Oxysterols in cultured bovine aortic smoth muscle cells and in the monocyte-like cell line U937

Oxysterols in cultured bovine aortic smooth muscle cells and in the monocyte-like cell line U937 were analyzed by gas-liquid chromatography. The following products were detected: 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, cholesterol-α-epoxide, cholesterol-β-epoxide, cholestanetriol, and 20- and 25-hydroxycholesterol. The proportions of oxidized cholesterol varied from 1 to 3% of total cholesterol. The concentrations of oxysterols were lower when the smooth muscle cells were maintained in a vitamin E-enriched medium, and were higher in U937 cells when the cells were activated with phorbol myristate acetate. The cell oxysterol content appears to be regulated by factors that inhibit or enhanced free radical generation. The concentrations of oxysterols found in cells may serve as an indicator of the extent of lipid peroxidation.     

舌鳞状细胞癌永生化细胞系细胞的侵袭性

目的比较从转移灶中富集出的舌鳞状细胞癌永生化细胞系Tca8113-Ml细胞与原发灶组织获得的Tca8113细胞系细胞侵袭能力的差异,探讨舌鳞状细胞癌永生化细胞系细胞侵袭特性与转移肿瘤干细胞(migrating cancer stem cell,MCSC)的相关性。方法采用Transwell小室侵袭试验和BALB/c裸鼠移植瘤试验分析Tca8113和Tca8113-Ml细胞的侵袭能力。结果 Tca8113-Ml组的穿膜细胞数(93.8±4.4)明显多于Tca8113组(34.0±1.6),且差异有统计学意义(P0.01);Tca8113-Ml组裸鼠更易形成移植瘤,且移植瘤形成较快,生长速度也较快,成瘤率为6/6,而Tca8113组成瘤率为4/6。结论舌鳞状细胞癌永生化细胞系Tca8113-Ml细胞的侵袭能力较强,表明从转移灶中富集出的细胞系细胞具有明显的干性,提示肿瘤转移与MCSC之间存在相关性。     

Gangliosides from rat cerebellum: Demonstration of considerable heterogeneity using a new solvent for thin layer chromatography

Using a new solvent (methyl acetate/n-propanol/chloroform/methanol/0.25% aqueous KCl, 25∶20∶20∶20∶17, v/v) and high performance silica gel thin layer chromatographic plates, all common gangliosides found in brain can be easily separated with 1 hr. This system is reproducible and “tailing” is negligible compared with previous solvents. When such a system is applied to separate the gangliosides found during the development of the rat cerebellum, a considerable heterogeneity is observed. Data are presented (using rechromatography experiments, fractionation on DEAE-Sephadex, treatment with neuraminidase or alkaline medium and carbohydrate analysis) suggesting that the complex profiles obtained with this chromatographic system are not due to chromatographic artifacts but result from the high resolving power of the system. After separation by ion-exchange chromatography, 28 gangliosides can be detected.     

Incorporation and metabolism of fatty acids by cultured dissociated cells from rat cerebrum

Dissociated brain cells in culture incorporate a variety of saturated and unsaturated fatty acids into their cellular lipids. Of the various fatty acids studied, uptake of radioactivity was greatest for stearic acid and decreased progressively with decreasing chain length. Incorporation of radioactivity from linoliec and linolenic acids was more extensive than from oleic acid. Cellular phospholipids and triacylglycerols were labeled preferentially from all fatty acid precursors, with the relative amount of label in phospholipids being greatest when cells were incubated with linolenic acid. Fatty acids underwent desaturation and chain elongation. Changes in the labeling pattern of phospholipid fatty acids in the course of incubation demonstrated precursor-product relationships for laurate (12∶0), myristate (14∶0), palmitate (16∶0), and stearate (18∶0) and for linolenate (18∶3), eicosapentaenoate (20∶5), docosapentaenoate (22∶5), and docosahexaenoate (22∶6). The appearance of label in 22∶5 and 22∶6 paralleled the entrance of label into the ethanolamine phosphoglyceride fraction. Conversion of linoleate (18∶2 ω6) to arachidonate (20∶4 ω6) could be demonstrated but did not proceed via 18∶3 ω6.     

