Effects of dietary supplementation with n-3 fatty acids compared with n-6 fatty acids on bronchial asthma

OBJECTIVE: The effects of perilla seed oil (n-3 fatty acids) on bronchial asthma were compared with the effects of corn oil (n-6 fatty acids) in relation to the pulmonary function and the generation of leukotriene B4 (LTB4) and C4 (LTC4) by leucocytes. METHODS AND SUBJECTS: 14 asthmatic subjects were divided randomly into two groups: one group (7 subjects) consumed perilla seed oil-rich supplementation and the other group (7 subjects) consumed corn oil-rich supplementation for 4 weeks. Generation of LTs by leucocytes and respiratory function were compared between the two groups. RESULTS: The generation of LTB4 and LTC4 by leucocytes tended to increase in subjects (N=7) with corn oil-rich supplementation, and decrease in subjects (N=7) with perilla seed oil-rich supplementation. Significant differences between the two groups were observed in the generation of LTB4 at 2 weeks (p<0.05) and LTC4 at 2 weeks (p<0.05) after dietary supplementation. Significant increases in the value of PEF (p<0.05), FVC (p<0.01), FEV(1.0) (p<0.05) and V(25) (p<0.05) were found in subjects who received perilla seed oil supplementation for 4 weeks. And significant differences in the value of FVC (p<0.05) and FEV(1.0) (p<0.05) were observed between the two groups after 4 weeks of dietary supplementation. CONCLUSION: These results suggest that perilla seed oil-rich supplementation is useful for the treatment of asthma in terms of suppression of LTB4 and LTC4 generation by leucocytes, and improvement of pulmonary function.     

Release of platelet-activating factor from HL-60 human leukemic cells following macrophage-like differentiation

Platelet-activating factors (PAF), a phospholipid mediator of anaphylaxis, is also known to be released in vitro from both phagocytic polymorphonuclear neutrophils (PMN) and monocytes in response to a variety of stimuli. The fact that human myeloid cells of the HL-60 line can be made to differentiate in vitro into macrophage-like cells by 12- O-tetradodecanoylphorbol-13-acetate (TPA) prompted us to investigate the generation and release of PAF during this transformation. Both passive release of PAF at pH 9.5, and active release, following phagocytosis of C3b- and C3d-opsonized yeast spores, and stimulation with C5a anaphylatoxin from untreated and TPA-treated HL-60 cells, PMN, and plastic-adherent normal human monocytes were studied. It was found that after 3 days of TPA treatment, HL-60 cells released PAF following phagocytosis of C3b- and C3d-opsonized yeast spores. Inhibition of PAF release by a selective inhibitor of phospholipase A2 and labeling of PAF with sodium 14C-acetate indicated that PAF generation is a two-step process: (1) release of PAF precursor from cell membranes and (2) its acetylation. A model for the in vivo study of mechanisms and metabolic events involved in PAF generation and release could perhaps be built on these findings.U     

The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression

The HL-60 cell line, derived from a single patient with acute promyelocytic leukemia, provides a unique in vitro model system for studying the cellular and molecular events involved in the proliferation and differentiation of normal and leukemic cells of the granulocyte/monocyte/macrophage lineage.     

cGMP-induced differentiation of the promyelocytic cell line HL-60.

cGMP is a second messenger that mediates numerous metabolic events; in the present work a role in myeloid cell differentiation was demonstrated. Nitroprusside and NaNO2, which activate cytosolic guanylate cyclase and increase the intracellular cGMP concentration, induced granulocytic differentiation of the human promyelocytic cell line HL-60; differentiation was measured by acquisition of the OKM1 antigen, morphological changes, and nitroblue tetrazolium reduction. When theophylline, a phosphodiesterase inhibitor, which by itself induced modest differentiation, was added to nitroprusside or NaNO2, differentiation increased in an additive fashion. The degree of differentiation correlated with the increase in the intracellular cGMP concentration. 8-Bromoguanosine 3',5'-cyclic monophosphate, a membrane-permeable cGMP analogue, also induced differentiation of HL-60 cells but was much more effective in the presence of theophylline, with the two agents interacting synergistically. The effect of theophylline in these studies could not be attributed to increasing the intracellular cAMP concentration. Dimethyl sulfoxide, and established inducer of differentiation of HL-60 cells, markedly enhanced the differentiation induced by nitroprusside and NaNO2.     

