore a reevaluation of the assignment of the CH stretching bands seemed necessary. Our Raman spectroscopic investigation of ω-deuterated dipalmitoyl lecithin liposomes improves the assignment of CH stretch vibrations to methylene and methyl groups. The deuteration displaces the methyl group vibrations to the 2050–2250 cm?1 region and produces gross intensity changes of the bands at 2883 and 2936 cm?1. These changes lead to the conclusion that both bands arise from vibrations which can be attributed simultaneously to the methylene and methyl groups of the fatty acid chains. The displacement of the CH3 group vibrations from their original positions enhances the intensity ratios (per centimeter), and , for the CH2- groups which are used to monitor the lipid phase transition, and implies that the contributions of the CH3 groups to the phase transition curves are unimportant. Our finding that the -CD3 groups reflect no phase transition supports this statement. 相似文献
11.
Iura M. Alves Lucas S. Abreu Cinara O. S. Costa Mireille Le Hyaric Maria Lenise S. Guedes Milena B. P. Soares Daniel P. Bezerra Eudes S. Velozo 《化学与生物多样性》2017,14(3)
One new chromone 3,3‐dimethylallylspatheliachromene methyl ether ( 1 ), as well as five known chromones, 6‐(3‐methylbut‐2‐enyl) allopteroxylin methyl ether ( 2 ), 6‐(3‐methylbut‐2‐enyl) allopteroxylin ( 3 ), 3,3‐dimethylallylspatheliachromene ( 4 ), 5‐O‐methylcneorumchromone K ( 5 ) and spatheliabischromene ( 6 ), two alkaloids, 8‐methoxy‐N‐methylflindersine ( 7 ) and 8‐methoxyflindersine ( 8 ), and two limonoids, limonin diosphenol ( 9 ) and rutaevin ( 10 ), were isolated from Dictyoloma vandellianum A. Juss (Rutaceae). Cytotoxic activities towards tumor cell lines B16‐F10, HepG2, K562 and HL60 and non‐tumor cells PBMC were evaluated for compounds 1 – 6 . Compound 1 was the most active showing IC50 values ranging from 6.26 to 14.82 μg/ml in B16‐F10 and K562 cell lines, respectively, and presented IC50 value of 11.65 μg/ml in PBMC cell line. 相似文献
12.
Wenhui Zhang Yuqing Chen Xiaohang Pei Yuzhu Zang Shuangyin Han 《Saudi Journal of Biological Sciences》2018,25(2):242-247
Objective
To investigate the role of DR4 gene in the occurrence, development and prognosis of acute myeloid leukemia (AML), find a new regulatory gene of Decitabine for the treatment of AML, namely DR4 gene, and explore the molecular mechanism of AML in the treatment of AML.Methods
The methylation level and the mRNA expression level of DR4 gene promoters of bone marrow mononuclear cells in 122 patients with newly diagnosed AML and 24 patients with iron deficiency anemia (IDA) were detected using Methylation specific PCR (MS-PCR) and Q-RT-PCR, respectively, and a correlation analysis of them was conducted. The effects of Decitabine on the proliferation of K562 cells were detected using CCK-8 assay. Then, the effects of Decitabine on the methylation level and the mRNA expression level of DR4 genes of K562 cells treated with Decitabine were detected using MS-PCR and Q-RT-PCR, respectively. The effects of Decitabine on the cell cycle and apoptosis of K562 cells were detected using flow cytometry.Results
Compared with the control group, the methylation level (P?=?.002) of DR4 genes of bone marrow mononuclear cells in patients with newly diagnosed AML was high. The methylation level (P?=?.01) of DR4 genes of bone marrow mononuclear cells in patients of the positive group of enlargement of liver, spleen and lymph node was lower than that of the negative group, and the methylation level (P?=?.006) of DR4 genes in patients of the high risk group of clinical stage was lower than that of the low risk group, and the methylation level (P?=?.03) of DR4 genes in patients of the group where patients did not achieve complete remission (CR1) after a course of induction chemotherapy was lower than that of the group where patients achieved complete remission (CR1) after a course of induction chemotherapy. There was a significant negative correlation (P?<?.01) between the methylation level and the mRNA expression level of DR4 genes of bone marrow mononuclear cells in 122 patients with newly diagnosed AML. After the K562 cells were treated with Decitabine for 48?h, the methylation level of DR4 gene promoters gradually decreased, while the mRNA expression level of DR4 genes gradually increased, both of which showed a concentration-dependent relationship. After the K562 cells were treated with 5?µmol/L Decitabine for 48?h, the K562 cells in G0/G1 phase and G2/M phase increased significantly, and the K562 cells in S phase decreased significantly.Conclusion
DR4 gene played an important role in the occurrence and development of AML. Decitabine can effectively inhibit the proliferation of K562 cells, which probably partly because it can terminate the methylation effect of DR4 gene promoters and restore the mRNA expression of DR4 genes. 相似文献13.
