Dopaminergic genes modulate response inhibition in alcohol abusing adults.

Compulsion in alcohol use disorders (AUD) has been attributed to impairment in response inhibition. Because genes that regulate dopamine (DA) have been implicated not only for risk for AUD but also for impulsivity based on behavioral studies, we set out to examine the underlying neural mechanisms associated with these effects. We collected functional magnetic resonance imaging images on 53 heavy drinking but otherwise healthy adults while performing the Go/NoGo task. We predicted that genetic variants previously reported in the literature to be associated with substance abuse, specifically the DRD2 rs1799732 and DRD4 VNTR, will modulate neural processes underlying response inhibition. Our results showed differential neural response for the DRD4 VNTR during successful inhibition in the inferior frontal gyrus (IFG) (cluster-corrected P<0.05, z=1.9). Similarly, DRD2 rs1799732 groups were significantly different in the precuneus and cingulate gyrus during successful response inhibition (cluster-corrected P<0.05, z=1.9). These findings provide further evidence for the role of DAergic genes in modulating neural response in areas that underlie response inhibition and self-monitoring processes. Variants within these genes appear to influence processes related to impulsive behavior, which may increase one's risk for alcohol abuse and dependence.

EGFR isoforms and gene regulation in human endometrial cancer cells.

BACKGROUND: Epidermal growth factor (EGF) and its receptor (EGFR) constitute a principal growth-promoting pathway in endometrial cancer cells. Pre-clinical studies were undertaken to compare the expression of EGFR isoforms and the downstream effects of activating or blocking EGFR function in Ishikawa H cells, derived from a moderately differentiated type I endometrioid adenocarcinoma, or in Hec50co cells, derived from a poorly differentiated type II adenocarcinoma with papillary serous sub-differentiation. RESULTS: We investigated whether EGFR mutations are present in the tyrosine kinase domain (exons 18-22) of EGFR and also whether EGFR isoforms are expressed in the Ishikawa H or Hec50co cell lines. Sequence of the EGFR tyrosine kinase domain proved to be wild type in both cell lines. While both cell lines expressed full-length EGFR (isoform A), EGFR and sEGFR (isoform D) were expressed at significantly lower levels in Hec50co cells compared to Ishikawa H cells. Analysis of gene expression following EGF vs. gefitinib treatment (a small molecule EGFR tyrosine kinase inhibitor) was performed. Early growth response 1, sphingosine kinase 2, dual specificity phosphatase 6, and glucocorticoid receptor DNA binding factor 1 are members of a cluster of genes downstream of EGFR that are differentially regulated by treatment with EGF compared to gefitinib in Ishikawa H cells, but not in Hec50co cells. CONCLUSIONS: Type I Ishikawa H and type II Hec50co endometrial carcinoma cells both express EGFR and sEGFR, but differ markedly in their responsiveness to the EGFR inhibitor gefitinib. This difference is paralleled by differences in the expression of sEGFR and EGFR, as well as in their transcriptional response following treatment with either EGF or gefitinib. The small cluster of differently regulated genes reported here in these type I vs. type II endometrial cancer-derived cell lines may identify candidate biomarkers useful for predicting sensitivity to EGFR blockade.

Mitochondrial DNA deletion and sarcopenia.

PURPOSE: Accumulation of mitochondrial DNA deletions and the resultant impaired oxidative phosphorylation may play a pathogenic role in the mediation of age-related sarcopenia. METHODS: Twenty four participants of the New Mexico Aging Process Study were classified as normal lean (n = 15) or sarcopenic (n = 9) based on body composition determined by Dual Energy x-ray Absorptiometry. Complex I and Complex IV activities were measured in the skeletal muscle samples obtained from gastrocnemius muscle. A two-stage nested polymerase chain reaction strategy was used to identify the mitochondrial DNA deletions in the entire mitochondrial genome in the skeletal muscle samples. RESULTS: Although Complex I activity was not significantly different (5.5 +/- 0.9 vs. 4.6 +/- 0.7 mU/mg protein, P > 0.05), Complex IV activity was higher in sarcopenic subjects (1.4 +/- 0.3 vs. 1.0 +/- 0.1 mU/mg protein, P < 0.05). Mitochondrial DNA deletions were mostly located in the region of Complex I and spanned from nicotinamide adenine dinucleotide dehydrogenase 1 to nicotinamide adenine dinucleotide dehydrogenase 6. Deletions in the 8,577-10,407 bp and 10,233-11,249 bp regions were associated with a significant decrease in Complex I activity (P < 0.05 and P = 0.02, respectively). Total cumulative deletion, defined as the sum of individual length of deletions in a subject, was comparable in subjects with and without sarcopenia (1760 +/- 726 vs. 1782 +/- 888 bp, P > 0.05). The magnitude of mitochondrial DNA deletion, however, correlated positively with lean body mass (r = 0.43, P < 0.05). CONCLUSION: Thus, mitochondrial DNA deletions are common in elderly subjects and are negatively related to Complex I activity. The positive association between mitochondrial DNA deletions and lean body mass needs to be confirmed by studies in a larger study population.

