Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.     

Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.     

Possible involvement ofL-glycero-phosphoryl-ethanolamine in the phospholipid methylation pathway

It is usually accepted that GPEtn is only an intermediate in glycerophospholipid degradation. However, some years ago J. P. Infante published a review-hypothesis concerning the possible involvement of GPEtn and GPCho in the biosynthesis of acyl-specific glycerophospholipids in mammalian tissues. This paper reports the results obtained by incubating brain cortex slices in the presence of labeled GPEtn. GPEtn enters the cells and the radioactivity of the precursor is found in both PE and PC. When these results were compared with those obtained by incubating brain slices in the presence of³H-Etn, a significantly higher amount of radioactivity was found in PC, mainly after short incubations in the presence of low concentrations of the precursors. The slopes of the methylated forms of PE further underline the above differences found by comparing the results obtained with the two precursors. The results presented in this paper suggest a possible role of GPEtn in phospholipid biosynthesis and methylation pathway of PE.     

Effect of alcohol consumption on peripheral blood Alu methylation in Korean men

Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create a substantial public health problem worldwide. To date, no therapeutic can effectively solve these problems. They are complex diseases characterized by both genetic and environmental factors. DNA methylation can act as a downstream effector of environmental signals and account for multi-factorial nature of the disease. Global DNA methylation of peripheral blood cells has recently been proposed as a potential biomarker for disease risk. Alu elements host one-quarter of CpG dinucelotides in the genome to function as proxies for global DNA methylation. In this study, we evaluated the Alu methylation in the peripheral blood DNA of healthy volunteers and AUD patients using the pyrosequencing technology. The Alu methylation level is significantly higher in AUD compared to healthy controls (23.4?±?1.6 versus 22.1?±?1.0, t?=?7.83, p?<?0.0001). Moreover, significant correlation was found between Alu methylation and alcohol use disorders identification test score (r?=?0.250, p?<?0.0001), alcohol problem (r?=?0.294, p?<?0.0001), and life position (r?=?–0.205, p?=?0.0005). Overall, these novel findings indicate that alcohol-related increase in Alu methylation might play a complex role in the etiology and pathogenesis of AUD. Further studies are required to elucidate the mechanisms underlying this relationship.     

趋化因子CXCL14在SLE患者外周血单个核细胞中的表达及启动子甲基化分析

目的：分析趋化因子CXCL14在系统性红斑狼疮（SLE）患者外周血单个核细胞（PBMC）中的表达及其启动子甲基化特征。方法：收集28例SLE患者和20名健康人外周血单个核细胞（PBMC）标本,抽提细胞中RNA,逆转录后,以GAPDH为内参,通过定量PCR检测PBMC中CXCL14的表达,统计分析CXCL14表达水平与SLE各种临床资料的相关性,探讨PBMC中CXCL14表达与SLE的关系,通过重亚硫酸盐修饰的全基因组DNA用以BSP测序来明确不同标本中CXCL14启动子中甲基化位点的甲基化率。结果：CXCL14在SLE患者和正常人PBMC中的表达量存在显著差异（P < 0.05）。与健康人PBMC中CXCL14的含量相比较,CXCL14在SLE患者PBMC中表达量显著降低。与进一步CXCL14表达水平与SLE各种临床资料的相关性分析显示,CXCL14表达水平与SSB抗体（干燥综合征B抗体）、蛋白尿及血小板计数相关。与SSB抗体阴性SLE患者比较,SSB抗体阳性患者CXCL14表达水平更低（P <0.05）;与蛋白尿阴性SLE患者相比,蛋白尿阳性患者CXCL14表达水平更低（P < 0.05）;而血小板升高患者的CX-CL14表达水平更高（P < 0.05）。CXCL14表达水平与SLE活动、肾损害指标、抗ds-DNA、C反应蛋白（CRP）、补体C3水平等指标未见显著相关性。CXCL14启动子区甲基化分析显示,SLE患者CpG岛甲基化明显高于正常对照。结论：PBMC中低表达的CXCL14与SLE发生发展相关,其启动子区CpG岛过度甲基化是SLE患者CXCL14低表达的重要机制。     

Toluene-induced alterations in rat synaptosomal membrane composition and function

