Effects of antidepressants on mRNA levels of antioxidant enzymes in human monocytic U-937 cells

Alterations of antioxidant enzyme activities have been described in a number of psychiatric disorders including major depression. Subsequently, the present study examined the effects of different types of antidepressants (desipramine, imipramine, maprotiline and mirtazapine) in different concentrations (10(-5), 10(-6) and 10(-7) M) on the mRNA levels of various enzymes of the antioxidant system, including both intracellular superoxide dismutase isoforms, glutathione peroxidase and catalase as well as several enzymes of the glutathione metabolism in monocytic U-937 cells after short- and long-term treatment (2.5 and 24 h) via RT-PCR. Results indicated mainly short-term decreases in the mRNA levels of antioxidant enzymes after treatment with these substances in all the concentrations used. In addition, after long-term treatment, significant increases in the mRNA levels were seen in the cases of Cu, Zn superoxide dismutase, gamma-glutamyl-cysteine synthetase, glutathione-S-transferase and glutathione reductase, including the impacts of all the antidepressants used in concentrations of 10(-6) M and 10(-7) M. Based on the large number of significant effects of all types of antidepressants tested on various antioxidant enzymes, we suggest that antioxidant enzymes may represent important targets in the course of antidepressive treatment.     

Relationships among endocrine and signaling-related responses to antidepressants in human monocytic U-937 blood cells: analysis of factors and response patterns

OBJECTIVE: Antidepressants (AD) (desipramine, imipramine, maprotiline, mirtazapine) and corticosteroid (CS) were examined for their effects on gene expression in human monocytic U-937 blood cells. Endocrine and signaling-related response patterns were determined by expression analysis of different factors, comprising endocrine (glucocorticoid receptor [GR], GR-alpha/beta/gamma; mineralocorticoid receptor [MR]) and signaling-related pathways (p105, STAT3, c-jun, c-fos, JNK1, GAPDH, TNF-alpha). METHODS: A semiquantitative RT-PCR for factor responses after 24 h of treatment was conducted and exploratory multivariate statistical procedures were applied for further analysis. RESULTS: Compared to controls, significant reduction of mRNA levels of GR-beta under imipramine and of c-jun under desipramine treatment were found. CS treatment significantly reduced mRNA levels of GR-alpha/beta, TNF-alpha, p105 and c-jun compared to controls. Compared to CS treatment, significantly increased mRNA levels were found for JNK1 under imipramine treatment and for GR-alpha after treatment with all AD examined. DISCUSSION: The multivariate approach meets the requirements of the complex situation of metabolic reactions induced by AD or CS treatment. Our data show that AD affect both, endocrine and signaling-related factors in human monocytic U-937 blood cells, although clearly not in a uniform manner. Hereby, GR is obviously playing a comparably central role. Overall, AD treatment might indeed normalize deviations of cellular endocrine and signaling-related pathways in major depressive disorder via the mechanisms examined.     

Mood‐stabilizers differentially affect housekeeping gene expression in human cells

Recent studies have revealed that antidepressants affect the expression of constitutively expressed “housekeeping genes” commonly used as normalizing reference genes in quantitative polymerase chain reaction (qPCR) experiments. There has yet to be an investigation however on the effects of mood‐stabilizers on housekeeping gene stability. The current study utilized lymphoblastoid cell lines (LCLs) derived from patients with mood disorders to investigate the effects of a range of doses of lithium (0, 1, 2 and 5 mM) and sodium valproate (0, 0.06, 0.03 and 0.6 mM) on the stability of 12 housekeeping genes. RNA was extracted from LCLs and qPCR was used to generate cycle threshold (C_t) values which were input into RefFinder analyses. The study revealed drug‐specific effects on housekeeping gene stability. The most stable housekeeping genes in LCLs treated: acutely with sodium valproate were ACTB and RPL13A; acutely with lithium were GAPDH and ATP5B; chronically with lithium were ATP5B and CYC1. The stability of GAPDH and B2M were particularly affected by duration of lithium treatment. The study adds to a growing literature that the selection of appropriate housekeeping genes is important for the accurate normalization of target gene expression in experiments investigating the molecular effects of mood disorder pharmacotherapies. Copyright © 2014 John Wiley & Sons, Ltd.     