《Saudi Journal of Biological Sciences》2020,27(8):2038-2046
Pennate diatom Nitzschia palea can be cultured in outdoor vertical-bed photobioreactors to produce biodiesel. To assess the production of biomass and lipids, non-axenic cultures of Nitzschia palea were grown outdoors, and the growth of these cultures was measured biweekly. During the annual cycle of algal culture, the culture temperature ranged from 17.3 °C to 33.5 °C, the dry weight biomass ranged from 0.11 g l−1 to 0.25 g l−1, light energy] ranged from 1.94 Wm−2 to 3.9 Wm−2 and intracellular lipid content ranged from 7.1% to 11.4% of biomass weight after drying at 60 °C. Gas chromatography/mass spectroscopy (GC/MS) analysis of n-hexane extracts showed that the intracellular lipids were primarily C14:0 myristic acid (9.01%), C15:0 pentadecyclic acid (8.26%) and two types of C16:0, palmitic acid (41.13%) and palmitoleic acid (29.25%). Gel permeation analysis showed that carboxylic acids comprised 28.9% of lipids, 16.3% of monoglycerides, 27.3% of diglycerides and 24.3% of triglycerides. Alcoholysis of lipids resulted in the conversion of about 93.9% of fatty acids to equivalent fatty acid methyl esters (FAME) or biodiesel, which, on basis of wt%, consisted primarily of C15:0 methyl myristate (8.3%), C16:0 methyl pentadecanoate] (7.2%), C17:1methyl palmitoleate (28.7%) and methyl palimtate](39.8%). 相似文献
14.
Aarthi Jayanthan Delphine Bernoux Pinaki Bose Karl Riabowol Aru Narendran 《Cancer cell international》2011,11(1):1-11
Background
Secondary therapy-related acute lymphoblastic leukemia might emerge following chemotherapy and/or radiotherapy for primary malignancies. Therefore, other alternatives should be pursued to treat leukemia.Results
It is shown that vitamin K3- or vitamin C- induced apoptosis in leukemia cells by oxidative stress mechanism involving superoxide anion radical and hydrogen peroxide generation, activation of NF-κB, p53, c-Jun, protease caspase-3 activation and mitochondria depolarization leading to nuclei fragmentation. Cell death was more prominent when Jurkat and K562 cells are exposed to VC and VK3 in a ratio 1000:1 (10 mM: 10 μM) or 100:1 (300 μM: 3 μM), respectively.Conclusion
We provide for the first time in vitro evidence supporting a causative role for oxidative stress in VK3- and VC-induced apoptosis in Jurkat and K562 cells in a domino-like mechanism. Altogether these data suggest that VK3 and VC should be useful in the treatment of leukemia. 相似文献15.
The effects of cadmium (Cd) stress on lipid composition and biosynthesis were investigated in young leaves of ten-day-old
tomato seedlings (Lycopersicon esculentum Mill. cv. Ibiza F1). Cd was found to be mainly accumulated in roots, but a severe inhibition of biomass production occurred
in leaves, even at its low concentration (1.0 μM). Seven days after Cd treatment, the membrane lipids were extracted and separated
on silica-gel thin layer chromatography (TLC). Fatty acid methyl esters were analyzed by FID-GC on a capillary column. Our
results showed that Cd stress decreased the quantities of all lipids classes (phospholipids, galactolipids and neutral lipids).
Likewise, there was also a decline in the levels of tri-unsaturated fatty acids, such as linolenic (C18:3) and hexadecatrienoic
(C16:3) acids. The linolenic acid (C18:3) decreased in monogalactosyldiacylglycerol (MGDG) and all phospholipids, while hexadecatrienoinic
acid (C16:3) declined mainly in MGDG. Moreover, Cd at high concentrations (25.0 and 50.0 μM) significantly enhanced the levels
of lipid peroxides. Radiolabelling experiments were carried out by laying down microdroplets of [1-14C]acetate–a major precursor of lipid biosynthesis–on attached leaves of the control and Cd-treated plants. After incubation
for 1, 2, 12 and 24 h, the leaves were harvested and lipids extracted and analysed. Cd stress was found to decrease the incorporation
of [1-14C]acetate in total lipids. The biosynthesis of total lipids was altered with 25.0 and 50.0 μM Cd. The decline in the incorporation
of [1-14C]acetate due to Cd stress was observed in all lipid classes. There was also a substantial decline in the incorporation of
[1-14C]acetate in tri-unsaturated fatty acids. The results indicate that Cd treatment induces an oxidative stress by inhibiting
the chloroplastic and extrachloroplastic lipid-biosynthesis pathways as well as lipid peroxidation. 相似文献
16.
