Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid-soluble drugs such as anticancer agents. The mdr1a P-gp-encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 microg/mL puromycin, a P-gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P-gp expression and activity were strongly increased. When GP8.3 and puromycin-starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable rat in vitro model for studying the regulation of mdr1a expression at the BBB level.     

Presenilin-1 protein specifically expressed in Leydig cells with its expression level increased during rat testis development

Presenilin-1, mutations of which cause the early-onset of Alzheimer's disease, was shown to be abundantly expressed in the testis as well as the brain. In spite of the high expression level of this protein in the testis, no further analysis has been undertaken. We aimed to study the distribution and developmental changes in presenilin-1 protein, and to provide clues so as to elucidate the role of this protein in the rat testis. To evaluate the specificity of the anti presenilin-1 antibody, rat presenilin-1 protein was expressed in COS-7 cells and the recombinant protein was used for western blot analysis. A positive band of approximately 20 kDa corresponding to the C-terminal fragment of proteolyzed presenilin-1 protein was observed. Using testis and brain tissue samples, a 20 kDa band was detected in both tissues suggesting a similar proteolytic process, but the expression level in the testis was higher than that in the brain. The expression level increased significantly during postnatal testis development. By an immunohistochemical analysis of the rat testis, a strong signal was observed in interstitial cells and further study with cultured TM3 murine Leydig cells revealed an abundant expression of presenilin-1 in Leydig cells. Our study suggests that presenilin-1 expression in Leydig cells may play an important role in Leydig cell function and testis development.     

神经特异性转录因子DAT1基因的表达

采用生物信息学分析、细胞培养和RT—PCR等方法,以初步解DAT1基因在小鼠主要组织及神经系统相关肿瘤细胞中的表达．生物信息学的基因表达谱分析发现与DAT1同源的EST有100多条,大多数EST分布于胎脑、成年脑、脑肿瘤、肺及肺部的良性肿瘤等组织．SAGE分析发现DAT1在脑及神经系统肿瘤组织中表达非常广泛;RT—PCR方法检测发现DAT1只在脑组织中特异表达。而其它组织如心脏、肺、肝脏、骨骼肌、肾脏、睾丸、卵巢等未见表达;DAT1在培养的正常星形胶质细胞C8中不表达,在胶质瘤、神经母细胞瘤等细胞株中有不同水平的表达．由于DAT1是一种LIM蛋白,而LIM蛋白在细胞的分化、发育调控和肿瘤形成中有重要作用,DAT1在脑及神经系统相关肿瘤中的较高表达提示,DAT1在神经系统中有重要功能。并可能与神经系统肿瘤的发生相关．     

小鼠不同组织及缺氧损伤后的大鼠心肌细胞中RIP3的表达

目的:检测小鼠组织中受体相互作用丝氨酸/苏氨酸蛋白激酶家族(RIPs)表达谱,并检测RIP3在大鼠心肌细胞缺氧损伤后的表达。方法:①采用荧光实时定量PCR分别检测RIPs家族基因在小鼠组织(心、肝、肺、肾、脑、小肠、骨骼肌、脾和主动脉)中的mRNA表达谱,并采用Western blot进一步检测RIP3在小鼠组织的蛋白表达谱。②将培养的大鼠心肌细胞分为缺氧组和对照组,缺氧组置于缺氧环境中培养48 h,采用western blot检测其中RIP3的表达变化。结果:①mRNA水平:RIP1 mRNA在脑组织中表达最高,心脏、肺、肾、骨骼肌较低;RIP2在心脏和肺表达量较其他组织高;RIP3在肠中表达较其他组织高出4倍以上,脑组织中未检测到RIP3表达;RIP4的表达以肺最高,而骨骼肌、脑和血管中表达量低。②蛋白水平:在小鼠组织中,RIP3表达以脑、骨骼肌中最高,心脏、肝、肺中表达较低。③培养的大鼠心肌细胞中,缺氧组心肌细胞的RIP3表达量显著高于对照组(P0.05)。结论:RIPs在小鼠组织中呈现差异表达,而在培养的大鼠心肌细胞缺氧损伤后RIP3表达升高。     

Effects of LPS stimulation on the expression of prostaglandin carriers in the cells of the blood-brain and blood-cerebrospinal fluid barriers.

