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Human exposure to polycyclic aromatic hydrocarbons (PAHs) results in adverse health implications. However, the specific impact of paternal preconception PAHs exposure has not been fully studied. In this study, a total of 219 men aged 24&ndash, 53 were recruited and an investigation was conducted using a questionnaire requesting information about age, occupation, education, family history, lifestyle, and dietary preferences. Urine and semen samples were examined for the levels of the hydroxyl metabolites of PAHs (OH-PAHs) using ultra-high-performance liquid chromatography&ndash, tandem mass spectrometry and sperm DNA methylation by pyrosequencing. The results from the correlation analysis using seven OH-PAHs and the average methylation levels of the imprinting genes H19, PEG3, and MEG3 indicated that 1-OHPH is positively correlated with H19/PEG3 methylation levels. We further examined the correlation between each OH-PAH and the methylation levels at the individual CpGs. The results showed 1-OHPH is specifically correlated with CpG4 and CpG6 of the imprinted gene H19, CpG1 and CpG2 of PEG3, and CpG2 of MEG3, whereas 1-OHP is positively correlated with PEG3 at CpG1. Multivariate regression model analysis confirmed that 1-OHPH and 1-OHP are independent risk factors for the methylation of H19. These data show that sperm DNA imprinting genes are sensitive to adverse environmental perturbations.

Background Benzo[a]pyrene (BaP) is an environmental pollutant known to cause teratogenesis. However, the mechanism underlying this teratogenic effect is not fully understood. Recently, the alteration of DNA methylation of imprinting genes has emerged as a specific epigenetic mechanism linking the impact of environmental pollutants on embryonic development to paternal exposures. The aim of this study was to investigate the transgenerational effects of paternal BaP exposure on the imprinting genes in mouse sperm DNA. Methods Male C57BL/6J mice received BaP (1.0 or 2.5 mg/kg) or olive oil twice a week for 12 weeks. The methylation status of 6 imprinting genes (H19, Meg3, Peg1, Peg3, Igf2 and Snrpn) was examined by bisulfite pyrosequencing of the sperm DNA of BaP-exposed F0 generation and their offspring. Results BaP exposure reduced the methylation levels in the imprinting genes H19 and Meg3 and increased the methylation levels of Peg1 and Peg3; however, no significant differences was observed for the methylation levels of Igf2 or Snrpn in the sperm DNA. Furthermore, BaP-exposed male mice were mated with unexposed female mice to generate F1-2 generations. The methylation levels of the 6 genes in the sperm DNA from F1-2 offspring showed a similar pattern as that of the F0 male. The effects were attenuated in F1-2 generations. Conclusions Paternal BaP exposure altered the methylation levels of imprinting genes, implicating that imprinting genes are susceptible to environmental toxicants. Furthermore, a similar alteration was observed in the F1-2 generations although the attenuated in methylation in F2 generation, revealing a potential transgenerational effect.

Aims Fully understanding the assembly patterns of plant functional traits in plant communities is a big challenge in the study of community ecology. The assembly patterns in alpline meadow and driving factors are still poorly known. In this study, experiments with different mowing intensities and various treatments of fertilization were conducted in alpline meadow for the purpose of improving the understanding of the assembly patterns of plant functional traits in plant community. Methods Data on functional diversity of 10 traits for 33 species from 108 quadrats (with varying combinations of mowing and fertilization) were analyzed by randomizations tests, ANCOVA and regression analysis, respectively, for the trait convergence/divergence. Important findings None of the traits studied showed consistently significant convergence or divergence pattern in integral gradients of fertilization and mowing treatments, while the ratios of quadrats assembled under deterministic and stochastic processes were 82.7% and 17.3%, respectively. Among the functional traits tested, growth form, life cycle, aboveground dry mass per plant, leaf area and leaf dry mass showed a stochastic pattern of assembly, but they were not affected by variations in mowing treatments and community characteristics (i.e. aboveground net primary productivity, biomass loss and vegetation height). Convergence or divergence patterns were shown in traits such as plant inclination, reproductive modes, nitrogen fixation, plant height and specific leaf area, which were affected by variations in mowing treatments and community characteristics. Among them, assembly patterns of plant inclination and specific leaf area were only affected by community characteristics, while nitrogen fixation, reproductive modes and plant height were affected by both mowing treatments and community characteristics. Moreover, the patterns of assembly varied by traits. Aboveground net primary productivity and biomass 吕美强等: 刈割与施肥干扰下高寒草甸植物功能性状的构建模式 917 doi: 10.3724/SP.J.1258.2014.00086 loss well explained variations in the assembly pattern of plant functional traits. Mowing and fertilization showed the reversed effects on the assembly pattern of plant height, while no significant interaction was found between mowing and fertilization. The results suggested that there were different assembly patterns of functional traits in the community: stochastic pattern of assembly was the primary, and the deterministic pattern was secondary. Deterministic pattern was trait-dependent and correlated with variations in mowing treatments and community characteristics. The balancing effect of opposite selection forces, on the other hand, could lead to stochastic assembly patterns in plant functional traits.