Effects of naltrexone on cortisol levels in heavy drinkers.

The primary objectives of this study were to: (a) examine the neuroendocrine effects of naltrexone vs. placebo by comparing serum cortisol levels; and (b) test the biobehavioral correlates of naltrexone-induced changes in cortisol. Non-treatment seeking heavy drinkers (n=37) completed two intravenous alcohol administrations, one after naltrexone (50 mg) and one after placebo. Cortisol levels were measured at baseline and after alcohol intake (BrAC=0.06 g/dl) on both sessions, as were subjective responses to alcohol. Analyses revealed that naltrexone significantly raised overall cortisol levels compared to placebo. Cortisol levels decreased during alcohol administration and a stronger decrease was observed in the naltrexone condition. Cortisol levels were, in turn, inversely related to some of alcohol's the reinforcing effects (i.e., alcohol 'high,' vigor) and positively associated with some of its unpleasant effects (i.e., sedation and subjective intoxication). These results suggest that naltrexone alters cortisol levels in heavy drinkers and that its effects on subjective responses to alcohol may be related, in part, to naltrexone's ability to activate the HPA-axis.

Glucose up-regulates HIF-1 alpha expression in primary cortical neurons in response to hypoxia through maintaining cellular redox status.

It has been suggested that hypoxia-inducible factor 1 (HIF-1), a key regulator in cell's adaptation to hypoxia, plays an important role in the fate of neurons during ischemia. However, the mechanism of HIF-1 regulation is still not fully understood in neurons subjected to ischemia. In this study, we demonstrated that glucose up-regulated the expression of HIF-1alpha, the oxygen-dependent subunit of HIF-1, in rat primary cortical neurons exposed to hypoxia. To understand the mechanism of glucose-regulated HIF-1alpha expression, we investigated the relationships between HIF-1alpha expression, reactive oxygen species (ROS), and redox status. Low levels of HIF-1alpha protein expression were observed in the neurons exposed to in vitro ischemic conditions that had high levels of ROS (oxidizing environments), and vice versa. The glutathione (GSH) precursor, N-acetyl cysteine, induced HIF-1alpha protein expression in hypoxic neurons while the GSH synthesis inhibitor, l-buthionine sulfoximine, inhibited the expression. Moreover, (-)-epicatechin gallate, a ROS scavenger, elevated HIF-1alpha expression in the neurons subjected to in vitro ischemia. Furthermore, results from a systemic hypoxia model showed that a reducing environment increased HIF-1alpha expression in rat brains. Taken together, these data presented the first evidence that glucose promoted HIF-1alpha stabilization through regulating redox status in primary neurons exposed to hypoxia. The results imply that hypoxia only may not be sufficient to stabilize HIF-1alpha and that a reducing environment is required to stabilize HIF-1alpha in neurons exposed to hypoxia.

Epigenetic profiles of pre-diabetes transitioning to type 2 diabetes and nephropathy.

AIM: To examine DNA methylation profiles in a longitudinal comparison of pre-diabetes mellitus (Pre-DM) subjects who transitioned to type 2 diabetes mellitus (T2DM). METHODS: We performed DNA methylation study in bisulphite converted DNA from Pre-DM (n = 11) at baseline and at their transition to T2DM using Illumina Infinium HumanMethylation27 BeadChip, that enables the query of 27578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14495 genes. RESULTS: There were 694 CpG sites hypomethylated and 174 CpG sites hypermethylated in progression from Pre-DM to T2DM, representing putative genes involved in glucose and fructose metabolism, inflammation, oxidative and mitochondrial stress, and fatty acid metabolism. These results suggest that this high throughput platform is able to identify hundreds of prospective CpG sites associated with diverse genes that may reflect differences in Pre-DM compared with T2DM. In addition, there were CpG hypomethylation changes associated with a number of genes that may be associated with development of complications of diabetes, such as nephropathy. These hypomethylation changes were observed in all of the subjects. CONCLUSION: These data suggest that some epigenomic changes that may be involved in the progression of diabetes and/or the development of complications may be apparent at the Pre-DM state or during the transition to diabetes. Hypomethylation of a number of genes related to kidney function may be an early marker for developing diabetic nephropathy.

Genetic variation in the alpha synuclein gene (SNCA) is associated with BOLD response to alcohol cues.