Prostaglandins produced in cerebral endothelial cells (CECs) are the final signal transduction mediators from the periphery to the brain during fever response. However, prostaglandins are organic anions at physiological pH, and they enter cells poorly using simple diffusion. Several transporters have been described that specifically transport prostaglandins across cell membranes. We examined the expression of the two principal prostaglandin carriers, prostaglandin transporter (PGT), and multidrug resistance-associated protein 4 (MRP4) in cells of the blood-brain barrier and in choroid epithelial cells in vitro as well as in vivo in rat brain in control conditions and after lipopolysaccharide (LPS) challenge. We detected PGT in primary cultures of rat CECs, astrocytes, pericytes, and choroid epithelial cells. LPS stimulation had no effect on the expression level of PGT in these cells; however, after LPS stimulation the polarized, dominantly luminal, expression pattern of PGT significantly changed. MRP4 is also expressed in CECs, and its level was not influenced by LPS treatment. In rat brain, PGT was highly expressed in the supraoptic and paraventricular nuclei of the hypothalamus, in the ependymal cell layer of the third ventricle, and in the choroid plexus. LPS treatment increased the expression of PGT in the supraoptic and paraventricular nuclei. Our results suggest that PGT and MRP4 likely play a role in transporting prostaglandins through the blood-brain and blood-cerebrospinal fluid barriers and may be involved in the maintenance of prostaglandin homeostasis in the brain and in the initiation of fever response.     

Tissue distribution and membrane localization of aquaporin-9 water channel: evidence for sex-linked differences in liver.

Aquaporin-9 (AQP9) is a water channel membrane protein also permeable to small solutes such as urea, glycerol, and 5-fluorouracil, a chemotherapeutic agent. With the aim of understanding the pathophysiological role of AQP9, we performed an extensive analysis by Western blotting, RT-PCR, and immunolocalization in rat tissues. Western blotting analysis revealed a major band of approximately 32 kD in testis, liver, and brain. Immunofluorescence showed strong expression of AQP9 in the plasma membrane of testis Leydig cells. In liver, AQP9 expression was found to be sex-linked. Male rats had higher levels of AQP9 than female in terms of both protein and mRNA. Moreover, in female livers the expression of AQP9 was mostly confined to perivascular hepatocytes, whereas males showed a more homogeneous hepatocyte staining. No differences in AQP9 expression level related to the age or to protein content of the diet were found, indicating that differences in the liver may be gender-dependent. In the brain, AQP9 expression was found in tanycytes mainly localized in the areas lacking a blood-brain barrier (BBB), such as the circumventricular organs (CVOs) of the third ventricles, the subfornical organ, the hypothalamic regions, and the glial processes of the pineal gland. AQP9 expression in the osmosensitive region of the brain suggests a role in the mechanism of central osmoreception. All these findings show a unique tissue distribution of AQP9 compared to the other known aquaporins.     

Splicing factor and exon profiling across human tissues

It has been shown that alternative splicing is especially prevalent in brain and testis when compared to other tissues. To test whether there is a specific propensity of these tissues to generate splicing variants, we used a single source of high-density microarray data to perform both splicing factor and exon expression profiling across 11 normal human tissues. Paired comparisons between tissues and an original exon-based statistical group analysis demonstrated after extensive RT-PCR validation that the cerebellum, testis, and spleen had the largest proportion of differentially expressed alternative exons. Variations at the exon level correlated with a larger number of splicing factors being expressed at a high level in the cerebellum, testis and spleen than in other tissues. However, this splicing factor expression profile was similar to a more global gene expression pattern as a larger number of genes had a high expression level in the cerebellum, testis and spleen. In addition to providing a unique resource on expression profiling of alternative splicing variants and splicing factors across human tissues, this study demonstrates that the higher prevalence of alternative splicing in a subset of tissues originates from the larger number of genes, including splicing factors, being expressed than in other tissues.     

Molecular Cloning of a Rat Brain cDNA,with Homology to a Tyrosine Kinase Substrate,that Induces Galactosylceramide Expression in COS-7 Cells

Abstract: A rat brain cDNA clone has been isolated, using a eukaryotic cell transient expression system in conjunction with an anti-galactosylceramide (anti-GalCer) monoclonal antibody that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). A good correlation between GalCer expression and the level of the enzyme activity of UDP-galactose:ceramide galactosyltransferase (CGT) was demonstrated. The cDNA insert encoded a polypeptide of 771 amino acids with a calculated molecular mass of 85,787 Da. The cDNA hybridized to a single mRNA of 3.1 kb in all rat organs examined, including brain, testis, and skeletal muscle. The cDNA product was determined to be a tyrosine-phosphorylated protein with a molecular mass of 110 kDa in transfected COS-7 cells and adult rat brain. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes: The transfected cells appeared to be fibroblast-like cells, whereas the parent COS-7 cells were typical epithelial-like cells. The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate but no homology to CGT. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer expression in the brain.     