Background Recently, an increasing number of human and animal studies have reported that exposure to benzo(a)pyrene (BaP) induces neurological abnormalities and is also associated with adverse effects, such as tumor formation, immunosuppression, teratogenicity, and hormonal disorders. However, the exact mechanisms underlying BaP-induced impairment of neurological function remain unclear. The aim of this study was to examine the regulating mechanisms underlying the impact of chronic BaP exposure on neurobehavioral performance. Methods C57BL mice received either BaP in different doses (1.0, 2.5, 6.25 mg/kg) or olive oil twice a week for 90 days. Memory and emotional behaviors were evaluated using Y-maze and open-field tests, respectively. Furthermore, levels of mRNA expression were measured by using qPCR, and DNA methylation of NMDA receptor 2B subunit (NR2B) was examined using bisulfate pyrosequencing in the prefrontal cortex and hippocampus. Results Compared to controls, mice that received BaP (2.5, 6.25 mg/kg) showed deficits in short-term memory and an anxiety-like behavior. These behavioral alterations were associated with a down-regulation of the NR2B gene and a concomitant increase in the level of DNA methylation in the NR2B promoter in the two brain regions. Conclusions Chronic BaP exposure induces an increase in DNA methylation in the NR2B gene promoter and down-regulates NR2B expression, which may contribute to its neurotoxic effects on behavioral performance. The results suggest that NR2B vulnerability represents a target for environmental toxicants in the brain.

Background: Increasing evidence indicates that repeated exposure to and withdrawal from alcohol can result in persistent molecular and cellular adaptations. One molecular adaptation that occurs is the regulation of gene expression, which is thought to lead to the functional alterations that characterize addiction: tolerance, dependence, withdrawal, craving, and relapse. MicroRNAs (miRNAs) have been recently identified as master regulators of gene expression through post-transcriptional regulation. However, the role of miRNAs in the neuroadaptations after alcohol removal has not yet been directly addressed. Methods: We employed a chronic intermittent ethanol (CIE) model in primary cortical neuronal cultures to examine the global extent of differential miRNA expression using a TaqMan real-time PCR miRNA array. Results: Sixty-two miRNAs were differentially expressed after 10 days of CIE (CIE10) treatment (n = 42 with false discovery rate [FDR] 2) and 5 days post-CIE (P5) treatment (n = 26) compared with untreated control values. Compared to CIE10, ethanol (EtOH) removal experience in P5 induced a distinct expression pattern, including 20 differentially expressed miRNAs, which did not exhibit a significant change at CIE10. The predicted target molecules of EtOH removal-induced miRNAs function mainly in the regulation of gene transcription, but also function in neuron differentiation, embryonic development, protein phosphorylation, and synaptic plasticity. Interestingly, some of the miRNAs differentially expressed 5 days after CIE treatment were found to cluster on chromosomes near CpG islands, suggesting that they share functional similarity by targeting alcohol-related genes. Conclusions: Taken together, these results suggest a potential role of differentially expressed miRNAs in mediating EtOH removal-related phenotypes.