Cytokines regulate alpha(1)-adrenergic receptor mRNA expression in human monocytic cells and endothelial cells

The family of adrenergic receptors (AR) plays a central role in regulation of the activity of many organ systems. Consequently, regulated expression of the various subtypes of AR is an important mechanism in maintaining homeostasis. Previously, we have shown that alpha(1)-AR triggering of peripheral blood mononuclear cells from patients with juvenile chronic arthritis results in increased IL-6 production. In contrast, alpha(1)-AR agonists do not alter cytokine production by cells of healthy individuals.The aim of the present study was to investigate whether pro-inflammatory cytokines can regulate the expression of mRNA encoding AR of the alpha(1)-family. We show that human THP-1 monocytic cells express mRNA encoding of two of the three cloned subtypes of alpha(1)-AR: alpha(1b)-AR and alpha(1d)-AR mRNA. The cytokines TNF-alpha and IL-1beta decrease level of mRNA for alpha(1d)-AR in THP-1 monocytic cells. In contrast, alpha(1b)-AR mRNA levels are not affected by these two cytokines. Interestingly, IL-1beta and TNF-alpha induce the expression of alpha(1a)-AR mRNA in THP-1 monocytic cells.In human umbilical vein endothelial cells (HUVEC), IL-1beta and TNF-alpha decrease both alpha(1b)-AR and alpha(1d)-AR mRNA levels in HUVEC. alpha(1a)-AR mRNA is not detectable in HUVEC.IL-6 and IL-8, two other pro-inflammatory cytokines tested in this study, do not change alpha(1)-AR subtype levels in HUVEC or monocytic cells. Our data demonstrate that TNF-alpha and IL-1beta can regulate expression of alpha(1)-AR mRNA and that cytokine regulation of alpha(1)-AR expression is subtype- and tissue-specific.     

Alcohol differentially affects c-Fos expression in the supraoptic nucleus of long-sleep and short-sleep mice

Ethanol administration in long-sleep (LS) and short-sleep (SS) mice results in a large number of Fos-IR neurons in the supraoptic nucleus (SON) in LS, and almost no Fos-IR neurons in the same nucleus in SS mice. In contrast, isotonic saline, hypertonic saline, with or without ethanol, resulted in a similar pattern of Fos-IR in both strains. These data indicate a differential effect of ethanol on c-Fos signaling specifically in the SON. Since the LS and SS mice were specifically selected for differential sensitivity to the sedative/hypnotic effects of ethanol, this differential in c-Fos activity may be causally implicated in their differential sensitivity to ethanol.     

Glutamate receptor subtype expression in human postmortem brain tissue from schizophrenics and alcohol abusers

Antibodies to functional AMPA/kainate (GluR1, GluR2, GluR3), and kainate binding sites (GluR5–7) were used as probes to characterize and quantitate glutamatergic receptor subtypes in human post-mortem brain tissue from schizophrenic subjects and non-psychotic control subjects, which included normal controls and subjects with a previous history of alcohol abuse. Crude membrane fractions from human hippocampi and cingulate cortices were fractionated by SDS-PAGE, electrotransferred to nitrocellulose, and probed for the various glutamate receptor subytpes. Western blots were developed with chemilluminescence and the images analyzed by densitometry. Significant reductions were observed in the hippocampal immunoreactivity of both GluR2 and GluR3 AMPA/kainate receptor subtypes in schizophrenic subjects compared to the entire group of non-psychotic control subjects. No significant changes were observed in schizophrenic hippocampal GluR1 and GluR5 receptor subtypes or in levels of the structural control proteins, NCAM and tau. Significant increases were observed for GluR2 and GluR3 in the hippocampi of subjects with alcohol abuse histories when compared to the non-psychotic normal control group. When subjects with alcohol abuse histories were removed from the non-psychotic control pool, schizophrenics were no longer statistically different from the remaining normal controls. An analysis of GluR2 and GluR3 immunoreactivity in the cingulate cortex revealed no changes in these receptor subtypes among any of the groups. No alterations were observed in the immunoreactivity of these various proteins due to confounding factors such as age, sex, postmortem interval, or smoking history, except in the cingulate cortex were GluR3 receptor subtype levels were significantly reduced in the brains of smokers. These results generally do not support a role for the non-NMDA type glutamatergic receptors in schizophrenia. However, the role of chronic ethanol exposure on the human brain needs to be further investigated, and underscores the importance of defined control tissue for human postmortem studies.     

