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.     

Norepinephrine transporter (NET) knock-out upregulates dopamine and serotonin transporters in the mouse brain

The noradrenaline, serotonin and dopamine transporters are three main transporters, which are the target of the antidepressant drugs. In the present study we demonstrate that the life-long deletion of the noradrenaline transporter (NET) induced up-regulation of two other monoamine transporters, dopamine and serotonin (DAT and SERT, respectively). An increase in the binding of [³H]paroxetine to the SERT and [³H]GBR12935 to the DAT was observed in various brain regions of NET-KO mice, without alterations of mRNA encoding these transporters, as measured by in situ hybridization. This important finding impacts the interpretation of previous data indicating the supersensitizity of NET-KO mice for psychostimulants or stronger effect of citalopram in behavioral tests. While using the NET-KO mice in various psychopharmacological studies is very important, one has to be aware that these mice lack NET from the earliest period of their existence, thus compensatory alterations do take place and have to be considered when it comes to interpretation of the obtained results.     

Quantitation of Rat Dopamine Transporter mRNA: Effects of Cocaine Treatment and Withdrawal

Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive nuclease protection assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a greater than 40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72-h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down-regulation of dopamine transporter gene expression in dopamine neurons.     

Insertion/Deletion Polymorphism of the Serotonin Transporter Gene and Neuroticism as a Temperament Trait in Affective Patients and Healthy Individuals

Some studies associate the insertion/deletion polymorphism of the serotonin transporter (5-HTT) gene with anxiety-related personality traits in mentally healthy people, the short (s) allele being associated with a higher neuroticism score. The 5-HTT genotype and neuroticism score were established for 114 affective patients, 87 healthy relatives of endogenous psychosis patients, and for 156 mentally healthy people without familial psychiatric history. The effects of sex and age on the association between the two parameters was studied. Neuroticism proved to be not associated with the 5-HTT genotype.     

Regional distribution and cellular expression of tryptophan hydroxylase messenger RNA in postmortem human brainstem and pineal gland

The characterization and cellular localization of tryptophan hydroxylase mRNA in the human brainstem and pineal gland were investigated by using northern blot analysis and in situ hybridization histochemistry. Northern analysis of human pineal gland revealed the presence of two mRNA species that were absent in RNA isolated from human raphe. In situ hybridization experiments revealed very dense hybridization signal corresponding to tryptophan hydroxylase mRNA in cells throughout the pineal gland. In contrast, tryptophan hydroxylase mRNA was heterogeneously distributed in neurons in the dorsal and median raphe nuclei. Within the dorsal raphe, the ventrolateral and interfascicular subnuclei contained the greatest number of tryptophan hydroxylase mRNA-positive neurons. Also, the cellular concentration of tryptophan hydroxylase mRNA varied widely within the dorsal and median raphe. Comparison of the cellular concentration of tryptophan hydroxylase mRNA between the pineal gland and the raphe nuclei revealed an 11- and 46-fold greater average grain density of tryptophan hydroxylase mRNA positive cells in the pineal gland compared with the dorsal and median raphe, respectively. These findings are the first to demonstrate the cellular localization of tryptophan hydroxylase mRNA in the human brain and pineal gland as well as heterogeneity in the cellular concentration within and between these tissues.     

Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization

Gene expression in diseased tissues can indicate the contribution to a disease process and potentially guide therapeutic decision-making. Archival tissues with associated clinical outcome may be useful to discover or validate the role of a candidate gene in a disease process or the response to therapy. Such archival tissues are commonly formalin-fixed and paraffin-embedded, restricting the methods available for gene expression analysis. Obviously, the detection of proteins in tissues requires adaptation for each protein and the detection of secreted proteins can prove difficult or of reduced value since the protein detected may not reflect the total amount produced. Thus, we describe here a reliable method for the detection of mRNA in archival tissues. The method for mRNA in situ hybridization (ISH) was adapted by us for >15 different genes and applied to several hundred tissue microarrays (TMAs) and full sections generating >10,000 expression data points. We also discuss the utility of TMAs to simultaneously analyze several hundred tissue samples on one slide to minimize variability and preserve valuable tissue samples. Experimental protocols are provided that can be implemented without major hurdles in a typical molecular pathology laboratory and we discuss quantitative analysis as well as advantages and limitations of ISH with a special focus on secreted proteins. We conclude that ISH is a reliable and cost effective approach to gene expression analysis in archival tissues that is amenable to screening of series of tissues or of genes of interest.     

茶树硝酸盐转运蛋白基因的克隆和表达分析

硝酸盐转运蛋白（NRT）是植物吸收和利用硝态氮的一种关键蛋白。运用RACE技术从茶树中扩增出NRT基因的cDNA,并利用实时荧光定量PCR检测了CsNRT基因在不同茶树器官与品种之间的差异表达。结果表明：CsNRT基因的cDNA全长2 061 bp,开放阅读框为1 818 bp,编码含由605个氨基酸组成的蛋白质,GenBank登录号为KJ160503,属于NRT2基因家族。CsNRT为组成型基因,对不同处理的水培茶苗进行定量表达分析显示,该基因在根、茎、叶中都有表达,其中在根部的表达水平最高,1.0 mmol·L-1的NO3-可诱导其表达量上升7.53倍。不同茶树品种中CsNRT基因的表达也有较大差异,‘龙井长叶’和‘凫早2号’的表达量较高,前者强烈响应0.5和1.0 mmol·L-1 NO3-的诱导,后者的响应浓度为1.0和2.0mmol·L-1,而‘舒茶早’在各浓度下的表达差异不明显。     

基因表达分析方法及其研究进展

近几年来,随着功能基因组学研究的兴起,基因表达研究的分析方法也在不断发展,主要有：差减杂交、差异显示、表达序列标签、基因表达的序列分析、微阵列杂交等。简要评述这五种方法的原理、优缺点等。     

Acute and Persistent Suppression of Preproenkephalin mRNA Expression in the Striatum Following Developmental Hypoxic-Ischemic Injury

Abstract: The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in conjunction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.     

Differential In Vivo Regulation of mRNA Encoding the Norepinephrine Transporter and Tyrosine Hydroxylase in Rat Adrenal Medulla and Locus Ceruleus

Abstract: To investigate the regulation of norepinephrine transporter mRNA in vivo, we analyzed the effects of reserpine on its expression in the rat adrenal medulla and locus ceruleus. First, PCR was used to clone a 0.5-kb rat cDNA fragment that exhibits 87% nucleotide identity to the corresponding human norepinephrine transporter cDNA sequence. In situ, the cDNA hybridizes specifically within norepinephrine-secreting cells, but in neither dopamine nor serotonin neurons, suggesting strongly it is a partial rat norepinephrine transporter cDNA. Reserpine, 10 mg/kg administered 24 h premortem, decreased steady-state levels of norepinephrine transporter mRNA in the adrenal medulla by ∼65% and in the locus ceruleus by ∼25%, as determined by quantitative in situ hybridization. Northern analysis confirmed the results of the in situ hybridization analysis in the adrenal medulla but did not detect the smaller changes observed in the locus ceruleus. Both analyses showed that reserpine increased tyrosine hydroxylase expression in the adrenal medulla and locus ceruleus. These results suggest that noradrenergic neurons and adrenal chromaffin cells can coordinate opposing changes in systems mediating catecholamine uptake and synthesis, to compensate for catecholamine depletion.