A novel liquid chromatography-atmospheric-pressure chemical ionization-mass spectrometry (LC-APCI/MS) method was developed and validated for the simultaneous determination of four sesquiterpene pyridine alkaloids (wilfortrine, wilfordine, wilforgine and wilforine) in human plasma. The chromatographic separation was performed on a Shim-pack XR-ODS column using an ammonium acetate buffer solution-acetonitrile in a gradient program. The detection was achieved by an ion trap mass spectrometry in the positive selected ion monitoring (SIM) mode. The method utilized acetonitrile as protein precipitation solvent and followed by solid-phase extraction (SPE). Calibration curves were linear for the four alkaloids over the range of 0.5-100.0 μg/L with the limits of quantification of 0.5 μg/L, while the method exhibited the recovery of 86.5-98.6%, intra- and inter-day RSDs of less than 8.2% and 12.8%, respectively. Methodology was validated in line with the EU requirements (Commission Decision 2002/657/EC). Results of incurred samples demonstrated excellent reproducibility. To our knowledge, this is the first analytical method for simultaneous determination of the four sesquiterpene pyridine alkaloids in plasma. The method was applicable to clinical pharmaceutical research of alkaloids in rheumatoid arthritis volunteer patients after oral administrations.

Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α- and β-tubulin exist as numerous isotypes, differing in their amino acid sequences and encoded by different genes. The differences are highly conserved in evolution, suggesting that they are functionally significant. Neurons are a potentially very useful system for elucidating this significance, because they are highly differentiated cells and rich in tubulin isotypes. We have examined the distribution of β-tubulin isotypes in mouse primary cultured cortical neurons from embryonic fetus, newborn pups and adults. Neurons from both embryonic and adult mouse brains express the βI, βII, and βIII isotypes, but apparently not βIV or βV. βI, βII, and βIII are found in both cell bodies and neurites. However, the situation is different in newborn mice. Although βI and βIII are present in these neurons in both cell bodies and neurites and βIV is absent, just like in embryonic and adult mice, two striking differences were noted in the neurons from newborn mice. First, βV is apparently present evanescently in the neurons in both cell bodies and neurites. Interestingly, the βV was expressed strongly in newborn neurons after one day of culture; expression became much weaker after 3 days, and almost disappeared after 5 days. Second, the distribution of βII is different from other isotypes. After newborn mouse neurons were cultured for 3 days, βII started to disappear partly from the cell bodies; this was much more pronounced after five days in culture. Our findings suggest that βII's major function may involve the neurites and not the cell body. They also raise the possibility that βV has a unique role in the neurons of newborn mice.

Expression of the NMDA receptor 2B (NR2B) gene is upregulated following chronic intermittent ethanol (CIE) treatment and withdrawal, which underlies behavioral alterations in addiction. The goal of this study was to characterize the changes of histone modifications induced by CIE treatment and its subsequent removal associated to the upregulation of NR2B gene transcription. To investigate the involvement of histone acetylation in the effect of ethanol on the NR2B gene, we examined the influence of CIE on histone acetylation in the 5′ regulatory region of NR2B using a qChIP assay. CIE treatment and its subsequent removal produced a remarkable and selected increase in histone H3K9 acetylation. Interestingly, the majority of the increased H3K9 acetylation occurred after ethanol removal, which was coincident with a decrease in H3K9 methylation in the same time duration. Further examination of the mechanisms of ethanol-induced alterations on the histone modifications revealed that CIE-induced acetylation of H3K9 was not due to the changes in global enzyme activities or the expression of histone acetyltransferases (HATs) and deacetylase (HDACs). Instead, we found a significant downregulation in some histone methyltransferases (HMTs) at both the global level and the local chromatin of the NR2B gene following CIE treatment. Moreover, our experiments also indicated a decrease of G9a, Suv39 h1 and HDAC1–3 in the chromatin of the NR2B gene promoter, which may be responsible for the altered H3K9 modifications. Taken together, the findings suggest a mechanism where the changes in H3K9 modifications in the local chromatin of the NR2B gene underlie alcohol-induced neuroadaptation.