Fatty acids and phospholipids of adult and newborn rat hearts and of cultured,beating neonatal rat myocardial cells

Fatty acids and phospholipids of adult and newborn rat hearts and of cultured, neonatal rat heart cells were determined by gas liquid and thin layer chromatographies. In adult heart, the proportion of linoleic acid was higher and that of palmitic acid lower than in newborn hearts or in cultured cells. The relative amounts of linoleic and arachidonic acids in adult heart were affected by the source and amount of dietary fat. In heart cells, after 3 days in culture, the proportion of arachidonic acid resembled that in the newborn and adult rat hearts but showed a gradual and significant decline with age. The gradual shift in fatty acid composition as the cells aged in culture was attributed to outgrowth of mesenchymal cells (fibroblasts and endothelioid cells) characterized by a low relative proportion of arachidonic acid. The amounts of phospholipids in heart cells after 3 days in culture differed from those in the newborn or adult rat hearts. Phosphatidylethanolamine was highest in adult heart (34% of lipid phosphorus) and lowest in cells (26%); lecithin was higher in newborn heart (43%) than in adult heart (37%) or in cells (39%), while sphingomyelin was higher in cells (8%) than in newborn (5%) or adult heart (3%). Phospholipid levels in cultured heart cells were unrelated to those of serum in the growth medium. The absence of a significant change in phospholipid composition after continued incubation of the heart cell cultures for periods up to 3 weeks reflected the major structural role of these lipid components in cell membranes.     

大鼠结缔组织生长因子基因miRNA表达质粒的构建及其稳定转染大鼠肝星状细胞系的建立

目的构建大鼠结缔组织生长因子(Connective tissue growth factor,CTGF)基因miRNA表达质粒,并建立稳定转染大鼠肝星状细胞(Hepatic stellate cell,HSC)系。方法根据大鼠CTGF基因mRNA序列,设计并合成3对寡聚单链DNA X191-1、X191-2和X191-3及1对阴性对照序列DNA X191-4,将4对寡聚单链DNA退火成双链后,分别与载体pcDNA6.2-GW/EmGFP-miR连接,构建CTGF基因miRNA重组表达质粒,分别转染HSC-T6细胞,荧光显微镜观察细胞的转染效率,RT-PCR检测转染细胞中CTGF基因mRNA的转录水平;取干扰效率最高的重组质粒及阴性对照质粒,分别转染HSC-T6细胞,经杀稻瘟菌素持续加压筛选。结果经测序鉴定,重组表达质粒构建正确,插入片段的碱基序列与设计相符;细胞的瞬时转染效率约为50%;3组干扰质粒转染的HSC-T6细胞中,CTGF基因mRNA的转录水平明显低于空白对照组(P<0.01),其中X191-2质粒对CTGF基因转录的干扰效率最高;获得了稳定转染的HSC-T6细胞。结论成功构建了CTGF基因miRNA表达质粒,并获得了稳定转染的肝星状细胞系,为进一步研究肝纤维化的形成机制及其治疗奠定了基础。     

Phospholipid composition of cultured human endothelial cells

Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay.     

Control of lipid metabolism in cultured cells

Several studies are presented which indicate that composition of cell lipid is regulated by interaction between intracellular metabolism and lipid transport processes. When the fatty acid composition of cells cultured in essential fatty acid deficient conditions was studied, activation of synthesis of unusual polyun-saturated fatty acids was observed for a number of cell lines. In addition cells contained persistent residual amounts of linoleic acid, presumably owing to efficient scavenging mechanisms. The source of cell lipids was studied in both chemically defined and serum-supplemented media. In the absence of exogenous lipid, cells synthesize lipids from simple precursors, a process which is inhibited by adding serum. When serum lipid is present, cells preferentially utilize fatty acids as a source of nonsterol lipid. These are subsequently esterified intracellularly to make glycerides and phospholipids. When triglyceride is utilized as a source of cell lipid, it is first hydrolyzed before being taken up. By use of a nonhydrolyzable cholesterol ester analog, it is confirmed that both free and ester cholesterol are taken up and excreted by cells. Intracellular cholesterol content is thus regulated by rates of uptake, hydrolysis and excretion as well as by biosynthesis. One of 13 papers presented at the symposium “Lipid Metabolism in Cells in Culture,” AOCS Meeting, Houston, May 1971.     

AgNPs loaded microemulsion using gallic acid inhibits MCF-7 breast cancer cell line and solid ehrlich carcinoma

Abstract

The objective of this present work is to optimize and prepare silver nanoparticles(AgNPs) in Dioctyl sodium sulfosuccinate (AOT) microemulsion (ME) for oral use and to investigate its antibacterial and anticancer activity in vitro and in vivo. In vitro drug release study confirmed that faster release of drug at the tumor cells compared to the blood circulation. It also showed a potential antibacterial activity against pathogenic bacteria. The optimized AgNPs loaded ME confirmed significant cytotoxicity against MCF-7 cancer cell line with IC50 16.72?±?0.014?μg/mL and significant reduction in solid Ehrlich tumor growth in compared to the control, placebo and pure drug.     

