大鼠酪氨酸羟化酶基因在肌母细胞中稳定表达

用电穿孔法将大鼠酪氨酸羟化酶（Ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）基因转染大鼠Ｌ－６ＴＧ成肌细胞株,经ＰＣＲ检测、免疫组织化学和荧光组织化学检测证明,ＴＨ基因能在细胞内稳定整合和表达,并在辅因子存在时将酪氨酸转化为多巴．移植于大鼠纹状体后可成活并表达ＴＨ。     

Chronic Cocaine Administration Increases CNS Tyrosine Hydroxylase Enzyme Activity and mRNA Levels and Tryptophan Hydroxylase Enzyme Activity Levels

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self-administration by rats when given ad libitum access to this dose. This chronic, response-independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striaturn and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response-independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.     

Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia

The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced PaO2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S-labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.     

Long-Term Induction of Tyrosine Hydroxylase Expression: Compensatory Response to Partial Degeneration of the Dopaminergic Nigrostriatal System in the Rat Brain

Abstract: The present study was undertaken to examine the adaptive changes occurring 1 and 6 months after moderate or severe unilateral 6-hydroxydopamine-induced lesions confined to the lateral part of the rat substantia nigra pars compacta (SNC). The expression of tyrosine hydroxylase (TH) enzyme was analyzed in the remaining dopaminergic nigral cell bodies and in the corresponding striatal nerve endings. In the cell bodies of the lesioned SNC, TH mRNA content was increased (+20 to +30%) 6 months after the lesion without changes in cellular TH protein amounts. The depletion of TH protein in the nerve terminal area was less severe than the percentage of cell loss observed in the SNC at 1- and 6-month postlesion intervals. Moreover, the decrease in TH protein in the ipsilateral striatum was less pronounced 6 months after lesion than 1 month after. That no corresponding change in TH protein content was observed in the cell bodies at a time when TH increased in nerve terminals suggests that the newly synthesized protein is probably rapidly transported to the striatal fibers. These results suggest the existence of a sequence of changes in TH expression between cell bodies and fibers, occurring spontaneously after partial denervation of the nigrostriatal pathway.     

Induction of Gene Expression of the Catecholamine-Synthesizing Enzymes by Insulin-Like Growth Factor-I

Abstract: The effects of insulin-like growth factor-I (IGF-I) on gene expression and the activities of the three enzymes specific for catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N -methyltransferase (PNMT), were determined in bovine adrenomedullary chromaffin cells primary cultured in serum-free medium. The mRNA level of TH was maximally elevated in the presence of IGF-I by 3.1 ± 0.4-fold after 48 h, DBH by 5.1 ± 0.3-fold in 24 h, and PNMT by 2.8 ± 0.5-fold in 72 h. In addition, the activity of TH was increased by 77%, DBH by 70%, and PNMT by 23% in IGF-I-exposed cultures. In the absence of the growth factor, the mRNA levels of TH and DBH were decreased to 45 ± 10% and 35 ± 12% of the time-zero control within 48 h while PNMT mRNA was decreased to 82 ± 5% only after 72 h. When the cells were cotreated with the protein tyrosine kinase inhibitor genistein, DBH induction by IGF-I was suppressed, confirming that the effect is mediated by tyrosine kinase. Cotreatment with the protein kinase A (PKA) inhibitor H89 caused complete reversal of the IGF-I-induced DBH increase and the effects of IGF-I treatment and PKA activation by forskolin were not additive, suggesting that PKA is involved in the signaling initiated by IGF-I in these cells.     

Expression of Tyrosine Hydroxylase Gene in Cultured Hypothalamic Cells: Roles of Protein Kinase A and C

Abstract: In hypothalamic cells cultured in serum-free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8-bromoadenosine cyclic AMP, 3-isobutyl-1-methylxanthine, or forskolin) or an activator of protein kinase C (12- O -tetradecanoylphorbol 13-acetate or sn -1,2-diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half-life = 14 ± 1 h) in these cells.     