Regional and cellular expression of the mannose receptor in the post-natal developing mouse brain

The mannose receptor, a glycoprotein expressed in a soluble and membrane form by macrophages, plays an important role in homeostasis and immunity. Using biochemical and immunohistochemical analyses, we demonstrate that this receptor, both in its soluble and membrane forms, is expressed in vivo in the post-natal murine brain and that its expression is developmentally regulated. Its expression is at its highest in the first week of life and dramatically decreases thereafter, being maintained at a low level throughout adulthood. The receptor is present in most brain regions at an early post-natal age, the site of the most intense expression being the meninges followed by the cerebral cortex, brain stem and the cerebellum. With age, expression of the mannose receptor is maintained in regions such as the cerebral cortex and the brain stem, whereas it disappears from others such as the hippocampus or the striatum. In healthy brain, no expression can be detected in oligodendrocytes, ependymal cells, endothelial cells or parenchymal microglia. The mannose receptor is expressed by perivascular macrophages/microglia and meningeal macrophages, where it might be important for the brain immune defence, and by two populations of endogenous brain cells, astrocytes and neurons. The developmentally dependent, regionally regulated expression of the mannose receptor in glial and neuronal cells strongly suggests that this receptor plays an important role in homeostasis during brain development and/or neuronal function.     

Molecular Cloning and Characterization of Galactosylceramide Expression Factor-1 (GEF-1)

A rat brain cDNA clone has been isolated using a eukaryotic cell transient expression system with anti-galactosylceramide (GalCer) monoclonal antibody (MAb), that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), but no homology to UDP-galactose: ceramide galactosyltransferase. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes and cell growth suppression. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of CGT increased significantly in MDCK/GEF-1 cells compared with control cells. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer and sulfatide expression in the epithelial cells as well as in the brain.     

Correlation between cell morphology and aggrecan gene expression level during differentiation from mesenchymal stem cells to chondrocytes

A morphological parameter of polygonal index was defined as the ratio of cell adhesion area versus the square of the major cell axis, and cells that had an adhesion area larger than 4000 mum(2) and a polygonal index larger than 0.3 were considered large polygonal cells. Cell morphology tended to change from fibroblast-like to polygonal and the percentage of the large polygonal cells increased almost in proportion to aggrecan mRNA expression level during the differentiation culture of mesenchymal stem cells (MSCs) to chondrocytes. Approximately 80% of the large polygonal cells were negative for MSC marker (CD90, CD166) expression and the aggrecan mRNA expression level of the large polygonal cells was markedly higher than that of cells with other morphologies.     

Expression of placental leucine aminopeptidase/oxytocinase in neuronal cells and its action on neuronal peptides.

The placental leucine aminopeptidase (P-LAP)/oxytocinase whose serum level increases with gestation is thought to contribute to the maintenance of normal pregnancy. P-LAP mRNAs are expressed in various tissues other than the placenta. In this study, we identified P-LAP protein in the brain. In contrast with the placenta where a significant portion of P-LAP is released, the enzyme was localized in the membrane fraction in brain and PC12 cells and no soluble form of the enzyme was detected. When PC12 cells were differentiated into neuronal cells by nerve growth factor (NGF), a significant increase in the expression level of P-LAP in the cell was observed. As in the case of insulin treatment of 3T3-L1 adipocytes, treatment of PC12 cells with forskolin caused the translocation of the enzyme from intracellular vesicle to the cell surface plasma membrane. In addition, P-LAP was shown to degrade several bioactive neuropeptides such as Met-enkephalin and dynorphin A (1-8). These results suggest that P-LAP plays an important role in the regulation of neuronal cell function in the brain.     

Selective down-regulation of the pro-enkephalin gene during differentiation of a multiple neuropeptide-co-expressing cell line

Regulation of co-expression of three neuropeptide genes, i.e. genes encoding enkephalin, cholecystokinin, and gastrin-releasing peptide, was studied in human neuroepithelioma cells. In nondifferentiated state, the continuous cell line SK-N-MC displayed an equally high level of expression of the enkephalin, cholecystokinin, and gastrin-releasing peptide genes. By culturing in medium containing endothelial cell growth supplement the SK-N-MC cells differentiated morphologically into a cell type with neurite-like processes. After 3 days the expression of the enkephalin gene in endothelial cell growth supplement-differentiated cells was significantly reduced by 75% as compared to the nondifferentiated cells, while there was no change in the expression of the cholecystokinin and gastrin-releasing peptide genes during differentiation. The results show that the enkephalin gene is selectively down-regulated during differentiation of neuroepithelioma cells. It is suggested that the down-regulation is related to the transient expression of the enkephalin gene in developing brain and other organs. Thus the neuroepithelioma cell line may provide a cellular model to study the underlying molecular mechanism.     