Acetaminophen has been used as an analgesic for more than a hundred years, but its mechanism of action has remained elusive. Recently, it has been shown that acetaminophen produces analgesia by the activation of the brain endocannabinoid receptor CB1 through its para-aminophenol (p-aminophenol) metabolite. The objective of this study was to determine whether p-aminophenol could be toxic for in vitro developing mouse cortical neurons as a first step in establishing a link between acetaminophen use and neuronal apoptosis. We exposed developing mouse cortical neurons to various concentrations of drugs for 24 hr in vitro. Acetaminophen itself was not toxic to developing mouse cortical neurons at therapeutic concentrations of 10-250 μg/ml. However, concentrations of p-aminophenol from 1 to 100 μg/ml produced significant (p < 0.05) loss of mouse cortical neuron viability at 24 hr compared to the controls. The naturally occurring endocannabinoid anandamide also caused similar 24-hr loss of cell viability in developing mouse cortical neurons at concentrations from 1 to 100 μg/ml, which indicates the mechanism of cell death could be through the cannabinoid receptors. The results of our experiments have shown a detrimental effect of the acetaminophen metabolite p-aminophenol on in vitro developing cortical neuron viability which could act through CB1 receptors of the endocannabinoid system. These results could be especially important in recommending an analgesic for children or individuals with traumatic brain injury who have developing cortical neurons.

Background Repeated alcohol exposure is known to increase subsequent ethanol consumption in mice. However, the underlying mechanisms have not been fully elucidated. One postulated mechanism involves epigenetic modifications, including histone modifications and DNA methylation of relevant genes such as NR2B or BDNF. Methods To investigate the role of epigenetic mechanisms in the development of alcohol drinking behavior, an established chronic intermittent ethanol exposure reinforced ethanol drinking mouse model with vapor inhalation over two 9-day treatment regimens was used. The DNA methyltransferase inhibitor, 5-azacytidine or the histone deacetylase inhibitor, Trichostatin A was administered (intraperitoneally) to C57BL/6 mice 30 min before daily exposure to chronic intermittent ethanol. Changes in ethanol consumption were measured using the 2-bottle choice test. Results The results indicated that systemic administration of Trichostatin A (2.5 µg/g) facilitated chronic intermittent ethanol-induced ethanol drinking, but systemic administration of 5-azacytidine (2 µg/g) did not cause the same effect. However, when 5-azacytidine was administered by intracerebroventricular injection, it facilitated chronic intermittent ethanol-induced ethanol drinking. Furthermore, the increased drinking caused by chronic intermittent ethanol was prevented by injection of a methyl donor, S-adenosyl-L-methionine. To provide evidence that chronic intermittent ethanol- or Trichostatin A-induced DNA demethylation and histone modifications of the NR2B promoter may underlie the altered ethanol consumption, we examined epigenetic modifications and NR2B expression in the prefrontal cortex of these mice. Chronic intermittent ethanol or Trichostatin A decreased DNA methylation and increased histone acetylation in the NR2B gene promoter, as well as mRNA levels of NR2B in these mice. Conclusions Taken together, these results indicate that epigenetic modifications are involved in regulating ethanol drinking behavior, partially through altering NR2B expression.