OBJECTIVE: Preclinical studies implicate the gene encoding the alpha synuclein protein (SNCA) in the pathophysiology of alcohol dependence and dopamine neuron function. Results from clinical studies are less conclusive. Using neurobiological phenotypes in genetic studies, rather than typical heterogeneous diagnostic categories derived from self-report, may improve reliability across studies. This study aimed to examine whether polymorphisms in the SNCA gene were associated with alcohol taste cue-elicited responses in the brain, one such intermediate phenotype. METHOD: A total of 326 heavy drinkers who underwent an alcohol taste task during functional magnetic resonance imaging (fMRI) also were genotyped. Analyses focused on two previously identified SNCA variants (rs2583985 and rs356168) as well as 27 other single nucleotide polymorphisms from the Illumina Human1M BeadChip that were used in an exploratory analysis of the whole gene. Neurobiological phenotypes were defined as fMRI blood oxygenation level-dependent (BOLD) responses to alcohol taste cue (vs. a control cue) in seven regions of interest known to be involved in cue processing and rich in dopaminergic axon terminals. RESULTS: Polymorphisms in the SNCA gene were significantly correlated with BOLD activation. Specifically, the largest effect sizes and significance were seen for rs2583985 in paracingulate and caudate (focused analysis) and for rs1372522 in paracingulate (exploratory analysis). Activation in all regions of interest was correlated with alcohol-dependence severity. CONCLUSIONS: SNCA genotype was found to be associated with the degree of fMRI BOLD response during exposure to the taste of alcohol versus a control taste. This study also further validates the use of this alcohol taste task as an intermediate phenotype for alcohol-dependence severity.

A study of the influence of sex on genome wide methylation.

Sex differences in methylation status have been observed in specific gene-disease studies and healthy methylation variation studies, but little work has been done to study the impact of sex on methylation at the genome wide locus-to-locus level or to determine methods for accounting for sex in genomic association studies. In this study we investigate the genomic sex effect on saliva DNA methylation of 197 subjects (54 females) using 20,493 CpG sites. Three methods, two-sample T-test, principle component analysis and independent component analysis, all successfully identify sex influences. The results show that sex not only influences the methylation of genes in the X chromosome but also in autosomes. 580 autosomal sites show strong differences between males and females. They are found to be highly involved in eight functional groups, including DNA transcription, RNA splicing, membrane, etc. Equally important is that we identify some methylation sites associated with not only sex, but also other phenotypes (age, smoking and drinking level, and cancer). Verification was done through an independent blood cell DNA methylation data (1298 CpG sites from a cancer panel array). The same genomic site-specific influence pattern and potential confounding effects with cancer were observed. The overlapping rate of identified sex affected genes between saliva and blood cell is 81% for X chromosome, and 8% for autosomes. Therefore, correction for sex is necessary. We propose a simple correction method based on independent component analysis, which is a data driven method and accommodates sample differences. Comparison before and after the correction suggests that the method is able to effectively remove the potentially confounding effects of sex, and leave other phenotypes untouched. As such, our method is able to disentangle the sex influence on a genome wide level, and paves the way to achieve more accurate association analyses in genome wide methylation studies.

Identification of genetic and epigenetic marks involved in population structure.

Population structure is well known as a prevalent and important factor in genetic studies, but its relevance in epigenetics is unclear. Very little is known about the affected epigenetic markers and their connections with genetics. In this study we assessed the impact of population diversity on genome wide single nucleotide polymorphisms (SNPs) and DNA methylation levels in 196 participants from five ethnic groups, using principle and independent component analyses. Three population stratification factors (PSFs) were identified in the genomic SNP dataset, accounting for a relatively large portion of total variance (6%). In contrast, only one PSF was identified in genomic methylation dataset accounting for 0.2% of total variance. This methylation PSF, however, was significantly correlated with the largest SNP PSF (r = 0.72, p<1E-23). We then investigated the top contributing markers in these two linked PSFs. The SNP PSF predominantly consists of 8 SNPs from three genes, SLC45A2, HERC2 and CTNNA2, known to encode skin/hair/eye color. The methylation PSF includes 48 methylated sites in 44 genes coding for basic molecular functions, including transcription regulation, DNA binding, cytokine, and transferase activity. Among them, 8 sites are either hypo- or hyper-methylated correlating to minor alleles of SNPs in the SNP PSF. We found that the genes in SNP and methylation PSFs share common biological processes including sexual/multicellular organism reproduction, cell-cell signaling and cytoskeleton organization. We further investigated the transcription regulatory network operating at these genes and identified that most of genes closely interact with ID2, which encodes for a helix-loop-helix inhibitor of DNA binding. Overall, our results show a significant correlation between genetic and epigenetic population stratification, and suggest that the interrelationship between genetic and epigenetic population structure is mediated via complex multiple gene interactions in shared biological processes, through possibly, SNP-dependent modulation and ID2 repressor function.

Estrogen receptors alpha (ESR1) and beta (ESR2) are expressed in circulating human lymphocytes.