Susanne B. Breitkopf Stéphane J. H. Ricoult Min Yuan Ying Xu David A. Peake Brendan D. Manning John M. Asara 《Metabolomics : Official journal of the Metabolomic Society》2017,13(3):30
Introduction
Advances in high-resolution mass spectrometry have created renewed interest for studying global lipid biochemistry in disease and biological systems.Objectives
Here, we present an untargeted 30 min. LC-MS/MS platform that utilizes positive/negative polarity switching to perform unbiased data dependent acquisitions (DDA) via higher energy collisional dissociation (HCD) fragmentation to profile more than 1000–1500 lipid ions mainly from methyl-tert-butyl ether (MTBE) or chloroform:methanol extractions.Methods
The platform uses C18 reversed-phase chromatography coupled to a hybrid QExactive Plus/HF Orbitrap mass spectrometer and the entire procedure takes?~10 h from lipid extraction to identification/quantification for a data set containing 12 samples (~4 h for a single sample). Lipids are identified by both accurate precursor ion mass and fragmentation features and quantified using LipidSearch and Elements software.Results
Using this approach, we are able to profile intact lipid ions from up to 18 different main lipid classes and 66 subclasses. We show several studies from different biological sources, including cultured cancer cells, resected tissues from mice such as lung and breast tumors and biological fluids such as plasma and urine.Conclusions
Using mouse embryonic fibroblasts, we showed that TSC2?/? KD significantly abrogates lipid biosynthesis and that rapamycin can rescue triglyceride (TG) lipids and we show that SREBP?/? shuts down lipid biosynthesis significantly via mTORC1 signaling pathways. We show that in mouse EGFR driven lung tumors, a large number of TGs and phosphatidylmethanol (PMe) lipids are elevated while some phospholipids (PLs) show some of the largest decrease in lipid levels from ~?2000 identified lipid ions. In addition, we identified more than 1500 unique lipid species from human blood plasma.17.
Characterization of blood mononuclear cells reacting with K 562 cells after yellow fever vaccination
At various times after yellow fever (17 D) vaccination mononuclear blood cells were tested for cytotoxic potential in conjugate with K 562. They were characterized with monoclonal reagents Leu2a, Leu4, and OKM1, using indirect immunofluorescent technique. The cytotoxicity against K 562 increased after vaccination with a peak on Days 8–12 when the [3H]thymidine uptake was also maximal. The majority of cells in conjugates with K 562 were then T cells. Later the conjugates contained more OKM1-positive cells. OKM1 reagent was found to react with K 562 and thereby cause a coagglutination between this target cell and the OKM1-positive monocytic cells. The surface moiety reacting with OKM1 reagent might be of lipid nature. 相似文献
18.
Alberto Canfrán-Duque Oscar Pastor Manuel Reina Milagros Lerma Alfonso J. Cruz-Jentoft Miguel A. Lasunción Rebeca Busto 《PloS one》2015,10(10)
Scope
First- and second-generation antipsychotics (FGAs and SGAs, respectively), both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.Methods
HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.Results
Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.Conclusion
Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids) accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials. 相似文献19.
Sparks LM Moro C Ukropcova B Bajpeyi S Civitarese AE Hulver MW Thoresen GH Rustan AC Smith SR 《PloS one》2011,6(7):e21068
Objective
Disturbances in lipid metabolism are strongly associated with insulin resistance and type 2 diabetes (T2D). We hypothesized that activation of cAMP/PKA and calcium signaling pathways in cultured human myotubes would provide further insight into regulation of lipid storage, lipolysis, lipid oxidation and insulin responsiveness.Methods
Human myoblasts were isolated from vastus lateralis, purified, cultured and differentiated into myotubes. All cells were incubated with palmitate during differentiation. Treatment cells were pulsed 1 hour each day with forskolin and ionomycin (PFI) during the final 3 days of differentiation to activate the cAMP/PKA and calcium signaling pathways. Control cells were not pulsed (control). Mitochondrial content, 14C lipid oxidation and storage were measured, as well as lipolysis and insulin-stimulated glycogen storage. Myotubes were stained for lipids and gene expression measured.Results
PFI increased oxidation of oleate and palmitate to CO2 (p<0.001), isoproterenol-stimulated lipolysis (p = 0.01), triacylglycerol (TAG) storage (p<0.05) and mitochondrial DNA copy number (p = 0.01) and related enzyme activities. Candidate gene and microarray analysis revealed increased expression of genes involved in lipolysis, TAG synthesis and mitochondrial biogenesis. PFI increased the organization of lipid droplets along the myofibrillar apparatus. These changes in lipid metabolism were associated with an increase in insulin-mediated glycogen storage (p<0.001).Conclusions
Activation of cAMP/PKA and calcium signaling pathways in myotubes induces a remodeling of lipid droplets and functional changes in lipid metabolism. These results provide a novel pharmacological approach to promote lipid metabolism and improve insulin responsiveness in myotubes, which may be of therapeutic importance for obesity and type 2 diabetes. 相似文献20.
Jiajiao Zhang Yi Li Jianping Guo Bo Du Guangcun He Yingjun Zhang Rongzhi Chen Jiaru Li 《Metabolomics : Official journal of the Metabolomic Society》2018,14(9):120