Toluene is a widely used organic solvent that can produce acute central nervous system (CNS) effects. Since toluene reaches relatively high concentrations in the CNS and is extremely lipophilic, we investigated its effects on rat brain membrane composition and function. Toluene (1 g/kg, lh) did not alter total brain microsomal phospholipid (PL) or cholesterol (CL) content. However, synaptosomal PL was decreased (24%), while synaptosomal CL was unaltered. The PL/CL ratio, an indirect index of membrane fluidity, did not change, suggesting that toluene did not affect membrane fluidity. Fluorescence polarization studies employing 1,6-diphenyl-1,3,5-hexatriene (DPH) showed that toluene did not alter synaptosomal membrane fluidity after administration in vivo (1 g/kg) or in vitro (0.5 to 5.0 mM). Dose-response and time-course studies showed that toluene maximally decreased synaptosomal PL after 1 g/kg, 1 h. The dose-response and time-course studies also showed that the toluene-induced decreases in PL were a result of specific decreases in phosphatidylethanolamine (PE). Since PE was decreased, we assessed whether toluene altered synaptosomal membrane function by investigating phospholipid methylation, a reaction which uses PE as its initial substrate. Toluene decreased the incorporation of methyl groups into lipid when [³H]-methionine was used as the methyl donor, but did not affect methylation when [³H]-adenosylmethionine was the methyl donor. These data suggest that toluene-induced specific decreases in synaptosomal PE and inhibition of phospholipid methylation may alter normal synaptic function and play a critical role in the mechanism(s) of action of toluene's CNS effects.     

Enhancement of phospholipid hydrolysis in vasopressin-stimulated BHK-21 and H9c2 cells

The hydrolysis of phospholipids in vasopressin-stimulated baby hamster kidney (BHK)-21 and H9c2 myoblastic cells was investigated. Phosphatidylcholine and phosphatidylethanolamine in these cells were pulse labelled with [³H]glycerol, [³H]myristate, [³H]choline or [³H]ethanolamine, and chased with the non-labelled precursor until linear turnover rates were obtained. When cells labelled with [³H]glycerol or [³H]myristate were stimulated by vasopressin, no significant decrease in the labelling of phosphatidylcholine was detected, but the labelling of phosphatidic acid was elevated. However, the labellings of phosphatidylethanolamine and its hydrolytic product were not affected by vasopressin stimulation. When the cells were pulse labelled with [³H]-choline, vasopressin stimulation caused a decrease in the labelled phosphatidylcholine with a corresponding increase in the labelled choline. The apparent discrepancy between the two types of labelling might be explained by the recycling of labelled phosphatidic acid back into phosphatidylcholine, thus masking the reduction in the labelled phospholipid during vasopressin stimulation. Alternatively, the labelled choline produced by vasopressin stimulation was released into the medium, thus reducing the recycling of label precursor back into the phospholipid and making the decrease in the labelling of phosphatidylcholine readily detectable. Further studies revealed that vasopressin treatment caused an enhancement of phospholipase D activity in these cells. The presence of substrate-specific phospholipase D isoforms in mammalian tissues led us to postulate that the differential stimulation of phospholipid hydrolysis by vasopressin was caused by the enhancement of a phosphatidylcholine-specific phospholipase D in both BHK-21 and the H9c2 cells.Abbreviations BHK-21 cells baby hamster kidney-21 cells     

Induction of alpha interferon by membrane interaction between viral surface and peripheral blood mononuclear cells

Cells infected with viruses and fixed when viral antigens appeared at the cell membrane induced much higher alpha interferon (IFN-alpha) levels in human peripheral blood mononuclear cells (PBMC) than free virions. Relatively few inducer cells were sufficient for triggering IFN production. Optimal IFN yields depended on inducer/producer cell ratio. The response was peculiar to PBMC as it was not found in other cells in which IFN can normally be induced by free virions. IFN inducing activity was also exerted by live virus-infected PBMC, showing that this type of induction may have physiological importance. These findings confirm that viral induction of IFN-alpha is activated by some interaction between viral components presented at the cell surface and PBMC membrane. Thus induction of IFN by circulating cells infected by viruses and presenting viral antigens at the surface may be an efficient host defense mechanism. Since IFN yields close to 10(6) international units per milliliter are obtained, this system has potential for large scale production of native IFN-alpha.     

Effect of exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.     