Lipids of cultured hepatoma cells: I. Effect of serum lipid levels on cell and media lipids

Minimal deviation hepatoma cells were cultured as monolayers to confluency in roller flasks containing modified Swim's medium, supplemented with decreasing amounts of serum, lipid-free serum, and lipid-free serum containing added fatty acids. Good cell growth was observed until serum levels fell below 5% of the medium. Media containing lipid-free serum or lipid-free serum plus linoleic or palmitic acids did not support good growth. Lipids were extracted from cells; media, obtained during the first and last half of the incubation period, resolved into neutral and phospholipid fractions; fatty acid composition of each fraction analyzed by gas liquid chromatography; and lipid class distributions compared by thin layer chromatography. The data showed that the media contained more neutral lipids and phospholipids after incubation than initially, indicating that minimal deviation hepatoma cells excreted lipids into the media. The class composition of the excreted lipids resembled that of the serum. A comparison of media, cells, and serum fatty acid compositions indicated that the lipids secreted into the media were of cellular origin. Although some differences were noted, in general, cells grown on the nine different media had the same ca. neutral lipid and phospholipid class and fatty acid compositions. In contrast, dramatic differences were observed in the class and fatty acid compositions of the serums from that of the cells and media. These results indicate that exogenous serum lipids had little influence on cellular class and fatty acid compositions of the minimal deviation hepatoma cells. This neoplasm did not contain detectable levels of glyceryl ether diesters, indicating that this compound is not characteristic of all tumors. Lipid class profiles and fatty acid compositions of cells grown on various media suggest that the minimal deviation hepatoma cells can synthesize most, if not all, neutral lipid and phosphoglyceride classes found in liver. Presented at the AOCS Meeting, New Orleans, April 1973.     

Cholesterol ester hydrolase activity in mammary tissue of the lactating rat

Cholesterol ester hydrolase activity has been studied in mammary glands of rats. Subcellular fractionation of the glands obtained in mid-lactation indicated that around 80% of the recovered activity was associated with particulate fractions. Two distinct cholesterol ester hydrolase activities were identified, one with an optimum pH of 7.5–9.0 and the second (approximately 5% of the total activity) with a more acidic pH optimum. Although the neutral cholesterol ester hydrolase had some properties in common with the lipoprotein lipase in mammary tissue, it was shown to be a separate entity by several criteria. Its activity could be increased following treatment with Mg-ATP and cAMP-dependent protein kinase, suggesting identity with the hormone sensitive lipase of adipose tissue. The cholesterol ester hydrolase activity in mammary glands just after parturition was greater than in glands obtained either from late-pregnant or midlactating animals. The subcellular distribution of the neutral cholesterol ester hydrolase suggested that it may have a different function to the neutral cholesterol ester hydrolase of adrenals and other tissues. Nevertheless the fact that the activity of the enzyme can be modulated by cAMP-dependent protein kinase suggests the possibility that hormonal control of this enzyme may be involved in the regulation of cholesterol metabolism in the mammary gland.     

The immunosuppressive substance 2-chloro-2-deoxyadenosine modulates lipoprotein metabolism in a murine macrophage cell line (P388 cells)

A recently developed immunosuppressive substance, 2-chloro-2-deoxyadenosine (2-CdA), was reported to inhibit monocyte functions at low concentration. Because macrophages play a key role in the formation of atherosclerotic plaques, it was of interest to study the effect of 2-CdA on cellular lipid metabolism. For this purpose we have used a macrophage cell line (P388) to perform incubation studies in the presence of acetylated low density lipoprotein (Ac-LDL) and 2-CdA. The addition of 2-CdA, in concentrations ranging from 5–20 nM, induced a dose-dependent decrease in cellular cholesterol content and in the amount of extracellular [¹⁴C]oleic acid (OA) incorporated into the cholesteryl ester (CE) fraction. The effect was maximized at 20 nM 2-CdA with an 86% reduction in cholesterol esterification compared to controls (P<0.008). To evaluate the mechanism of interaction of 2-CdA with cellular lipid metabolism, deoxycytidine (dCyt) and 3-methoxybenzamide (3-MOB), substances known to antagonize the effect of 2-CdA in different ways, were co-administered with 2-CdA. dCyt, a competitive inhibitor of dCyt kinase, which catalyzes phosphorylation to the active metabolite, antagonized the effects of 20 nM 2-CdA, producing significantly greater incorporation of extracellular [¹⁴C]OA into the CE fraction than in the presence of 2-CdA alone (P<0.0086). Co-incubation with 2-CdA and the poly-ADP-ribose synthetase inhibitor 3-MOB, which is known to render cells resistant to 2-CdA toxicity by preventing cellular nicotinamide adenine dinucleotide (NAD)- and adenosine triphosphase-depletion, also reversed the effect of 2-CdA on lipid accumulation. However, incubation of P388 cells with 20 nM 2-CdA did not result in a decrease in cellular NAD content. As 20 nM 2-CdA showed no effect on intracellular cholesterol synthesis based on measurement by 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, the decrease in cellular cholesterol content and in [¹⁴C]OA incorporation seems to be primarily due to an interference with Ac-LDL metabolism.     