Influence of healthy human bone marrow fibroblasts on differentiation and proliferation of human leukemic cell line HL-60

The influence of bone marrow fibroblasts from healthy subjects on differentiation and clonal proliferation of HL-60 cells was studied. Clonal proliferation of HL-60 cells was examined by using agar culture method, DNA synthesis was done by counting 3H-thymidine incorporation into the cells, and differentiation of the cells was checked by non-specific esterase staining. The bone marrow fibroblasts and their conditioned-medium significantly stimulated colony formation and DNA synthesis of HL-60 cells, but they inhibited differentiation of the HL-60 cells. From these results, it was suggested that the bone marrow fibroblasts stimulated clonal proliferation of HL-60 cells and inhibited differentiation of the cells through humoral factors secreted by the fibroblasts.     

罗格列酮抑制HL-6O细胞增殖和诱导分化作用

目的:研究罗格列酮对人急性髓性白血病细胞(HL-60)增殖和诱导分化的作用。方法:用不同浓度的罗格列酮处理HL-60细胞24~72h,MTT观察细胞的增殖抑制作用,瑞特-姬姆萨染色法及硝基蓝四氮(NBT)还原比色法测定细胞分化,流式细胞仪检测细胞周期分布和凋亡率,免疫组化检测PPAR-γ表达。结果:不同浓度罗格列酮处理细胞后,HL-60细胞增殖明显受抑,抑制率最高达77%,抑制作用呈现时间、剂量和效应依赖性;细胞周期分析,罗格列酮能诱导HL-60细胞G1期阻滞;同时罗格列酮能促进HL-60细胞分化。免疫组化发现,HL-60细胞表达PPAR-γ,罗格列酮作用HL-60细胞后,发生核转位现象。结论:罗格列酮能抑制HL-60细胞增殖,诱导细胞分化和细胞周期G1期阻滞,其作用机制可能与激活PPAR-γ有关。     

Competition of n-3 and n-6 polyunsaturated fatty acids in the isolated perfused rat heart

When perfused with exogenous arachidonic acid (AA) or eicosapentaenoic acid (EPA), the rat heart incorporated these fatty acids into phospholipids, chiefly as phosphatidylcholine. The pattern of fatty acid incorporation at any given concentration of fatty acid in the perfusate was not different between n-3 and n-6 polyenoates. When rat hearts were perfused with the same amounts but different mixtures of EPA and AA, the incorporation of EPA showed a marked increase proportional to the EPA/AA ratio present in the perfusate. Results indicated that cardiac muscle phospholipids incorporate n-3 and n-6 polyenoates equally effectively and hence enrichment of n-3 polyenoate can displace AA in the cardiac phospholipid pool.     

Specific changes in the surface glycoprotein pattern of human promyelocytic leukemic cell line HL-60 during morphologic and functional differentiation.

The human promyelocytic leukemia cell line HL-60 can be induced to undergo morphological and functional differentiation in vitro by various low molecular weight compounds. The cellular morphology changes from blastoid appearance to that of granulocytes and the cells acquire the ability to phagocytize. We here report that the surface glycoproteins specifically change during this differentiation, as shown by the neuraminidase/galactose oxidase/NaB3H4 surface-labeling technique followed by polyacrylamide slab gel electrophoresis. The most prominent change is the loss of the major glycoprotein band typical for the blast cells which has an apparent molecular weight of 160,000 and the appearance of a major surface glycoprotein band with an apparent molecular weight of 130,000. Expression of the 130,000 molecular weight band correlates with the appearance of phagocytic and chemotactic activities of the cells. It has the same molecular weight as the major surface glycoprotein of freshly isolated human blood granulocytes.     

Differentiation-linked expression of cell surface markers on HL-60 leukemic cells

The cell surface antigenic phenotype of HL-60, a human acute promyelocytic leukemia cell line, has been analyzed before and after maturation induction with dimethylsulfoxide (DMSO) using a panel of markers including a "library" of monoclonal antibodies and "conventional" antisera in conjunction with the fluorescene-activated cell sorter. HL-60 cells express granulocyte and "leukocyte" differentiation antigens but not antigens of the lymphoid, platelet, and erythroid lineages. DMSO-induced morphological maturation was found to be associated with a decrease in the proportion of cells in mitotic cycle, induction of C3d receptors, increased expression of granulocytic and leukocyte antigens, and diminished expression of HLA-A,B,C and beta 2-microglobulin determinants. HL-60 cells have no detectable expression of HLA-DR-associated determinants as assayed by rabbit anti-p28,33 monoclonal anti-HLA-DR (monomorphic determinant), and HLA-DRw typing alloantisera. The relationship of these changes in cell surface properties to normal granulocytic differentiation is discussed.     