Blood glutathione redox status and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults

Oxidative stress and DNA methylation are metabolically linked through the relationship between one-carbon metabolism and the transsulfuration pathway, but possible modulating effects of oxidative stress on DNA methylation have not been extensively studied in humans. Enzymes involved in DNA methylation, including DNA methyltransferases and histone deacetylases, may show altered activity under oxidized cellular conditions. Additionally, in vitro studies suggest that glutathione (GSH) depletion leads to global DNA hypomethylation, possibly through the depletion of S-adenosylmethionine (SAM). We tested the hypothesis that a more oxidized blood GSH redox status is associated with decreased global peripheral blood mononuclear cell (PBMC) DNA methylation in a sample of Bangladeshi adults. Global PBMC DNA methylation and whole blood GSH, glutathione disulfide (GSSG), and SAM concentrations were measured in 320 adults. DNA methylation was measured by using the [³H]-methyl incorporation assay; values are inversely related to global DNA methylation. Whole blood GSH redox status (E_h) was calculated using the Nernst equation. We found that a more oxidized blood GSH E_h was associated with decreased global DNA methylation (B ± SE, 271 ± 103, p = 0.009). Blood SAM and blood GSH were associated with global DNA methylation, but these relationships did not achieve statistical significance. Our findings support the hypothesis that a more oxidized blood GSH redox status is associated with decreased global methylation of PBMC DNA. Furthermore, blood SAM does not appear to mediate this association. Future research should explore mechanisms through which cellular redox might influence global DNA methylation.     

Implications of sterol structure for membrane lipid composition, fluidity and phospholipid asymmetry in Saccharomyces cerevisiae

Sterols are essential components of the plasma membrane in eukaryotic cells. Nystatin-resistant erg mutants were used in the present study to investigate the in vitro effects of altered sterol structure on membrane lipid composition, fluidity, and asymmetry of phospholipids. Quantitative analyses of the wild type and mutants erg2, erg3 and erg6 revealed that mutants have lower sterol (free)-to-phospholipid molar ratios than the wild type. Phosphatidylcholine content was decreased in erg2 and erg3 mutants; however, it was increased in erg6 strains as compared to normals. Phosphatidylserine content was increased in the erg6 mutant only. Fluorescence anisotropy decreased with temperature in both probes, and was lower for mutants than for the wild type, suggesting an increased freedom in rotational movement due to decreased membrane order. Investigation of changes in the aminophospholipid transbilayer distribution using two chemical probes, trinitrobenzene sulfonic acid and fluorescamine, revealed that the amounts of phosphatidylethanolamine derivatized by these probes were quite similar in both the wild type and various erg strains. The present findings suggest that adaptive responses in yeast cells with altered sterol structure are possibly manifested through changes in membrane lipid composition and fluidity, and not through transbilayer rearrangement of aminophospholipids.     

类风湿性关节炎患者外周血单个核细胞 IL-10 基因启动子甲基化状态研究

白细胞介素-10（interleukin-10,IL-10）是在类风湿性关节炎中发挥重要免疫调节作用的细胞因子,其基因失活与已分化的Th1和Th2细胞染色质结构重塑有关。为了探讨IL-10基因启动子甲基化及基因失活在类风湿性关节炎(Rheumatoid Arthritis,RA)发病和进展中的作用,采用逆转录聚合酶链反应(RT-PCR)、酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)及甲基化特异性聚合酶链反应(MSP), 分别检测34例类风湿性关节炎患者和30例健康人外周血单个核细胞 IL-10 mRNA、蛋白表达水平及基因启动子甲基化状态。结果显示,病例组IL-10 mRNA及蛋白表达均低于健康对照组,无统计学差异（P>0.05）。病例组甲基化率(85.29%)高于健康对照组(43.33%), 具有统计学差异(x² =12.439,P=0.000)。IL-10基因启动子甲基化状态与其mRNA表达呈显著负相关(r=-0.579, P=0.001), 与所累关节数显著相关,但与血沉（ESR）、C反应蛋白（CRP）、类风湿因子(RF)、年龄均无相关性(P>0.05)。IL-10 mRNA表达与年龄、所累关节数、ESR、CRP及RF均无相关性(P>0.05)。上述结果提示,启动子甲基化可能是IL-10基因失活的重要机制,IL-10基因异常高甲基化状态可能参与了RA的发生发展。     

Effect of Bacillus anthracis lethal toxin on human peripheral blood mononuclear cells

Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage-like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.     

