Lack of an allelic association between polymorphisms of the dopamine D2 receptor gene and alcohol dependence in the German population

Our study examined recent claims of an association of the TaqI A1 allele and the functional -141C Ins allele of the dopamine D2 receptor (DRD2) gene with alcohol dependence. Genotypes of the TaqI A and -141C insertion/deletion polymorphisms were assessed in 196 German nonalcoholic controls and 310 German alcohol-dependent subjects, including two more homogeneous subgroups selected by either a history of parental alcoholism (n = 108) or by a history of severe physiological alcohol withdrawal symptoms (n = 99). We found no association between any of the biallelic variants or the A1/Ins haplotype and alcohol dependence (p > 0.05). Our present association results failed to replicate evidence that either the TaqI A1 allele or the functional Ins allele or the A1/Ins haplotype of the DRD2 gene confers vulnerability to alcohol dependence in the German population.     

The role of STAT, AP-1, E-box and TIEG motifs in the regulation of hepcidin by IL-6 and BMP-9: lessons from human HAMP and murine Hamp1 and Hamp2 gene promoters

Hepcidin, the principal regulator of the iron metabolism, is up-regulated in response to inflammatory stimuli, bone morphogenic proteins (BMPs) and iron excess. There are two murine hepcidin genes: hepcidin-1 (Hamp1) and hepcidin-2 (Hamp2). Hamp1 gene responds to both IL-6 and BMPs while Hamp2 responds to neither. We replaced the putative functional regulatory motifs of the Hamp1 promoter with the corresponding putative "non-functional" Hamp2 motifs and vice versa in reporter constructs. Conversion of the Hamp1 STAT site into the Hamp2 site reduced the basal level of reporter expression but did not affect IL-6 and BMP responsiveness; replacing Hamp2 site with the Hamp1 site only resulted in partial responsiveness. These data are in contrast to the role of the STAT site in the human hepcidin promoter which is important in both basal level and IL-6 inducible promoter activity. The murine AP1, E-box and TIEG motifs were found to neither influence the basal level of expression of Hamp1 and HAMP promoters nor play a critical role in the IL-6 and BMP-9 induced response. Our data suggest that the STAT site (nt -148 to -130) is important for the regulation of basal level expression of Hamp1 but there are additional regions that are responsible for the IL-6 and BMP-9 responsiveness within the Hamp1 promoter.     

贲门组织中多巴胺受体基因的分布与表达

目的探讨人贲门组织中多巴胺受体（DR）基因（D4mRNA,D5mRNA）的表达,揭示D4mRNA,D5mRNA在贲门组织中的抗溃疡机制。方法应用RT-PCR方法检测贲门组织中D4mRNA,D5mRNA的分布与表达。结果在食管环行肌、胃底环行肌、钩状纤维、套索纤维中D4mRNA分别为0．112±0．053、0．124±0．047、0．122±0．03、60．125±0．027,D5mRNA分别为0．116±0．023、0．118±0．035、0．121±0．026、0．116±0．078,两者均呈低表达（P〉0．05）。在贲门的食管黏膜和胃黏膜中,D4mRNA分别为0．416±0．082和0．423±0．094,D5mRNA分别为0．248±0．068和0．252±0．070,均呈高表达（P〉0．05）。两种黏膜与4条肌束中D4mRNA和D5mRNA的表达均存在显著性差异（P〈0．01）。结论在贲门肌肉组织和黏膜中D4mRNA和D5mRNA的表达不同,推测在贲门、胃黏膜中的D4mRNA、D5mRNA受体有着重要的抗溃疡作用。     

多巴胺受体D2基因TaqI位点多态性与高血压病的关系

目的 探讨多巴胺受体D2基因TaqI位点多态性与原发性高血压病的相关性。方法 运用聚合酶链反应一限制性片段长度多态性法（PCR－RFLP）分析TaqI位点A1A1，A1A2，A2A2基因型在原发性高血压病组和对照组的分布情况。结果 等位基因A1，A2在原发性高血压病组和对照组的分布频率分别为0．34，0．66和0．43，0．57。基因频率分布符合Hardy-Weinberg平衡，样本具有群体代表性，两组人群的基因型和等位基因频率比较，差异有显著性（P＜0．05）。原发性高血压病组中A2等位基因舒张压明显高于A1等位基因舒张压（P＜0．05）。结论 在湖北省人群中，多巴胺受体D2基因TaqＩ位点多态性与原发性高血压病显著相关。     

Molecular cloning of bullfrog D2 dopamine receptor cDNA: Tissue distribution of three isoforms of D2 dopamine receptor mRNA