Polyamines in 1 alpha, 25-dihydroxycholecalciferol-induced differentiation of human promyelocytic leukemia cells, HL-60

The human promyelocytic leukemia cell line, HL-60, differentiated into macrophage/monocytes in the presence of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], as assessed by the percentage of morphologically mature cells and their ability to reduce nitroblue tetrazolium. In this study of the mechanism involved, the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase (SAT), the rate-limiting enzymes of polyamine metabolism, as well as the cellular levels of polyamine were measured. ODC activity reached a peak 24 h after the addition of 1 alpha,25(OH)2D3 and then decreased, while SAT activity gradually increased as differentiation commenced. An increase in putrescine and decreases in spermidine and spermine were also observed. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, with or without methylglyoxalbis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, caused no effect on 1 alpha,25(OH)2D3-induced cell differentiation, although the cellular levels of putrescine and spermidine decreased markedly. Addition of alpha-difluoromethylornithine markedly suppressed cell proliferation; this effect was reversed by the addition of exogenous putrescine. Addition of exogenous spermidine or spermine to overcome activation of SAT also had no effect on 1 alpha,25(OH)2D3-induced cell differentiation. These results suggest both that polyamine metabolism is not important in 1 alpha,25(OH)2D3-induced differentiation of HL-60 cells, but that it is intimately involved in the proliferation of these cells.     

Synthesis of eosinophil-associated enzymes in HL-60 promyelocytic leukemia cells

Eosinophils derived from HL-60 cells share many of the abnormalities of granule histochemistry and morphology frequently seen in eosinophils of patients with certain malignancies, especially those seen in acute myelomonocytic leukemia with abnormal eosinophils (FAB class M4eo). In order to understand the pathogenesis of these abnormalities, four enzymes, characteristic of the eosinophil, were studied in HL-60 promyelocytic leukemia cells at various stages of eosinophilic differentiation. Using biochemical and ultrahistochemical techniques, the following differences from normal eosinophil development were demonstrated. First, both myeloperoxidase and eosinophil peroxidase coexisted in the population of maturing HL-60 eosinophils. Second, the granules formed from the condensation of material in vacuoles which were derived from dilated segments of the endoplasmic reticulum; the role of the Golgi apparatus in processing of peroxidase appeared minimal. Third, low levels of lysophospholipase and arylsulfatase were present in the cells compared to normal eosinophils. Finally, crystallizations resembling precursor structures of Auer rods appeared in the granules of about 5% of the cells. These findings suggest that several disorders of the control of protein synthesis and processing exist in HL-60 eosinophils which may be responsible for the abnormal granule morphology and histochemistry.     

Biosynthesis of polyunsaturated fatty acids of (n-6) and (n-3) series in isolated adrenocortical cells of rats. Effect of ACTH

Both the capacity of isolated adrenocortical cells to incorporate and transform [1-14C]linoleic and [1-14C]alpha-linolenic acids and the effect of ACTH on the biosynthesis of polyunsaturated fatty acids from [1-14C]alpha-linolenic acid were investigated. The cells were able to incorporate both labeled precursor acids and convert them into higher homologs. This transformation increases along the incubation time tested. When linoleic acid was the precursor, the biosynthesis of higher homologs was carried out following the desaturating-elongating route. Both pathways, the desaturating-elongating and the elongating, were detected when the substrate was alpha-linolenic acid. The results proved the existence of delta 6, delta 5 and delta 4-desaturases in this type of cells. Isolated adrenocortical cells obtained from rats treated with ACTH showed an increase in the amount of [1-14C]alpha-18:3 that remained in the cells without metabolization and, consequently, a decrease in the last product formed (20:5 n-3) was evident compared to the controls. Simultaneously, the desaturation-elongation products decreased significantly. Similar results were obtained when cells isolated from untreated rats were incubated for 3 h in the presence of ACTH. In this case, the values obtained returned to normal levels 6 h after incubation. These results were mimicked by dibutyryl-cyclic AMP. It can be concluded that the effect of ACTH on the biosynthesis of polyunsaturated fatty acids from alpha-linolenic acid was mediated through an enhancement of the intracellular levels of cAMP.     