Dexamethasone selectively regulates the activity of enzymatic markers of cerebral endothelial cell lines

Summary Two endothelial cell lines were derived from grafts of the central nervous system using retrovirus mediated gene transfer to introduce the polyoma middle-T oncogene into fetal rat brain endothelial cells and transplantation of these cells into adult rat brain. In this report, we further characterize these cells and the effect of dexamethasone on the expression of specific enzymatic markers. These cells take up acetylated low density lipoprotein, leucine, and glucose, and express Factor VIII-related antigen, angiotensin converting enzyme, alkaline phosphatase, gamma-glutamyltranspeptidase, and as yet undescribed aminopeptidase A and B-like enzymes. When grown on semi-permeable membranes, these transformed cells do not spontaneously retain small hydrophilic molecules. In culture, one of the lines (EC 193) forms a confluent monolayer of spindle-shaped cells homogenously expressing gamma-glutamyltranspeptidase at a level comparable to primary cells. The other cell line (EC 219) grows as clusters of elongated cells, and gamma-glutamyltranspeptidase activity is expressed mainly in cells forming the clusters. This clustered pattern changes to a confluent one after culture on type-I collagen. Dexamethasone increases angiotensin-converting enzyme activity, and decreases the expression of gamma-glutamyltranspeptidase and aminopeptidase A, whereas the aminopeptidase B activity is little modified. Inhibition of aminopeptidase A activity by amastatin, potentiates angiotensin II effects on DNA synthesis. These results indicate that retrovirally transformed brain endothelial cells are a useful model for studying the blood-brain barrier in vitro and that dexamethasone, an agent with the potential to reduce brain edema, directly affects some blood-brain barrier properties in these endothelial cell lines.     

The Noncompetitive AMPAR Antagonist Perampanel Abrogates Brain Endothelial Cell Permeability in Response to Ischemia: Involvement of Claudin-5

The blood–brain barrier (BBB) is formed by brain endothelial cells, and decreased BBB integrity contributes to vasogenic cerebral edema and increased mortality after stroke. In the present study, we investigated the protective effect of perampanel, an orally active noncompetitive AMPA receptor antagonist, on BBB permeability in an in vitro ischemia model in murine brain endothelial cells (mBECs). The results showed that perampanel significantly attenuated oxygen glucose deprivation (OGD)-induced loss of cell viability, release of lactate dehydrogenase, and apoptotic cell death in a dose-dependent manner. Perampanel treatment did not alter the expression and surface distribution of various glutamate receptors. Furthermore, the results of calcium imaging showed that perampanel had no effect on OGD-induced increase in intracellular Ca²⁺ concentrations. Treatment with perampanel markedly reduced the paracellular permeability of mBECs after OGD in different time points, as measured by transepithelial electrical resistance assay. In addition, the expression of claudin-5 at protein level, but not at mRNA level, was increased by perampanel treatment after OGD. Knockdown of claudin-5 partially prevented perampanel-induced protection in cell viability and BBB integrity in OGD-injured mBECs. These data show that the noncompetitive AMPA receptor antagonist perampanel affords protection against ischemic stroke through caludin-5 mediated regulation of BBB permeability.     

Localization of lysosomal acid phosphatase mRNA in mouse tissues.

We studied the expression of lysosomal acid phosphatase (LAP) in mouse by hybridizing Northern blots and tissue sections with the mouse LAP cDNA. Three mRNA species of 2.3, 3.2 and 5.2 KB were identified, which differ in the length of their 3' untranslated region (UTR). The 3.2 KB mRNA is expressed in equal amounts in all tissues and represents the major species in most tissues, whereas the amounts of the 2.3 and 5.2 KB species differ. In situ hybridization of different tissues of adult mice showed a uniform expression of LAP, as expected for a housekeeping gene, except in testis and brain. In testis we found an increase in the LAP mRNA level in spermatocytes. By Northern blot analysis of young mouse testis, this increase could be attributed to late pachytene primary spermatocytes or secondary spermatocytes. In brain tissue the neurons were predominantly labeled, especially the Purkinje and pyramidal cells, whereas glial cells expressed only low amounts of LAP mRNA. Very high LAP expression was also found in the epithelial cells of the choroid plexus. Analysis of LAP expression during mouse embryonic development between Days 9.5 and 17.5 revealed a prominent expression relative to other tissues in the neural tube from Day 9.5 to Day 13.5.