The cDNA encoding D2 dopamine receptor was cloned from the distal lobe of the bullfrog pituitary. The deduced amino acid sequence of the bullfrog D2 dopamine receptor (bfD2A) spanned 444 amino acids and exhibited typical features of those of D2 dopamine receptors cloned in other animals to date. It showed a high similarity of 75-87% with rat, turkey, Xenopus and tilapia counterparts. Further analysis of nucleotide sequence of the cDNA revealed the presence of putative truncated D2 dopamine receptor isoforms, bfD2B and bfD2C, of which nucleotide sequences lacked 12 and 99 nucleotides of the coding region for bfD2A, respectively. The alignment analysis indicated that putative bfD2C isoform was close to D2_S subtype cloned in mammals and birds, whereas bfD2A and putative bfD2B isoforms were close to mammalian and avian D2_L subtype and homologous to two isoforms of Xenopus. This is the first report of the presence of mRNAs for two D2_L-like isoforms and one D2_S-like isoform in a single species. The amino acid sequence responsible for producing isoforms is present in the third intracellular loop, which has been shown to play an important role in the coupling with G protein. Accordingly, differences in the mode of coupling with G protein among three isoforms were suggested. The expression of three isoforms mRNA in organs and tissues was analyzed by RT-PCR. In the brain, pars distalis and pars neurointermedia, mRNAs for three isoforms were invariably expressed, whereas only putative bfD2C mRNA was expressed in peripheral organs and tissues.     

Central overexpression of angiotensin AT(1A) receptors prevents dopamine D(2) receptor regulation of alcohol consumption in mice

BACKGROUND: While angiotensin receptors are found on the soma and terminals of dopaminergic neurons, controversy surrounds the potential role of angiotensin in alcohol consumption. METHODS: Using a transgenic mouse with a brain-specific overexpression of angiotensin AT(1A) receptors (NSE-AT(1A) mice), we have examined the role of angiotensin in alcohol consumption and alcohol-induced regulation of the dopaminergic system. RESULTS: The functional relevance of the overexpressed AT(1A) receptors was confirmed by an exaggerated rehydration response following 24-hour dehydration. NSE-AT(1A) mice showed a high preference for alcohol (similar to wild-type mice); yet, raclopride treatment had no effect on alcohol consumption in NSE-AT(1A) mice, while significantly reducing consumption in wild-type mice. In contrast, NSE-AT(1A) mice showed enhanced sensitivity to raclopride compared with wild types in terms of D(2) receptor up-regulation within the ventral mesencephalon. In addition, striatal D(2) receptors in NSE-AT(1A) mice were sensitive to up-regulation by chronic alcohol consumption. CONCLUSIONS: Collectively, these data imply that while expression of angiotensin AT(1A) receptors on striatal neurons has no impact upon basal alcohol consumption or preference, AT(1A) receptors do modulate the sensitivity of dopamine D(2) receptors to regulation by alcohol and the ability of a D(2) receptor antagonist to reduce consumption.     

N-甲基-D-天冬氨酸受体2B基因(GRIN2B)启动子区多态性与散发性阿尔茨海默病的相关性分析

目的探讨中国北方汉族人群N-甲基-D-天冬氨酸受体2B(N-methyl-D-aspartate receptor2B,NR2B)基因GRIN2B启动子区变异与散发性阿尔茨海默病(sporadic Alzheimer′s disease,SAD)的关系。方法利用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)的方法对中国北方汉族362例SAD患者和334名正常对照者进行GRIN2B启动子多态性及载脂蛋白E(ApoE)分型。结果中国北方汉族人群中GRIN2B启动子区存在-200T/G(rs1019385)、-421C/A(rs3764028)、-1447T/C(ENS10557853)和-1497G/A(rs12368476)多态性位点;-421C/A多态性在SAD和正常对照组中的基因型频率(P=0.029)和等位基因频率(P=0.010)差异显著;不携带ApoEε4的人群中-421C/A多态性的基因型频率(P=0.012)及等位基因频率(P=0.004)差异亦显著;所发现的4个多态性位点之间存在连锁不平衡关系,但其单体型分布在AD组和正常对照组中差异不显著。结论-421C/A多态性与AD发病有明显的相关性,在ApoEε4阴性的人群中这种相关性更为明显,-421C/A多态性可能独立于ApoEε4而影响SAD发病。     

人细胞色素P450 4A11基因核心启动子区的鉴定

目的研究细胞色素P4504A11基因启动子区对该基因转录活性的影响。方法运用生物信息学的方法预测细胞色素P4504A11基因启动子区的重要调控序列，然后采用5’侧翼区缺失的方法将包含重要调控序列的启动子克隆到双报告基因载体中，用脂质体转染HepG2细胞，24h后检测荧光素酶相对活性。然后使用Alibaba 2．0Transfae4．0预测增强子区域可能存在的转录因子结合位点。结果·36--720-＋1、-972～＋1、-1039-＋1区域的启动子活性较高，其中-972～＋1区域最强，与此相比-536-＋1、-720～＋1荧光素酶相对活性分别下降了96．9％和70．1％。结论人CYP4A11基因5’侧翼区的影响转录活性的启动子关键区域位于-972--720bp范围内。     

Temporal expression of G-protein-coupled receptor 54 (GPR54), gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus

The G-protein-coupled receptor 54 (muGPR54) cDNA was cloned from the brain of the grey mullet, and its expression level, as well as those of the gonadotropin-releasing hormones (GnRH1, GnRH2, GnRH3) and dopamine receptor D2 (drd2), in the brain, pituitary and ovary of pubertal fish (early, intermediate, advanced) were determined by real-time quantitative RT-PCR (QPCR). The muGPR54 cDNA has an open reading frame of 1140 bp with a predicted 380 amino acid peptide, containing seven putative transmembrane domains and putative N-glycosylation and protein kinase C phosphorylation sites. QPCR results showed that the early stage of puberty in grey mullet is characterized by significantly high levels of expression of GPR54, GnRH and drd2 in the brain relative to the intermediate and advanced stages, except for GnRH1 that increased at the advanced stage of puberty. In the pituitary, drd2 expression declined significantly at the advanced stage relative to levels at the intermediate stage. Ovarian expression of GPR54 significantly increased from the intermediate stage of puberty relative to the early stage while that of GnRH1 acutely increased at the advanced stage of puberty. The ovarian expression of drd2 decreased as puberty progressed, but the changes were not significant. The results suggest the possible role of GPR54 and GnRH in positively regulating pubertal development in grey mullet and the dopaminergic inhibition of reproductive function mediated by drd2.     

Endogenous kappa opioid receptor systems modulate the responsiveness of mesoaccumbal dopamine neurons to ethanol

Background: Endogenous κ‐opioid receptor (KOPR) systems modulate the actions of several drugs of abuse. Their role in modulating the effects of ethanol is unknown. An increase in nucleus accumbens extracellular dopamine (DA) has been implicated in mediating the rewarding and locomotor‐activating effects of ethanol and virtually all drugs of abuse. The present microdialysis studies were conducted to determine whether the lack of KOPR alters ethanol‐evoked DA levels in the nucleus accumbens of naïve mice and whether a similar effect is observed in mice repeatedly exposed to ethanol. Methods: Gene deletion techniques were used in conjunction with in vivo microdialysis to examine the influence of lack of KOPR on ethanol‐evoked DA in the nucleus accumbens. To determine whether pharmacological inactivation of KOPR produces similar effects in naïve mice and those repeatedly exposed to ethanol, the KOPR antagonist norbinaltorphimine (n‐BNI) was administered in wild‐type mice before repeated air or ethanol vapor inhalation. Microdialysis was conducted 24 hours later. Results: Acute ethanol administration increased DA levels in the nucleus accumbens of wild‐type mice. In littermates lacking the KOPR gene, ethanol‐evoked DA levels were enhanced. Prior ethanol exposure reduced ethanol‐evoked DA levels in vehicle‐treated and n‐BNI–treated mice. Statistical analysis, however, revealed a significant main effect of n‐BNI, indicating that KOPR blockade increased ethanol‐evoked DA levels in naïve mice and repeated ethanol exposure attenuated, but did not abolish, this effect. Conclusions: These findings demonstrate that inhibition of KOPR leads to increased sensitivity to the DA‐releasing effects of ethanol in the nucleus accumbens.     

The molecular regulation of Chinook salmon gonadotropin β-subunit gene transcription

Expression of the gonadotropin β-subunit genes is tightly regulated both cell-specifically and by the regulatory hormones to achieve the appropriate gonadotropic hormone levels required for reproductive development and function. Although the cDNA sequences of these genes are highly conserved across species, their promoter sequences are not and few functional studies have been carried out to understand the molecular mechanisms through which their expression is regulated. We and others have carried out several studies on the LHβ gene promoter of Chinook salmon (Oncorhynchus tschawytscha), and also isolated the FSHβ gene from the same species. We present here a review of these studies and also novel data pertaining to both genes, in an attempt to collate the current understanding of the molecular regulation of the gonadotropin β-subunit genes in these fish.     

Examining a pathway for hormone mediated maternal effects--yolk testosterone affects androgen receptor expression and endogenous testosterone production in young chicks (Gallus gallus domesticus)

In vertebrates maternal androgens can substantially influence developing offspring, inducing both short and long term changes in physiology and behavior, including androgen sensitive traits. However, how the effects of maternal hormones are mediated remains unknown. Two possible pathways are that maternal androgens affect parts of the hypothalamus-pituitary-gonadal axis (HPG axis) or the sensitivity to androgens by affecting androgen receptor (AR) densities within the brain. To investigate both pathways, testosterone within the physiological range or vehicle only was injected into the egg yolk of unincubated chicken eggs and AR mRNA expression in different brain nuclei as well as plasma testosterone levels were measured in two week old male and female chicks that had hatched from these eggs. Our results showed a significant sex difference in plasma testosterone levels with males showing higher levels than females. Furthermore, AR mRNA expression as well as plasma testosterone levels were significantly lower in chicks hatched from testosterone treated eggs. These results suggest a compensatory mechanism for avoiding potential detrimental effects of high testosterone levels.