Neuron-restrictive silencer factor (NRSF) is a transcriptional repressor of multiple neuronal genes. This study addressed the role of NRSF in N-methyl-D-aspartate (NMDA) receptor NR2B promoter activity and the molecular mechanisms of ethanol-induced NR2B up-regulation in fetal cortical neurons. The 5'-flanking region of the NR2B gene contains five NRSE-like elements. Functional analysis of the upstream regions of the NR2B gene by transient transfection of neurons revealed that neuron-restrictive silencer element (NRSE) motifs located between base pair -1407 and -2741 represses transcription of the gene. Analysis by electrophoretic mobility shift assay and reporter gene assay identified NRSE2 and 3 as responsible for repressing NR2B gene transcription. The identity of NRSF as the functional binding factor is suggested by the specific binding of in vitro synthesized NRSF or cell lysate to the labeled probes and the specific antibody-induced supershift. Furthermore, whereas mutations of NRSE2 and 3 motifs increased the promoter activity, overexpression of NRSF reduced it significantly. The pattern of NRSF expression during development was investigated and demonstrated that the highest expression is on embryonic day 14 with moderate expression on postnatal day 0, reflecting a possible role of NRSF as a regulator during development. Treatment of cultured cortical neurons with 100 mM ethanol for 5 days caused a significant decrease in the NRSF mRNA and protein levels, NRSF/NRSE binding activity, and an increase in the promoter activity. Therefore, our studies suggest that NRSF is a negative regulator of NR2B expression and may contribute to the ethanol-induced up-regulation of the NR2B gene in fetal cortical neurons.

Prenatal alcohol exposure produces many developmental defects of the central nervous system (CNS), such as in the corpus callosum (CC). This study was designed to observe the effect of prenatal alcohol exposure during the second trimester equivalent on the development of dendritic arbors of CC projection neurons (CCpn) in rat visual cortex. In addition, the effect of second trimester equivalent prenatal alcohol exposure on brain weight was determined. Pregnant dams received 1.2-6.0 g/kg ethanol (EtOH) during gestational day (G) 11-20. Controls consisted of normal and nutritionally matched pairfed (PF) dams. Pups were sacrificed on the day of birth, G26, G29 and G33. DiI crystals were placed in the midsagittal CC bundle to retrogradely label CCpn. Images of visual cortex were obtained from tissue slices using a confocal laser scanning microscope. The number and length of apical and basilar dendrite branches were determined. The results show that prenatal alcohol exposure restricted to the second trimester equivalent alters the development of the CCpn dendritic arbor and the brain weight in a blood alcohol concentration (BAC)-dependent manner. The alteration in the EtOH CCpn is manifested as an increase in the number and length of CCpn apical and basilar dendrite branches, while brain weight is reduced compared with Controls.

This study investigated the effect of NO on c-Fos expression using immunocytochemical techniques, following training in the one-trial passive avoidance response and Morris water maze tasks. Fos-like immunoreactive (FLI) neurons were examined in the hippocampus and cerebral cortex of rats in which intracerebroventricular (i.c.v.) injection of Nomega-nitro-L-arginine (NAME) was given 30 min before training in the experimental groups. The results showed that: (1) when compared with untreated rats or rats receiving i.c.v. saline injection, fos-like immunoreactive neurons were significantly increased in both brain regions following training in either task, and (2) preceding i.c.v. injections of NAME blocked both the formation of memory and the increased expression of c-Fos in both brain regions subjected to training on the two models. These results suggest that NO production may be a critical factor for the formation of memory and the enhanced c-Fos expression, and so might contribute to the long-term plasticity of synaptic organization in the two brain regions.

The number and distribution of Fos-like-immunoreactive neurons in different supraspinal brain areas induced by formalin injection into one hindpaw was estimated in rats with transected dorsal half of the spinal cord at the thoracic level in an attempt to avoid most of the descending modulatory actions. The results showed that: (i) after spinal lesion, the peripheral noxious inputs, going up mainly through the ventral spinal cord, elicited a more widespread and densely located Fos-like-immunoreactive neurons in subcortical areas, many of them showed no Fos expression when noxious stimulation was given in rats with intact spinal cord; (ii) at the same time, a small number of subcortical areas, such as the lateral ventroposterior thalamic nucleus and dorsal raphe nucleus, exhibited no significant increase of nociceptive Fos-like immunoreactive neurons after spinal lesion as compared to that with intact spinal cord; and (iii) there appeared a prominent expansion of cortical areas with densely located Fos-like-immunoreactive neurons in spinal-lesioned rats as compared with the limited labelled areas in the control group with intact spinal cord. These results indicate that: (i) in avoiding the spinally descending modulatory mechanisms, more widespread supraspinal and cortical neurons will be recruited and activated in response to the noxious stimulation; and (ii) the descending systems exert differential actions on the spinal targets which project nociceptive signals to different supraspinal regions. The implication of these facts is discussed.