Arsenic affects expression and processing of amyloid precursor protein (APP) in primary neuronal cells overexpressing the Swedish mutation of human APP

Arsenic poisoning due to contaminated water and soil, mining waste, glass manufacture, select agrochemicals, as well as sea food, affects millions of people world wide. Recently, an involvement of arsenic in Alzheimer's disease (AD) has been hypothesized (Gong and O’Bryant, 2010). The present study stresses the hypothesis whether sodium arsenite, and its main metabolite, dimethylarsinic acid (DMA), may affect expression and processing of the amyloid precursor protein (APP), using the cholinergic cell line SN56.B5.G4 and primary neuronal cells overexpressing the Swedish mutation of APP, as experimental approaches.Exposure of cholinergic SN56.B5.G4 cells with either sodium arsenite or DMA decreased cell viability in a concentration- and exposure-time dependent manner, and affected the activities of the cholinergic enzymes acetylcholinesterase and choline acetyltransferase. Both sodium arsenite and DMA exposure of SN56.B5.G4 cells resulted in enhanced level of APP, and sAPP in the membrane and cytosolic fractions, respectively. To reveal any effect of arsenic on APP processing, the amounts of APP cleavage products, sAPPβ, and β-amyloid (Aβ) peptides, released into the culture medium of primary neuronal cells derived from transgenic Tg2576 mice, were assessed by ELISA. Following exposure of neuronal cells by sodium arsenite for 12 h, the membrane-bound APP level was enhanced, the amount of sAPPβ released into the culture medium was slightly higher, while the levels of Aβ peptides in the culture medium were considerably lower as compared to that assayed in the absence of any drug. The sodium arsenite-induced reduction of Aβ formation suggests an inhibition of the APP γ-cleavage step by arsenite. In contrast, DMA exposure of neuronal cells considerably increased formation of Aβ and sAPPβ, accompanied by enhanced membrane APP level. The DMA-induced changes in APP processing may be the result of the enhanced APP expression. Alternatively, increased Aβ production may also be due to stimulation of caspase activity by arsenic compounds, or failure in Aβ degradation.In summary, the present report clearly demonstrates that sodium arsenite and DMA affect processing of APP in vitro.     

T-type calcium channel expression in cultured human neuroblastoma cells

Human neuroblastoma cells (SH-SY5Y) have similar structures and functions as neural cells and have been frequently used for cell culture studies of neural cell functions.Previous studies have revealed L-and N-type calcium channels in SH-SY5Y cells.However,the distribution of the low-voltage activated calcium channel (namely called T-type calcium channel,including Cav3.1,Cav3.2,and Cav3.3) in SH-SY5Y cells remains poorly understood.The present study detected mRNA and protein expres-sion of the T-type calcium...     

SNCA基因过表达慢病毒质粒构建及稳定转染293T细胞系

目的构建SNCA基因过表达慢病毒质粒,转染293T细胞,建立稳定转染细胞系。方法应用PCR技术扩增目的基因,并将扩增产物插入慢病毒载体质粒pGC-FU上,并对阳性克隆进行基因测序鉴定。pGC-FU-SNCA-GFP重组质粒包装293T细胞,转染24小时后,用荧光显微镜观察标签GFP绿色荧光蛋白的表达,并用West blotting法测定目的蛋白的表达。结果成功构建了pGC-FU-SNCA-GFP慢病毒过表达质粒,获得了稳定转染的293T细胞株。结论人SNCA基因过表达慢病毒载体成功,构建和稳定转染293T细胞系的建立,为进一步体外研究α-突触核蛋白的功能奠定了基础。     

Tumor necrosis factor and interleukin-1 down-regulate receptors for substance P in human astrocytoma cells