Effect of thyrotropin-releasing hormone on immune functions of peripheral blood mononuclear cells

The tripeptide thyrotropin-releasing hormone (TRH) works as a hypothalamic hormone, but is found also outside the brain in intrinsic nerve fibers of the gastrointestinal tract. There is evidence that TRH modulates the activity of immunocompetent cells, although there are only very few data on TRH-mediated immune effector functions. Since we could recently show that TRH inhibits monocyte activities we were also interested in other possible TRH modulated immune functions. Peripheral blood mononuclear cells (PBMC) from ten healthy subjects were cultured for 7 days and pulsed with 0.125 and 0.250 microgram/ml Pokeweed mitogen (PWM). 10(-12) to 10(-6) M TRH was added simultaneously with PWM. Lymphocyte proliferation [(3H]thymidine incorporation), interferon-gamma (IFN-gamma) activity (RIA) and immunoglobulin activities (IgG, IgM, IgA; ELISA) were determined in the supernatants. We could demonstrate a TRH-dependent decrease in PWM-pulsed IgG activity with significant (alpha = 0.05) values at 10(-8) and 10(-10) M (-29 +/- 6%/-16 +/- 3% for PWM 0.125 microgram/ml and -17 +/- 9%/-11 +/- 9% for PWM 0.250 microgram/ml). This inhibitory effect could be abolished by an anti-TRH antiserum. There was no TRH effect on IgM and IgA activities, IFN-gamma activity and lymphocyte proliferation compared with the PWM stimulated values alone. The described TRH effect on the polyclonal IgG response by PBMC gives further evidence for a functional link between the immune system and the endocrine system, although its underlying mechanism is not yet clear.     

Stimulation of phospholipid methylation by glucose in pancreatic islets

A two fold stimulation in the incorporation of [3H-methyl] groups from [3H-methyl] methionine into phospholipids was seen in intact pancreatic islets within six minutes of exposure to a glucose concentration that stimulates insulin release. Nonstimulatory sugars, L-glucose and D-galactose, as well as dibutyryl cAMP, did not affect phospholipid methylation in islet cells. A calcium channel blocker, verapamil, inhibited methylation. These studies suggest that the signal for glucose-induced insulin release could involve phospholipid methylation.     

Effect of acute heat shock on gene expression by human peripheral blood mononuclear cells.

We studied the effect of heat shock on gene expression by normal human cells. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adults. Paired samples from each subject were subjected to either 20 min of heat shock (43 degrees C) or control (37 degrees C) conditions and then returned to 37 degrees C. RNA was isolated 160 min later, and five representative samples were analyzed on Affymetrix gene chip arrays containing approximately 12,600 probes. A biologically meaningful effect was defined as a statistically significant, twofold or greater difference in expression of sequences that were detected in all five experiments under control (downregulated sequences) or heat shock (upregulated sequences) conditions. Changes occurred in 395 sequences (227 increased by heat shock, 168 decreased), representing 353 Unigene numbers, in every functional category previously implicated in the heat shock response. By RT-PCR, we confirmed the findings for one upregulated sequence (Rad, a G protein) and one downregulated sequence (osteopontin, a cytokine). We conclude that heat shock causes extensive gene expression changes in PBMCs, affecting all functional categories of the heat shock response.     

Proteomic map of peripheral blood mononuclear cells

In the field of proteomics extensive efforts have been focused on the knowledge of proteins expressed by different cell types. In particular, enormous progress has been done in the characterization of blood cellular components. In this work, we have established a public 2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two hundred and forty-six spots corresponding to 174 different proteins have been identified on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins have been classified in thirteen categories on the basis of their differential functions or cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of several proteins has been discussed in relation to their biological function. We intend to show the potentiality of PBMCs to investigate the proteomics changes possibly associated with a large number of pathologies such as autoimmune, neurodegenerative and cancer diseases.     

Lindane-induced modifications to membrane lipid structure: Effect on membrane fluidity after subchronic treament

Chronic lindane intoxication by injecting subcutaneously the toxicant, resulted in an altered lipid pattern in rat ventral prostate membranes. An increase of membrane fluidity was also observed using a fluorescence polarization technique. Whenin vitro experiments were carried out with both treated and untreated rats, an interesting lack of parallelism was found, which could indicate the development of a resistance to membrane disordering by lindane. The observed changes in cholesterol and phospholipid composition are also consistent with the hypothesis that lindane perturbs the lipid matrix of membranes, possibly inducing complex compensatory changes in the membrane lipid composition.