Alteration of Gene Expression in Rice Hybrid F1 and Its Parental Seedlings

Hybrid rice ( Oryza sativa L. ) seedling is more vigorous in root development and plant growth than its parental lines in the tested indica rice of hybridized combination (Shanyou 63 (Fl): Zhenshan 97A × ♂Minghui 63). Analysis of the difference in gene expression between the hybrid Fl and its parental seedlings by means of mRNA differential display indicated that gene expression of the parental lines was obviously altered the hybrid Fl both in quantity and quality., Quantitatively, there were over-expression and under-expression of genes in hybrid Fl with genetic expression trend forwards a single parent. Qualititatively, hybrid Fl could have specific gene expression, single parem (maternal or paternal) gene silence, co-suppression of paternal genes, and single paternal gene expression. The relationship between heterosis formation and alteration of gene expression of parental lines in hybrid Fl was also discussed.     

三棱内酯B调控人冠状动脉内皮细胞基因表达谱的生物信息学分析

目的:分析三棱内酯B在人冠状动脉内皮细胞中的表达谱数据集,寻找三棱内酯B调控血管内皮功能的关键作用靶点。方法:基于GEO公共数据库,下载原始表达谱数据集(GSE44598),经过差异基因筛选,功能注释,通路富集,信号通路网络以及基因互作网络分析,找出三棱内酯B对人冠状动脉内皮细胞基因表达谱产生影响的关键基因和信号通路。结果:同对照组相比,三棱内酯B给药组共有5224个基因有显著性差异,包括2628个上调基因和2596个下调基因。基因功能注释和信号通路富集分析表明,差异基因主要参与了细胞周期过程。网络分析显示,MAPK信号通路、细胞周期通路以及PLCG2,PRKACA和ADCY4等为关键信号通路和基因。结论:三棱内酯B通过影响PLCG2,PRKACA,ADCY4等基因的表达,参与MAPK和细胞周期等信号通路,从而调节人冠状内皮细胞的功能。这些关键基因和信号通路是三棱内酯B在心血管疾病治疗应用中潜在的作用靶点。     

Both Short- and Long-Term Effects of Nerve Growth Factor on Tyrosine Hydroxylase in Calf Adrenal Chromaffin Cells Are Blocked by S-Adenosylhomocysteine Hydrolase Inhibitors

We have previously shown that primary cultures of calf chromaffin cells respond to nerve growth factor (NGF) treatment with a selective induction of tyrosine hydroxylase (TH), which takes 48 h to be manifested. In the present study, we report that short exposure of calf chromaffin cells to NGF (5-60 min) results in TH activation, which involves a change in the Vmax of the enzyme with no change in the number of enzyme molecules, similar to an effect that has been previously reported in PC12 cells. This activation is markedly potentiated when the chromaffin cells are plated on a laminin substrate, such that after 5 min of NGF exposure, there is an approximately fourfold increase in the TH activity. Both short-term activation and long-term TH induction brought about by NGF treatment are blocked by 5'-deoxy-5'-methylthioadenosine and other drugs that act as S-adenosylhomocysteine (SAH) hydrolase inhibitors to block methylation by end-product inhibition. These drugs did not inhibit cyclic AMP-mediated TH activation or increases in the levels of TH. However, measurements of the degree of blockade of methylation in cells treated with these drugs, taken together with conceptual information regarding the nonregulatory nature of methylation in eukaryotic cells, were not consistent with inhibition of methylation as the crucial effect of the drugs to block the effects of NGF. Nonetheless, since SAH hydrolase inhibitors selectively inhibited NGF-mediated effects, and not comparable effects triggered by other stimuli, these compounds provide useful tools in future studies of the biochemical signalling mechanism of NGF.     