This study examined the influence of cytokines on substance P (SP) receptors (NK1 subtype) in the human astrocytoma cell line UC11. Following trypsinization and passage, the density of SP receptors in these cells was rather low but gradually increased several fold over the course of a few days in culture. Frequent replacement of the growth medium enhanced the density of receptors even more, suggesting that growth factors in the culture medium may determine the levels of receptor. Exposure of the cells to sub-nanomolar concentrations of tumor necrosis factor (TNFα) or interleukin-1β (IL1β), but not interleukin-2 or interleukin-6, decreased the density of SP receptors. This was accompanied by a decrease in the ability of SP to stimulate inositolphosphate formation. The ability of histamine to activate inositolphosphate formation was not influenced by the cytokines. The decrease in SP receptor density was readily reversible on washout of the cytokines. The EC₅₀ for TNFα was approximately 0.5 ng/ml, the EC₅₀ for IL1β was approximately 0.1 ng/ml. Radioligand binding studies with [¹²⁵I]TNFα indicated the presence of a low density of high affinity binding sites for this ligand: K_d = 2.5 ± 0.6ng/ml, B_max = 14.8 ± 2.7fmol bound/mg protein (assuming trimeric form of ligand bound). The most likely explanation for the cytokine effect is an inhibition of the synthesis of new receptors.     

促自噬剂AZD8055对U87起始细胞Notch通路相关蛋白的表达和成球能力的影响

目的研究促自噬剂AZD8055对U87细胞株起始细胞Notch通路相关蛋白的表达和成球能力的影响。方法应用免疫磁珠法分选获得U87细胞株CD133阳性的胶质瘤起始细胞（GICs）。采用免疫荧光染色技术鉴定GICs细胞球干性标志物CDl33和Nestin,以及自噬相关蛋白LC3B和p62的表达。以CCK8检测AZD8055对U87起始细胞存活率的影响。采用Western blot检测AZD8055不同浓度处理组Notch1、Notch胞内域、Hes1、p62、LC3B蛋白的表达。通过单细胞成球实验观察AZD8055对GICs成球能力的影响。结果免疫荧光染色结果显示,GICs细胞球中CD133和Nestin呈阳性表达。CCK8检测结果提示,随着AZD8055浓度的增高,U87起始细胞的存活率逐渐降低。Western blot结果提示,与溶媒对照组相比,随着AZD8055浓度的升高,药物处理组的自噬水平增高,而Notchl通路相关蛋白的表达降低,且各浓度组之间的蛋白表达差异均有统计学意义（均P〈0．05）。免疫荧光染色结果提示,经AZD8055处理后U87起始细胞的LC3B表达明显升高,p62表达降低。单细胞成球实验显示,随着AZD8055药物浓度的增高,各组成球数目和细胞球体积递减,且差异有统计学意义（均P〈0．05）,说明U87GICs的成球能力降低。结论AZD8055通过诱发自噬抑制了Notch通路相关蛋白的表达,并且AZD8055能够抑制U87GICs的成球能力。     

pEGFP-ING4真核表达载体的构建及在人脐静脉内皮细胞中的表达

目的构建携带生长抑制因子4(ING4)基因的真核表达载体pEGFP-ING4并转染人脐静脉内皮细胞(HUVEC),为进一步研究ING4对胶质瘤血管生成影响提供基础。方法提取人胎盘细胞中总RNA,RT-PCR扩增ING4并克隆至pEGFP-C2载体,酶切电泳鉴定出阳性克隆送测序,利用靶向转染试剂jetPEI-HUVEC转染HUVEC,通过免疫组化检测转染细胞中ING4的表达。结果 成功扩增ING4基因,基因全长747 bp。重组质粒pEGFP-ING4的酶切鉴定及测序结果均证明ING4基因成功克隆到真核表达载体中。酶切片段电泳结果表明:目的条带与ING4的开放读码框大小相符;测序结果和ING4的开放读码框比对相似性为100%。转染重组质粒后的HUVEC中ING4蛋白表达水平增强。结论本实验成功构建携带ING4基因的真核表达载体pEGFP-ING4,并可在HUVEC中获得ING4蛋白的增强表达。     

Suppression of MYC by high expression of NMYC in human neuroblastoma cells

Members of the MYC gene family, including MYC, NMYC, and LYMC, have been found amplified and expressed at high level in various human cancers. We have analysed the expression of two members of the MYC gene family, NMYC and MYC, in human and murine neuroblastoma cells. Whenever NMYC and MYC are co-expressed, MYC expression predominates. Cells carrying high expression of NMYC as result of amplification lack MYC expression. The same is ture for neuroblastoma cells in which expression of a single-copy NMYC is upregulated or into which a vector forcing high expression of an exogenous NMYC had been introduced by transfection. Our studies indicate a regulatory interaction between MYC and NMYC in neuroblastoma cells.     