Compared effect of n-3 and n-6 dietary fatty acids on rat intestinal acyl-CoA:cholesterol acyltransferase activity

Polyunsaturated fatty acids are known to lower plasma cholesterol concentrations. We studied their effect on intestinal acyl-CoA:cholesterol acyltransferase (ACAT) activity in rats fed either salmon oil or corn oil (17% fat) with or without 1% cholesterol. After an 8-week feeding period we confirmed the hypolipidemic effect of salmon oil and we established its ability to stimulate ACAT activity in rats fed low-cholesterol diets. The most striking effect of 1% dietary cholesterol on ACAT activity was obtained in the control group (34% enhancement), whereas cholesterol supplementation had no effect on ACAT activity in the salmon oil group. The results enable us to suggest that n-3 fatty acids have an effect per se on ACAT activity; the regulation of enzyme activity by dietary cholesterol probably involves independent processes.     

PTS对HL-60白血病细胞的抑制及其与化疗药物协同性研究

目的　探讨人参三醇 (PTS)对 HL - 6 0白血病细胞生长的影响及其与化疗药的协同作用。方法　在液体培养、半固体集落形成培养中 ,以不同浓度的 PTS处理 HL- 6 0细胞 2 4、4 8、72小时 ,用细胞生长曲线和集落形成率观察 PTS的增殖和抑制效应 ,MTT法检测 PTS对化疗药物的协同性。结果　低浓度 (10 μg/ ml) PTS能显著地刺激 HL- 6 0细胞增殖 ,中浓度 (5 0 μg/ ml)使细胞增殖受抑 ,并呈时间 -效应依赖性。PTS分别与化疗药物高三尖杉酯硷 (HHr)、阿糖胞苷 (Ara- c)联用能增加对 HL- 6 0细胞的杀伤作用 ,抑制率为 (5 6 .5± 2 .3) %和 (5 3.1±6 .2 ) % ,明显高于对照组的 (39.3± 7.1) %和 (38.2± 6 .4 ) % (P<0 .0 5 ) ;PTS与足叶乙甙 (Vp- 16 )联用的抑制率为 33.8%。结论　不同剂量的 PTS对 HL - 6 0细胞具有刺激增殖和抑制生长的双向效应 ,并能增强 HL - 6 0细胞对化疗药物的敏感性 ,是人参皂甙内抑制白血病细胞的有效成分 ,可为临床应用提供依据     

n-3多聚不饱和脂肪酸与心血管疾病

在发达国家,心血管疾病为其主要死亡原因,在发展中国家,心血管疾病的发病率逐年增加。根据2000年中国卫生年鉴公布的资料显示,心血管（包括脑血管）病的死亡率无论在城市还是农村均居于首位。如何预防心血管疾病,控制其病情,改善其预后为当代医学的主要研究课题之一。在西方国家的研究中,鱼油被认为具有心血管保护作用。     

Autocrine proliferative effect of ghrelin on leukemic HL-60 and THP-1 cells

Ghrelin is a 28 amino acid peptide hormone that is mainly produced by the stomach, but also by several tissues and tumors. Ghrelin is octanoylated on the Ser(3), but is also detected as a des-acylated form. Only the acylated ghrelin activates the GH secretagogue receptor (GHS-R) type 1a to stimulate GH release, and regulate food intake and energy metabolism. For the first time, we report that ghrelin and des-acyl ghrelin are present in human promyelocytic HL-60, monocytic THP-1 and lymphoblastic SupT1 cell lines. The human leukemic cell lines did not express the functional GHS-R 1a, whereas they expressed GHS-R 1b, a truncated variant of the receptor. Leukemic cell proliferation was not modified by the addition of octanoylated or des-acyl ghrelins. However, THP-1 and HL-60 cell proliferations were inhibited by SB801, an antibody directed against the N-terminal octanoylated portion of ghrelin, suggesting that octanoylated ghrelin stimulates cell proliferation via an autocrine pathway involving an as yet unidentified ghrelin receptor. Both octanoylated and des-acyl ghrelins did not alter the basal adenylate cyclase activity. Treatments of THP-1 and SupT1 cells by both octanoylated and des-acyl ghrelins did not modify the adenylate cyclase activity in response to vasoactive intestinal peptide, suggesting that ghrelin is unlikely to modulate the anti-inflammatory and differentiating properties of vasoactive intestinal peptide.     

Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of gamma-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.