Opioid peptide gene expression in rat trigeminal nucleus caudalis neurons: normal distribution and effects of trigeminal deafferentation

Preproenkephalin (preproenkephalin A) and preprodynorphin (preproenkephalin B) are the opioid peptide genes expressed in neurons of the nucleus caudalis of the trigeminal nuclear complex. We have used recently developed techniques for quantitative in situ hybridization to identify the neurons in laminae I and II of the nucleus caudalis that display the mRNA products of each of these genes. The specificity of these hybridization patterns is supported by several biochemical features, and by qualitative and quantitative parallels with previous immunohistochemical results. In animals killed 4 days after unilateral lesions of the trigeminal ganglion, neuronal expression of both preproenkephalin and preprodynorphin is altered in the nucleus caudalis. Decreases in preproenkephalin mRNA are due to a decline in the number of neurons that appear to express this gene. Conversely, preprodynorphin mRNA increases by adding a significant population of expressing neurons. These deafferentation-induced changes in gene expression may provide clues to the role of primary afferent information in modulating the functions of nucleus caudalis neurons containing opioid peptides.     

Differential expression of preproenkephalin and preprodynorphin mRNAs in striatal neurons: high levels of preproenkephalin expression depend on cerebral cortical afferents

Preproenkephalin and preprodynorphin mRNAs can be detected by in situ hybridization in medium-sized striatal neurons in normal rats and in rats with unilateral cerebral cortical lesions. Hybridization of 35S-labeled oligonucleotide cDNAs complementary to specific regions of each mRNA reveals that preproenkephalin-expressing neurons are more numerous than cells expressing preprodynorphin. Hybridization densities above enkephalin-positive neurons are also more than twice those noted above preprodynorphin-expressing cells. Northern analyses of mRNA extracted from the striatum are consistent with these relationships. The striatal preproenkephalin hybridization densities are decreased ipsilateral to cerebral cortical lesions; this change evolves largely between 1 and 5 d following the lesion. Striatal preproenkephalin mRNA is thus more prominent than preprodynorphin mRNA and depends on cerebral cortical inputs for its full expression.     

Identification of a cDNA clone specific for the neural cell adhesion molecule AMOG

A cDNA clone of the neural cell adhesion molecule AMOG was isolated from a lambda gt10 library constructed from 8-day-old mouse brain poly(A) + RNA with a 17mer oligonucleotide probe designed from a nonapeptide sequence obtained from tryptic peptides of AMOG. The cDNA clone expressed as a fusion protein that is recognized by polyclonal AMOG antibodies; conversely, polyclonal antibodies prepared against the fusion protein react with AMOG. The clone contains the full sequence derived from the nonapeptide. Of all tissues tested, only brain expresses detectable levels of AMOG by ELISA and Northern blot analyses, indicating a high correlation in expression at the protein and mRNA levels. Both brain and astrocytes express a 3 kb long mRNA, which appears to be encoded by a single gene.     

Isolation and characterization of the mouse homolog of the preprodynorphin (Pdyn) gene

We have isolated and sequenced the mouse preprodynorphin gene (Pdyn). The Pdyn gene can encode for six biologically active dynorphin peptides. The predicted mouse preprodynorphin has 90%, 67%, and 66% identity with the predicted rat, porcine, and human preprodynorphins, respectively. Using an RT-PCR technique, we show that the Pdyn gene starts being expressed at embryonic day 12.5, with a steep increase of expression by embryonic day 14.5; in the adult mouse it is expressed in the brain, but not in liver, heart, spleen, or kidney.     

RLM-RACE法确定胰岛素降解酶基因的转录起始位点

目的精确定位胰岛素降解酶基因的转录起始位点,为今后研究该基因启动子区的转录调控及其参与疾病的发病机制奠定基础。方法采用5'cDNA末端快速扩增技术,得到多个胰岛素降解酶基因cDNA 5'末端序列,连接至PUC19 T载体中,鉴定阳性克隆并测序。结果根据两次实验结果,确认胰岛素降解酶基因的5'UTR序列长度为32bp。结论胰岛素降解酶基因存在单一的转录起始位点,位于翻译起始点上游32bp处,碱基为G。     

Acute intermittent morphine increases preprodynorphin and kappa opioid receptor mRNA levels in the rat brain

We determined the effects of morphine on mRNA levels for the opioid ligands preprodynorphin (PPD) and preproenkephalin (PPE) and the kappa opioid receptor (KOR). Rats received six injections of morphine (6.25 mg/kg/injection) every 2 h, and were sacrificed 30 min later. mRNA levels were measured in brain tissue after removal of the cortex, cerebellum and brainstem. There were increases in PPD and KOR mRNA levels (P<0.05 and P<0.005, respectively), with no alteration of PPE. These alterations in the kappa/dynorphin system may counter morphine-induced effects on the brain.