Per and neuropeptide expression in the rat suprachiasmatic nuclei: compartmentalization and differential cellular induction by light

Per1 and Per2, two clock genes rhythmically expressed in the suprachiasmatic nucleus (SCN), are implicated in the molecular mechanism of the circadian pacemaker and play a major role in its entrainment by light. To date, it is not known if every cell of the SCN, a heterogeneous structure in respect of neuropeptide content, expresses clock genes equally. The aim of this study was to identify, by single and double non-radioactive and/or radioactive hybridizations, the cell types (AVP, VIP and GRP) expressing Per1 or Per2 in the SCN of rats, (1) when Per are highly expressed during the daytime, and (2) after induction of Per expression by a light pulse at night. Our results indicate that, during the daytime, Per1 and Per2 genes are both mainly expressed in the AVP cells of the dorso-median part of the SCN, whereas only a few VIP cells in the ventral part of the SCN exhibit Per gene expression. In contrast, following a light pulse at night, there is differential induction of the two Per genes. Per1 expression essentially occurs in the ventro-lateral GRP cells, while Per2 expression is not restricted to the retinorecipient part of the SCN as it also occurs in AVP cells. Altogether, our results suggest that Per1 and Per2 are mainly expressed in AVP cells during the daytime and suggest that GRP cells play an important role in resetting of the clock by light.     

Stable expression of the alkaline phosphatase marker gene by neural cells in culture and after transplantation into the CNS using cells derived from a transgenic rat

Multipotent stem cells and more developmentally restricted precursors have previously been isolated from the developing nervous system and their properties analyzed by culture assays in vitro and by transplantation in vivo. However, the variety of labeling techniques that have been used to identify grafted cells in vivo have been unsatisfactory. In this article we describe the characteristics of cells isolated from a transgenic rat in which the marker gene human placental alkaline phosphatase (hPAP) is linked to the ubiquitously active R26 gene promoter. We show that hPAP is readily detected in embryonic neuroepithelial stem cells, neuronal-restricted precursor cells, and glial-restricted precursor cells. Transgene expression is robust and can be detected by both immunocytochemistry and histochemistry. Furthermore, the levels of hPAP on the cell surface are sufficient for live cell labeling and fluorescence-activated cell sorting. Expression of hPAP is stable in isolated cells in culture and in cells transplanted into the spinal cord for at least 1 month. We submit that cells isolated from this transgenic rat will be valuable for studies of neural development and regeneration.     

铅对大鼠C6细胞GRP78表达的影响

目的观察不用时间点暴露于不同剂量醋酸铅后大鼠神经胶质瘤C6细胞葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)表达量的影响,探讨不同时间不同剂量铅暴露对内质网应激反应的影响。方法Wistar大鼠神经胶质瘤C6细胞培养于含醋酸铅的培养液中,分别于不同时间终止染铅,用Western印迹法检测内质网GRP78表达量。结果(1)0.2μmol/L染铅组:7和30 d GRP78蛋白表达显著增高,其余各个时间点均无显著性变化;(2)1.0μmol/L染铅组:1 d后GRP78蛋白表达量开始显著增高,染铅30 d时已达到染铅前的6.3倍;(3)2.0μmol/L染铅组:0.5 h起GRP78表达量即显著增高,染铅7 d时达到高峰,是染铅前的4.3倍,到30 d时表达量又下降为染铅前的2.6倍。结论铅可以使Wistar大鼠神经胶质瘤细胞内质网上的GRP78蛋白应激性表达增加,内质网上的GRP78是铅的重要蓄积库。     

Grp78用于胸部良恶性肿瘤鉴别诊断的实验研究

目的探讨分子伴侣Grp78用于胸部良恶性肿瘤鉴别诊断的可能性。方法应用免疫印迹技术对36例疑似肺癌病人胸水脱落细胞中Grp78的表达水平进行检测，并将检测结果与胸水脱落细胞学检查结果和临床病理资料对照。结果免疫印迹结果表明Grp78高表达的21例病人，经过临床观察和病理学检查，有19例被临床诊断为肺癌，GW7S表达水平较低15例病人中，没有病例被诊断为肺癌，灵敏度为90％，特异度为85．7％；而应用传统的检测方法对36例肺癌疑似病人胸水进行脱落细胞学检查，有12例病人怀疑为肺癌，经过临床及病理学检查有4例被排除，灵敏度为47％，特异度为85％。结论检测肺癌疑似病人胸水脱落细胞中Grp78的表达水平可以提高肺癌鉴别诊断的灵敏度，有利于胸部恶性肿瘤的早期诊断及鉴别诊断。     

X线机容量保护电路的故障检修

F78-Ⅲ型X线机在摄影过程中，开机后或开机曝光一两次后，机器过载，曝光不能进行，关机后重新开机，机器恢复正常，之后又重复出现此故障现象。由上述现象初步判断容量保护电路有故障。     

GRP94、EIF2α在原发型闭角性青光眼小梁中的作用研究

目的 通过检测内质网应激的相关因子 GRP94、EIF2α在原发性慢性闭角型青光眼( PCACG)患者小梁组织中的表达,研究内质网应激在PCACG小梁组织氧化损伤中的作用,探讨PCACG的发病机制。 方法 分别用HE染色法观察实验组及对照组小梁组织镜下显微形态的变化、免疫组化法测定实验组及对照组小梁组织中内质网应激相关因子 GRP94、 EIF2α的表达水平。 结果 HE染色显示,PCACG 患者小梁组织排列不规则,小梁网眼窄小,Schlemm 管腔变窄且不规则。免疫组化结果显示,对照组的小梁网组织GRP94、EIF2α少量表达,实验组小梁组织中GRP94、EIF2α的表达明显增加,差异均具有统计学意义( P<0.05)。 结论 PCACG 患者小梁组织中存在内质网应激,内质网应激EIF2α通道参与了小梁组织的氧化损伤,促进了眼内压的升高。     