The absorption of fatty acids by functional bovine mammary cells

Freshly dispersed bovine mammary cells rapidly absorbed long chain fatty acids from the culture medium. Differences in the rates of absorption were observed, i.e., palmitic > stearic > oleic > myristic > linoleic acid. The preponderance of the fatty acids absorbed were esterified into triglycerides (>75%) and the remainder were mostly incorporated into phospholipids. The cells secreted triglycerides into the culture medium. Of the phospholipid classes, phosphatidylcholine always contained most of the radioactivity in all experiments with labeled fatty acids. These observations are related to the metabolism of mammary cells in vivo.     

The influence of dietary medium chain triglycerides on rat mammary tumor development

The N-nitrosomethylurea rat mammary tumor model was used to compare the tumor-promoting effects of a highfat (HF) diet containing a 3:1 mixture of medium chain triglycerides (MCT) and corn oil with that of a HF and a low-fat (LF) corn oil diet. The serum and tumor lipid content and fatty acid (FA) composition were also determined in the three dietary groups. It was found that the MCT-containing diet failed to promote tumor development compared with the HF corn oil group. Tumor incidence in the HF-MCT group was similar to that of the LF corn oil group (5% fat, w/w), but significantly decreased compared to the HF corn oil group. Total serum cholesterol levels were significantly depressed in the HF corn oil group compared to the HF-MCT and LF corn oil groups. Analysis of serum and tumor FA profiles indicated that the HF corn oil group exhibited approximately twice the amount of linoleic acid (LA) as the other two treatment groups. Differences among the three groups in the major FA metabolite of LA, arachidonic acid, were minimal. These results are consistent with the hypothesis that tumor promotion by dietary fat is more a function of the type than the amount of fat ingested. In addition, they indicate that MCT, due at least in part to their unique structural and physiological properties, exert markedly different effects on mammary tumor development than conventional long chain unsaturated fatty acids. Presented at the symposium on “Specialty Lipids and Their Biofunctionality” at the annual meeting of the American Oil Chemists' Society, Philadelphia, May 1985.     

Effects of dietary fish oil on human mammary carcinoma and on lipid-metabolizing enzymes

The growth rate of a human mammary carcinoma, MX-1, was significantly reduced in athymic “nude” mice fed fish oil. Tumors from the fish oil-fed animals also showed a greater sensitivity to two anti-neoplastic agents, mitomycin C and doxorubicin. Mitochondria were isolated from control livers, host livers and tumors from fish oil-and corn oil-fed animals, and increased levels of 20∶5n−3 and 22∶6n−3 were found in mitochondrial lipids in all three tissues from the fish oil-fed animals. To investigate the effect of dietary n−3 fatty acids on lipid metabolism, the activity of the acyl-CoA:carnitine acyltransferase and three acyl-CoA desaturases were measured. Carnitine acyltransferase activity toward all four acyl-CoA substrates tested was markedly increased in mitochondria from liver by feeding fish oil. In mitochondria from tumors, feeding fish oil resulted in an increased activity toward only 18∶3n−3. These data suggest that fish oil may induce an increase in the oxidation of fatty acids. The Δ⁹-desaturase activity was decreased in microsomes from liver and tumor from fish oil-fed animals. However, both the Δ⁶ and Δ⁵ desaturases were increased in tumor and in control liver as a result of feeding fish oil. The Δ⁵ desaturase was not altered in microsomes from the host animals. The effect of fish oil on the Δ⁵ and Δ⁶ desaturases may involve alterations to metabolism of specific polyunsaturated fatty acids especially in the tumor tissue.     