人sTRAIL基因载体构建及其在真皮干细胞的表达

背景：基于肿瘤坏死因子(tumour necrosis factor，TNF)可溶性相关凋亡诱导配体(soluble related apoptosis inducing ligand, sTRAIL)抗瘤的生物特性，克隆sTRAIL基因，构建其真核表达载体，为肿瘤细胞凋亡的实验研究奠定基础。 目的：构建真核表达载体pEGFP-N1/sTRAIL，并转染真皮干细胞，以探究sTRAIL基因导入真皮干细胞的表达情况。 方法：采用RT-PCR二步法，以人胎盘组织总RNA为模板，扩增sTRAIL的cDNA序列，将其克隆入pMD18-T载体，随后亚克隆入载体pEGFP-N1中，构建其真核瞬时表达载体pEGFP-N1/sTRAIL，以Fugene 6转染技术，将sTRAIL基因导入真皮干细胞。倒置显微镜下观测转染后真皮干细胞生长变化及sTRAIL在其中的表达等情况。RT-PCR法对目的基因转染的真皮干细胞进行鉴定。 结果与结论：克隆到sTRAIL基因的cDNA序列，成功构建了其真核表达载体，该真核表达载体能携带sTRAIL基因在真皮干细胞中正常表达。倒置显微镜下观测到转染后真皮干细胞生长状态与未转染的真皮干细胞无差异。以经sTRAIL基因转染的真皮干细胞该基因组DNA为模板，用RT-PCR法能扩增到sTRAIL基因cDNA序列。     

Differential expression of heat shock protein HSP27 in human neurons and glial cells in culture

HSP27 expression was investigated in cultured neurons and glial cells isolated from fetal human brains using immunoblotting and immunocytochemistry. Under unstressed conditions, HSP27 was identified at a high level in astrocytes (>99%), at a low level in neurons (7%), and at a minimally detectable level in microglia (<1%), whereas it was undetectable in oligodendrocytes. Under these conditions, HSP27 was located in the cytoplasm, fractionated into the Triton X-100-soluble phase, and composed chiefly of the basic isoform (HSP27a). After exposure to heat stress (43°C90 min), the level of HSP27 exproion ryas not altered in astrocytes but was elevated significantly in neurons (11–21%) and microglia (4–7%) during 8–48 hr postrecovery periods, while it remained undetectable in oligodendrocytes. In addition, various human neural cell lines exhibited differential patterns of HSP27 expression under unstressed and heat-stressed conditions. Following heat shock treatment (45°C/30 min), granular aggregates of HSP27 were identified in the cytoplasm of astrocytes. Under heat-stressed conditions, HSP27 was distributed within the Triton X-100-insoluble fraction associated with an increase in two more acidic isoforms (HSP27b and HSP27c). HSP27 and αβ-crystallin were coexpressed in astrocytes under unstressed and heat-stressed conditions. When astrocytes were exposed to known HSP27 inducers, hydrogen peroxide and cysteamine reduced the synthesis of HSP27, while estradiol showed no effects. The differential patterns of constitutive and heat-induced expression of HSP27 in cultured human neurons and glial cells suggest that the cellular mechanisms by which HSP27 expression is regulated are different among various cell types in the human central nervous system. © 1995 Wiley-Liss, Inc.     

MHC Class II expression by human glioma cells after in vitro incubation with soluble antigens

Recent studies have suggested that astrocytes share with the macrophages several properties in vitro, among which is the ability to express MHC Class II molecules and to present some antigens to syngeneic primed lymphocytes (MHC-restricted presentation). It has been claimed in the literature that astrocytes cannot start the presentation and cannot express the related MHC Class II molecules if not previously stimulated with gamma IFN. In this paper we report that 2 human GFAP + glioma cell lines, incubated in culture with various soluble antigens for at least 24 h, were able, in the absence of gamma IFN or of activated lymphocytes, to express the MHC Class II and to expose the antigens on their surfaces. Moreover, when a lysosomotropic agent such as chloroquine was added during the incubation, no MHC Class II expression was observed. This last datum suggests that in astrocytes, as is already known in macrophages, the processing of antigens and their assembling with MHC Class II molecules probably involves the lysosomal apparatus.