Bone marrow thymocytes in part mediate the bone-preserving effects of estrogen by decreasing their production of osteoclast growth factors such as interleukin-1 and -6 and tumor necrosis factor alpha in the presence of physiological amounts of estradiol. Although several in vitro studies implicate the T-lymphocyte as a candidate mediator of estrogen signaling in the skeleton, whether these cells or any lymphocytes ordinarily express one or both nuclear estrogen receptors was previously unresolved. The purpose of our investigation was therefore to ascertain, by using real-time PCR, immmunoblotting, and cytometric techniques, if any of the nuclear estrogen receptors could be detected in normal peripheral blood mononuclear cells (PBMNC) collected from healthy volunteers. The results of immunoblotting experiments revealed that both estrogen receptor alpha (ESR1) and beta (ESR2) proteins are expressed in nuclei, but not in the cytoplasm of PBMNC harvested from all of the 15 healthy male and female volunteers (aged 23-50 years) we tested. PBMNCs contained mRNA coding for the two major full-length isoforms of ESR2 and the expression of ESR2 protein was localized within a lymphocyte subpopulation by cytometric analysis. Our data provide further evidence that lymphocytes and monocytes are responsive to estrogen and underscore its importance in modulating the immune response, as well as the vascular and skeletal health of men and women.

Models representing type I and type II human endometrial cancers: Ishikawa H and Hec50co cells.

OBJECTIVE: Endometrial cancer models are critical to the advancement of investigation, and Ishikawa H and Hec50co cells have been used as research tools. The purpose of these studies is to verify the degree to which these commonly used cell models share the molecular characteristics of the two major in vivo endometrial cancer subtypes, I and II. METHODS: The studies reported include an analysis of pathologic features, tumor suppressor mutations, detailed karyotyping, and cell cycle regulation. RESULTS: Ishikawa H cells are hormone responsive and have lost PTEN expression. In addition they have lost RB1 expression due to a deletion in exon 9. Hec50co cells have lost p53 expression due to a deletion at the junction of exon 6 and intron 6-7. Compared to Ishikawa H cells, Hec50co cells harbor many more chromosomal rearrangements (29 versus seven), and the doubling time is more rapid. The percent of cells in each phase of the cell cycle is reported and linked to cell cycle regulators. CONCLUSION: We present extensive data indicating that Ishikawa H cells are excellent models for type I endometrial cancers, and Hec50co cells faithfully replicate the molecular characteristics of type II endometrial cancers. These studies allow testing of new therapeutic regimens using appropriate cell models.

Skeletal muscle, cytokines, and oxidative stress in end-stage renal disease.

BACKGROUND: End-stage renal disease (ESRD) is a state of microinflammation, with increased activation of cytokines and augmented oxidative stress. While peripheral blood mononuclear cells are an established source of reactive oxygen species and inflammatory cytokines during hemodialysis (HD), skeletal muscle is also capable of generating these biomolecules. METHODS: Femoral arterio-venous (A-V) balance of interleukin-1 (IL-1), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), malonyldialdehyde (MDA), and carbonyl protein (CP) were measured in 17 ESRD patients and 9 healthy volunteers. ESRD patients were studied before (pre-HD) and during HD. mRNA levels of cytokines, heme oxygenase-1 (HO-1), and suppressors of cytokine signaling-2 (SOCS-2) were quantitated in the skeletal muscle by real-time polymerase chain reaction (PCR). RESULTS: Arterial concentration of MDA (pmol/mL) was higher pre-HD (325.5 +/- 19.6) compared to controls (267.7 +/- 14.7), but decreased intradialysis (248.8 +/- 16.1) (P < 0.01). Dialysis clearance of MDA was 16.9 +/- 3.1 mL/min. CP concentration (nmol/mg protein) in the artery was significantly higher pre-HD (2.29 +/- 0.09) than in controls (1.92 +/- 0.05), and remained stable during HD (2.23 +/- 0.07). Plasma cytokines increased to a variable degree in the artery and vein during HD. A-V balance studies demonstrated that the MDA (17.8%) and CP (5.1%) concentrations increased significantly in the vein intradialysis. Venous concentration of IL-6 was higher than that in the artery during dialysis (16.27 +/- 2.42 vs. 11.29 +/- 2.17 pg/dL, P < 0.01). mRNA levels of IL-6 (0.028 +/- 0.02 vs. 6.69 +/- 0.21), HO-1 (0.96 +/- 0.01 vs. 5.08 +/- 1.11), and SOCS-2 (0.63 +/- 0.12 vs. 0.82 +/- 0.14) in the muscle increased during HD (P < 0.01). Immunohistochemical studies confirmed the increase in IL-6 protein in the skeletal muscle during HD. The intradialytic increase in IL-1, IL-10, and TNF-alpha gene expression was not significant. CONCLUSION: Skeletal muscle may also contribute to the circulating plasma IL-6 and increased oxidative stress during HD.