Uptake and oxidation of malonaldehyde by cultured mammalian cells

Primary cultures of rat skin fibroblasts were used as a model system to investigate the cellular uptake and oxidation of malonaldehyde (MA). The cells were grown in a medium containing 10⁻⁵ M, 10⁻⁴ M or 10⁻³ M concentrations of [1,3-¹⁴C]MA. There was a limited, concentration-dependent uptake of MA by 24 hr (∼4% at all concentrations). The uptake of [1,2-¹⁴C]acetate by 24 hr was ∼24%; 83–89% of the¹⁴C in the MA taken up was oxidized to¹⁴CO₂ by 24 hr and ∼5% was recovered in the major lipids. Despite its low uptake and rapid oxidation to CO₂, pretreatment of the cells with 10⁻³ M MA for 24 hr produced a latent inhibition of [¹⁴C]glucose oxidation. Limited cellular uptake of MA may explain the tolerance of cells grown in culture to relatively high MA concentrations.     

Growth characteristics of a Bombyx mori insect cell line in stationary and suspension cultures

The growth characteristics of the BM-5 insect cell line of Bombyx mori (silkworm) have been experimentally investigated in order to develop optimal growth protocols when these cells are used to produce large quantities of biopesticides or human proteins by recombinant baculoviruses. Experiments were performed in 2 mL wells and 200 mL spinner flasks. Spinner flasks were operated at 80 rpm with 0.3% methyl cellulose (MCL) added to the medium in order to protect the cells from liquid shear stress. In addition to the effect of agitation rate and amount of MCL added to the medium, the cell response during the adaptation to growth in suspension from stationary cultures is reported. Exposure of the cells to varying nutrient and metabolite concentrations is accomplished through batch and repeated-batch modes in 2 mL wells. The results imply that glutamine is a limiting nutrient and lactate has an inhibitory effect on cell growth. Ammonia depletion from the medium was accompanied by uric acid accumulation, suggesting that ammonia is converted to this metabolic product by the “uricotelic” and “nucleicolytic” metabolic pathways.     

Microsomal phosphatidic acid phosphohydrolase of rat mammary tissue: I. General properties

The microsomal bound phosphatidic acid phosphohydrolase from lactating rat mammary tissue had a specific activity of six nmoles per mg protein per minute. The optimum pH was 7.0; magnesium at 1.3 mM was required for maximum activity, and at low substrate concentrations magnesium lowered the Km of the enzyme for phosphatidic acid. Diglycerides exerted little effect while diglyceride ether stimulated enzyme activity. Inorganic salts, i.e., potassium phosphate and potassium chloride, enhanced rates of phosphatidic acid hydrolysis under standard assay conditions.     

Effect of phosphatidylcholine structure on the adenylate cyclase activity of a murine fibroblast cell line

To determine which structural characteristics of membrane phospholipids influence adenylate cyclase activity, we measured basal and sodium fluoride- or forskolin-stimulated activity in a murine fibroblast cell line,i.e., Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid-depleted FCS (LD-FCS) or LD-FCS complexed with different phosphatidylcholine (PC) molecular species. Cells grown in the presence of LD-FCS showed a substantial decrease in their basal and NaF-stimulated adenylate cyclase activities; however, their forskolin-stimulated activity was not altered, suggesting that the enzyme's catalytic site is not affected by changes in membrane lipids. Media supplemented with different LD-FCS/PC complexes were shown to prevent the LD-FCS-mediated reduction of basal and NaF-stimulated adenylate cyclase activity to different extents. Addition ofcis-9-16∶1/cis-9-16∶1,cis-9-18∶1/cis-9-18∶1 orcis-9-18∶1/cis-9,12-18∶2sn-glycerophosphocholine (GPC) completely restored adenylate cyclase activity, whilecis-11-18∶1/cis-11-18∶1 GPC was not effective and only a partial recovery was observed with 16∶0/16∶0, 16∶0/cis-9-18∶1 andtrans-9-18∶1 GPC. Considering the structural features of these seven PC molecular species, the findings suggest that an optimal lipid environment is conferred to the enzyme by the presence of thecis double bonds, each located in Δ9 position of the PC acyl chains. The limited effect ofcis-9-16∶1/cis-9-18∶1 GPC andcis-9-18∶1/cis-9-16∶1 GPC suggests that an equal length of the terminal hydrocarbon chains extending bevond the Δ9 double bonds is also important. Moreover, complete restoration of adenylate cyclase activity in cells exposed to 16∶0/cis-9,12-18∶2 GPC suggests that twocis-9,12 double bonds located on the same chain are as effective as twocis-9 double bonds each located on two different chains of PC. As the four double bonds of 16∶0/cis-5,8,11,14-20∶4 GPC had no effect, a mere increase in the number of double bonds seems insufficient to build an optimal lipid microenvironment for the enzyme.