NOX1, a member of the NOX family of NADPH oxidases, is expressed primarily in colon epithelium, where it may function in host defense and growth regulation. We investigated factors responsible for its transcriptional regulation in vitro and its expression in vivo. Analysis of promoter constructs in the CaCo2 cell line identified a complex element between -422 and -291 critical for promoter activity. This element contained four sites that bound GATA-4, -5, and -6 in vitro with varied affinities. One site also bound the caudal-related homeodomain proteins Cdx1 and Cdx2, whereas another also bound hepatocyte nuclear factor-1alpha (HNF-1alpha). GATA-6, HNF-1alpha, and Cdx2 also bound to this region in the intact chromatin of CaCo2 cells. These factors demonstrated cooperativity when transactivating the NOX1 promoter. NOX1 mRNA was detected in human colon epithelial cells along the crypt-villus axis. A gradient of NOX1 mRNA expression was seen in the colons of normal as well as germ-free mice, with significantly higher levels in distal compared with proximal segments. The expression gradients of NOX1 mRNA in the colon paralleled those of GATA-6, HNF-1alpha, and Cdx1. These data indicate that developmental, tissue-restricted transcription factors play a key role in NOX1 regulation in vivo.

Macrophages have the potential to deliver therapeutic genes to many target tissues. Macrophage-specific synthetic promoters (SPs) generated by random ligation of myeloid/macrophage cis elements had activity up to 100-fold that of a native macrophage promoter in macrophage cell lines, but were minimally active in nonmyeloid cells. Mouse bone marrow cells (BMCs) transduced ex vivo with lentivectors expressing green fluorescent protein (GFP) driven either by an SP (SP-GFP) or a cytomegalovirus (CMV) promoter (CMV-GFP) were used for syngeneic transplantation of lethally irradiated mice. Blood leukocytes showed stable GFP expression for up to 15 months after transplantation. SP-GFP expression was selective for CD11b+ macrophages, whereas CMV-GFP expression was observed in erythrocytes, as well as in both CD11b+ and CD11b- leukocytes. Furthermore, SP-GFP expression was much stronger than CMV-GFP expression in CD11b+ macrophages. apoE-/- BMCs transduced with the lentiviral vector encoding human apoE were used to transplant apoE-/- mice. Macrophage expression of apoE from 10 to 26 weeks of age significantly reduced atherosclerotic lesions in recipient apoE-/- mice. Thus, the novel SPs, especially when combined with lentivectors, are useful for macrophage-specific delivery of therapeutic genes.

We have shown previously that long-term ethanol treatment causes an up-regulation of N-methyl-D-aspartate (NMDA) receptor 2B subunit (NR2B) number and function in cultured fetal mouse cortical neurons. To examine the intracellular signaling pathways involved in this NR2B gene transcription, we have subjected fetal cortical neurons to long-term treatment with ethanol and studied its effect on cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) levels by Western blot and enzyme-linked immunosorbent assay. We find a significant increase in phosphorylated CREB, without change in total CREB protein, in cells treated with ethanol for 5 days. Long-term ethanol treatment did not increase levels of both total and phospho-ERK in serum-free medium, whereas it did increase ERK phosphorylation in medium containing serum, without affecting total ERK levels. CREB phosphorylation was increased by ethanol treatment in both media, irrespective of the presence of serum. Electrophoretic mobility shift assay, using a 25-base pair (bp) double-stranded DNA fragment containing the cyclic AMP response element (CRE)-like sequence of the NR2B promoter as (32)P-labeled probe, showed an increase in specific CRE binding to nuclear proteins isolated from cells undergoing long-term ethanol treatment. A 467-bp DNA fragment of the NR2B promoter containing the CRE sequence cloned into the luciferase vector exhibited high reporter activity in transient cotransfection assay of mouse cortical neurons, and ethanol treatment increased this activity. Introducing site-directed mutation in the CRE sequence significantly reduced the reporter activity relative to the wild-type construct, and it also abolished the stimulatory effect by ethanol. Our results indicate that CREB is probably involved in mediating ethanol-induced up-regulation of NR2B gene.