The Roles of CRE, TRE, and TRE-Adjacent S1 Nuclease Sensitive Element in the Regulation of Tyrosine Hydroxylase Gene Promoter Activity by Angiotensin II

Abstract: The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bovine adrenal medullary cells. Angiotensin II-responsive elements are located within −54/+25-bp and −269/−55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12- O -tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.     

Short-term Effects of Endothelins on Tyrosine Hydroxylase Activity and Expression in the Olfactory Bulb of Normotensive Rats

The olfactory system in rats is part of the limbic region with extensive afferent connections with brain areas involved in the regulation of behaviour and autonomic responses. The existence of the endothelin system and catecholaminergic neurons in the olfactory bulb suggests that endothelins may modulate noradrenergic transmission and diverse olfactory mediated processes. In the present work we studied the effect of endothelin-1 and -3 on neuronal norepinephrine release and the short-term regulation of tyrosine hydroxylase in the olfactory bulb. Results showed that both endothelins increased tyrosine hydroxylase activity through the activation of a non-conventional endothelin G-protein coupled receptor, coupled to the stimulation of protein kinase A and C, as well as Ca²⁺/calmodulin-dependent protein kinase II. On the other hand, neither endothelin-1 nor endothelin-3 modified tyrosine hydroxylase total protein levels, but both peptides increased the phosphorylation of serine residues of the enzyme at sites 19 and 40. Furthermore, endothelins enhanced norepinephrine release in olfactory neurons suggesting that this event may contribute to increased tyrosine hydroxylase activity by reducing the feedback inhibition. Taken together present findings show a clear interaction between the endothelin system, and the catecholaminergic transmission in the olfactory bulb. Additional studies are required to evaluate the physiological functions regulated by endothelins at this brain level.     

Elevation of RNA Coding for Tyrosine Hydroxylase in Rat Adrenal Gland by Reserpine Treatment and Exposure to Cold

When rats are treated daily with reserpine or maintained at 4 degrees C, the level of a specific RNA coding for tyrosine hydroxylase is elevated in the adrenal gland. The increase in this specific RNA temporally precedes and is quantitatively equal to the increase in adrenal tyrosine hydroxylase enzyme activity elicited by these treatments. These results suggest that prolonged stress may lead to changes in the levels of specific RNA species in the adrenal gland.     

An HSV-1 Vector Containing the Rat Tyrosine Hydroxylase Promoter Enhances Both Long-Term and Cell Type-Specific Expression in the Midbrain

Abstract: A defective herpes simplex virus type one (HSV-1) vector that contains a 6.8-kb fragment of the rat tyrosine hydroxylase promoter (pTHlac-7kb) was examined for its capability to target catecholaminergic cell type-specific expression in the CNS. Cell type-specific expression was assessed by comparison with a control vector (pHSVlac) that uses the HSV-1 immediate early 4/5 promoter to support expression in multiple cell types. In initial experiments comparing expression in catecholaminergic and noncatecholaminergic cell lines, pTHlac-7kb supported a seven- to 20-fold increase in reporter gene expression in catecholaminergic cell lines. Four days after stereotactic injection into the midbrain of adult rats, pTHlac-7kb supported a 10-fold targeting of β-galactosidase expression to tyrosine hydroxylase-expressing neurons in the substantia nigra pars compacta compared with pHSVlac. Expression from pTHlac-7kb was stably maintained for 6 weeks with no significant changes in the pattern of expression. Long-term expression from pTHlac-7kb was confirmed by RNA and DNA analysis. In contrast, reporter gene expression in the midbrain from pHSVlac decreased ∼30-fold between 4 days and 6 weeks after gene transfer. Thus, within the context of this HSV-1 vector system, the tyrosine hydroxylase promoter enhanced cell type-specific expression and contributed to stable, long-term expression of a recombinant gene product in neurons. The capability to target recombinant gene expression to catecholaminergic neurons in specific brain areas may be useful for studies on the roles of these neurons in brain physiology and behavior.