Transforming growth factor-β1 in the urine of young children with urinary tract infection

Urinary tract infection (UTI) is a frequent cause of morbidity during the first years of life and may lead to renal insufficiency. Transforming growth factor-1 (TGF-) is both immunoregulatory and an important mediator of interstitial fibrosis. TGF- was detected in the urine of 52% of 48 children aged 1–24 months with a first episode of UTI (94% due to Escherichia coli) and no obstructive nephropathy compared with 0 of 20 healthy young children (P<0.001). TGF- was detected in the urine only during the early stage (<1 day) after initiation of treatment. It was detected more frequently (P=0.06) and in significantly higher concentrations (P=0.046) in children with a normal ^99m Tc-dimercaptosuccinic acid scan compared with those with abnormal scans performed 3–14 days after the diagnosis of UTI, suggesting a regulatory role in fibrogenesis and outcome of pyelonephritis in childhood.     

The unfolded protein response is activated in Alzheimer’s disease

Alzheimers disease (AD) is, at the neuropathological level, characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR) that may protect the cell against the toxic buildup of misfolded proteins. In this study we investigated the activation of the UPR in AD. Protein levels of BiP/GRP78, a molecular chaperone which is up-regulated during the UPR, was found to be increased in AD temporal cortex and hippocampus as determined by Western blot analysis. At the immunohistochemical level intensified staining of BiP/GRP78 was observed in AD, which did not co-localize with AT8-positive neurofibrillary tangles. In addition, we performed immunohistochemistry for phosphorylated (activated) pancreatic ER kinase (p-PERK), an ER kinase which is activated during the UPR. p-PERK was observed in neurons in AD patients, but not in non-demented control cases and did not co-localize with AT8-positive tangles. Overall, these data show that the UPR is activated in AD, and the increased occurrence of BiP/GRP78 and p-PERK in cytologically normal-appearing neurons suggest a role for the UPR early in AD neurodegeneration. Although the initial participation of the UPR in AD pathogenesis might be neuroprotective, sustained activation of the UPR in AD might initiate or mediate neurodegeneration.     

Stimulation of proliferation and migration of a colorectal cancer cell line by amidated and glycine-extended gastrin-releasing peptide via the same receptor

Although amidated forms of gastrin-releasing peptide (GRP) have been identified as autocrine growth factors in small cell lung cancer, their role in the development and progression of colorectal carcinoma is less clear. In addition, the biological activity of non-amidated gastrin-releasing peptide has not been investigated in colorectal carcinoma cells. We therefore investigated the effect of bombesin (a homologue of gastrin-releasing peptide) on proliferation, migration and inositol phosphate production in the human colorectal carcinoma cell line DLD-1, and determined the ability of gastrin-releasing peptide receptor antagonists to inhibit these effects. We also compared the biological activities of amidated and non-amidated GRP in the same assays. Treatment with either bombesin, or amidated or non-amidated GRP resulted in significant increase in proliferation, and in migration in a wound-healing assay. Both the mitogenic and migratory effects of amidated and non-amidated forms were inhibited by the GRP receptor antagonist [d-Phe⁶, Leu-NHet¹³, des-Met¹⁴]-bombesin(6-13). The presence of GRP receptor mRNA and GRP binding sites in three colorectal carcinoma cell lines was demonstrated by RT-PCR and by binding of radiolabelled bombesin, respectively. Transfection of DLD-1 cells with a dominant negative phosphatidylinositol 3-kinase did not affect bombesin-stimulated cell proliferation, but inhibited bombesin-stimulated cell migration. Bombesin and GRPgly activated phospholipase C, mitogen-activated protein kinase and focal adhesion kinase. We conclude that both amidated and non-amidated forms of gastrin-releasing peptide accelerate proliferation and migration of DLD-1 human colorectal carcinoma cells via the gastrin-releasing peptide receptor, but that phosphatidylinositol 3-kinase is only involved in the cell migration signalling pathway. Our results suggest a potential role for gastrin-releasing peptide receptor antagonists in the management of colorectal carcinoma.     

Two types of VIP neuronal components in rat suprachiasmatic nucleus

Vasoactive intestinal peptide (VIP) neurons constitute a large group in the suprachiasmatic nucleus (SCN) and it is thought that they are involved in the generation and entrainment of circadian rhythm. We have characterized these VIP-expressing neurons in rat SCN by their ability to induce the mammalian Period1 (Per1) gene in response to light exposure, innervation of retinal afferents, day-night variations in VIP mRNA, and coexpression of gastrin releasing peptide (GRP). VIP neurons in the ventrolateral SCN (SCNVL) were subdivided into two groups, light-evoked Per1-inducible main SCNVL (SCNVLmain) and non-Per1-inducible medially located SCNVL (SCNVLmed). Retinal innervation was abundant in the SCNVLmain but nearly absent in the SCNVLmed. Day-night variation in VIP mRNA expression level was observed in the SCNVLmain but not in the SCNVLmed. GRP mRNA was seen in rarely SCNVLmed but abundant in SCNVLmain, where some neurons coexpressed VIP mRNA. These findings indicate that VIP neurons in the SCN can be divided into two topographically and functionally distinct groups.