Inhibitors of apoptosis (IAPs) antagonize cell death and regulate the cell cycle. One mechanism controlling IAP expression is translation initiation through the internal ribosome entry sites. Alternatively, IAP expression can be regulated at the transcription level. We showed recently the activation of IAP2 transcription by severe hypoxia. To pursue this regulation, we have cloned the full-length cDNA of rat IAP2, and have isolated and analysed the promoter regions of this gene. The cDNA encodes a protein of 589 amino acids, exhibiting structural features of IAP. In rat tissues, a major IAP2 transcript of approximately 3.5 kb was detected. We subsequently isolated 3.3 kb of the proximal 5'-flanking regions of this gene, which showed significant promoter activity. Of interest, 5' sequential deletion of the promoter sequence identified an enhancer of approximately 200 bp. Deletion of cAMP-response-element-binding protein (CREB) sites in the enhancer sequence diminished its activity. Finally, the IAP2 gene promoter was activated significantly by severe hypoxia and not by CoCl(2) or desferrioxamine, pharmacological inducers of hypoxia-inducible factor-1. In conclusion, in this study we have cloned the full-length cDNA of rat IAP2, and for the first time we have isolated and analysed promoter sequences of this gene, leading to the identification of enhancer elements. Moreover, we have demonstrated activation of the gene promoter by severe hypoxia, a condition shown to induce IAP2. These findings provide a basis for further investigation of gene regulation of IAP2, a protein with multiple functions.

Macrophages, derived from hematopoietic stem cells (HSC), play a key role in atherosclerosis. To treat atherosclerosis, we plan to alter expression of relevant genes in macrophages by ex vivo transduction of HSC with lentiviral vectors carrying a macrophage-specific promoter. Myeloid promoter cis-elements were randomly ligated and the resultant DNA inserted into a reporter vector upstream of a myeloid basal promoter. Luciferase activity of some synthetic promoters was 10-200-fold over that of the CSF1R macrophage promoter in Thp-1 and other macrophage cell lines, but quite low in non-macrophage cells compared with the CMV promoter. Mouse bone marrow was collected, transduced ex vivo with lentiviral vectors expressing GFP driven either by a synthetic promoter (SP-GFP) or CMV promoter (CMV-GFP), and transplanted into lethally irradiated recipients. At various time points (1.5, 4, 8, 11, 15 months) post-transplant, peripheral blood analyzed by FACS and GFP expression was quite stable over time. SP-GFP expression was detected in most CD11b+ leukocytes, but only in a small fraction of CD11b- cells and not at all in erythrocytes, while CMV-GFP expression was observed ubiquitously. To test the promoters in macrophage gene therapy for atherosclerosis, we transduced apoE-/- BM cells with a lentiviral vector encoding human apoE (apoE) and transplanted them into apoE-/- mice. Comparable levels of apoE were measured in culture media of peritoneal macrophages from either SP-apoE or CMV-apoE recipient mice. However, serum apoE of SP-apoE recipients was only one fourth of that of CMV-apoE, implying that SP activity was restricted to the monocyte/macrophage lineage. Macrophage expression of apoE from 10 to 26 weeks of age significantly reduced atherosclerotic lesions in recipient mice. In conclusion, the combination of the synthetic macrophage promoter, lentiviral vectors, and bone marrow stem cell transplantation ameliorates atherogenesis and leads to a novel treatment of this disease.

Background The NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5′-regulatory area following CIE treatment. Methods Primary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5′-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP) assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA) and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity. Results Analysis of individual CpG methylation sites within the NR2B 5′regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced DNA demethylation is accompanied by increased occupation by transcription factors. Conclusions These results suggest an important role of DNA demethylation in mediating CIE-induced NR2B gene up-regulation, thus implicating a novel molecular site of alcohol action.