ABSTRACTcDNAs representing the a subunit of polyomavirus enhancer binding protein 2 (PEBP2; also cafled PEA2) were isolated. The products of the cDNAs are highly homologous to that of Drosophila segmentation gene runt (run) for an N-proximal 128-amino acid region showing 66% identity. The run homology region encompasses the domain capable of binding to a specific nucleotide sequence motif and of dimerizing with the companion 13 subunit. The human AMLI gene related to t(8;21) acute myeloid leukemia also has a run homology region. Together with the 18 subunit, which increases the affinity of the a subunit to DNA without binding to DNA by itself, PEBP2 represents a newly discovered family of transcription factor. The major species of PEBP2a mRNA was expressed in T-cell lines but not in B-cell lines tested. Evidence indicated that PEBP2 functions as a transcriptional activator and is,involved in regulation of T-ceil-specific gene expression.Potential use of polyomavirus (Py) as a probe of mouse development has been recognized since it was observed that the wild-type Py did not grow in embryonal carcinoma cells but that it grew when the cells were induced to differentiate (1)(2)(3)(4)(5). Through the analysis of the Py enhancer, which determines the differentiation-dependent and cell-typespecific expression of the viral genes, a number of transcription factors involved in developmental regulation have been identified (6-10).PEBP2/PEA2, which binds to both the A and B cores of the Py enhancer (11), is undetectable in embryonal carcinoma F9 cells and becomes detectable after the cells are induced to differentiate (6,7). It specifically recognizes a consensus sequence, R/TACCRCA (R, purine), which was originally identified in the Py enhancer (11) and is also compatible with the core motif of murine leukemia virus enhancers (12,13). In addition, many T-cell-specific genes, such as T-cell receptor (TCR) a, X3, yand 8 and CD3e genes, contain potential PEBP2 binding sites, suggesting the possibility that PEBP2 is involved in T-cell-specific gene expression (13 [708][709][710][711][712][713][714]. The mutations are the same as the M2 mutation (14). For expression in mammalian cells, the coding regions of al, a2, and 831 cDNAs were cloned into the Xho I site of pCDMPy, in which the Py origin and enhancer region were deleted from pCDM8 (Invitrogen), resulting in pCDMPy-al, pCDMPy-a2, and pCDMPy-31. The bacterial expression plasmids pETal and pETa2, carrying the whole coding regions of al and a2 cDNAs, were constructed in the same way as pETf82 (16). To produce deletion mutants of a2, the whole coding region of a2 cDNA was inserted into the BamHI/HindIII fragment of plasmid pQE9 (Qiagen) resulting in pQE9-a2 encoding a fusion protein tagged with an N-terminal histidine cluster. Various deletions were introduced into pQE9-a2 using appropriate restriction sites: N94C306 encodes a region of aa 94-306; N1C226 encodes aa 1-226; N1C158 encodes aa 1-158; N80C226 encodes aa 80-226. N1C226, N1C158, and N80C226 proteins have one, two, o...
OBJECTIVENatriuretic peptides (NPs) have been characterized as vascular hormones that regulate vascular tone via guanylyl cyclase (GC), cyclic GMP (cGMP), and cGMP-dependent protein kinase (cGK). Recent clinical studies have shown that plasma NP levels were lower in subjects with the metabolic syndrome. The present study was conducted to elucidate the roles for NP/cGK cascades in energy metabolism.RESEARCH DESIGN AND METHODSWe used three types of genetically engineered mice: brain NP (BNP) transgenic (BNP-Tg), cGK-Tg, and guanylyl cyclase-A (GCA) heterozygous knockout (GCA+/−) mice and analyzed the metabolic consequences of chronic activation of NP/cGK cascades in vivo. We also examined the effect of NPs in cultured myocytes.RESULTSBNP-Tg mice fed on high-fat diet were protected against diet-induced obesity and insulin resistance, and cGK-Tg mice had reduced body weight even on standard diet; surprisingly, giant mitochondria were densely packed in the skeletal muscle. Both mice showed an increase in muscle mitochondrial content and fat oxidation through upregulation of peroxisome proliferator–activated receptor (PPAR)-γ coactivator (PGC)-1α and PPARδ. The functional NP receptors, GCA and guanylyl cyclase-B, were downregulated by feeding a high-fat diet, while GCA+/− mice showed increases in body weight and glucose intolerance when fed a high-fat diet. NPs directly increased the expression of PGC-1α and PPARδ and mitochondrial content in cultured myocytes.CONCLUSIONSThe findings together suggest that NP/cGK cascades can promote muscle mitochondrial biogenesis and fat oxidation, as to prevent obesity and glucose intolerance. The vascular hormone, NP, would contribute to coordinated regulation of oxygen supply and consumption.
a b s t r a c tInduced pluripotent stem (iPS) cells were recently established from human fibroblasts. In the present study we investigated the adipogenic differentiation properties of four human iPS cell lines and compared them with those of two human embryonic stem (ES) cell lines. After 12 days of embryoid body formation and an additional 10 days of differentiation on Poly-L-ornithine and fibronectincoated dishes with adipogenic differentiation medium, human iPS cells exhibited lipid accumulation and transcription of adipogenesis-related molecules such as C/EBPa, PPARc2, leptin and aP2. These results demonstrate that human iPS cells have an adipogenic potential comparable to human ES cells.
Our study indicates that the ACTH stimulation test is useful in the diagnosis of an APA among patients suspected of PA. This test can be used to select patients who are highly suspected of an APA and definitely require adrenal venous sampling.
Mineralocorticoid receptors (MRs) are classically known to be expressed in the distal collecting duct of the kidney. Recently it was reported that MR is identified in the heart and vasculature. Although MR expression is also found in the brain, it is restricted to the hippocampus and cerebral cortex under normal condition, and the role played by MRs in brain remodeling after cerebral ischemia remains unclear. In the present study, we used the mouse 20-min middle cerebral artery occlusion model to examine the time course of MR expression and activity in the ischemic brain. We found that MR-positive cells remarkably increased in the ischemic striatum, in which MR expression is not observed under normal conditions, during the acute and, especially, subacute phases after stroke and that the majority of MR-expressing cells were astrocytes that migrated to the ischemic core. Treatment with the MR antagonist spironolactone markedly suppressed superoxide production within the infarct area during this period. Quantitative real-time RT-PCR revealed that spironolactone stimulated the expression of neuroprotective or angiogenic factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), whereas immunohistochemical analysis showed astrocytes to be cells expressing bFGF and VEGF. Thereby the incidence of apoptosis was reduced. The up-regulated bFGF and VEGF expression also appeared to promote endogenous angiogenesis and blood flow within the infarct area and to increase the number of neuroblasts migrating toward the ischemic striatum. By these beneficial effects, the infarct volume was significantly reduced in spironolactone-treated mice. Spironolactone may thus provide therapeutic neuroprotective effects in the ischemic brain after stroke.
Although there have been reports of the differentiation of mesenchymal stem cells and mouse embryonic stem (ES) cells into steroid-producing cells, the differentiation of human ES/induced pluripotent stem (iPS) cells into steroid-producing cells has not been reported. The purpose of our present study was to establish a method for inducing differentiation of human ES/iPS cells into steroid-producing cells. The first approach we tried was embryoid body formation and further culture on adherent plates. The resultant differentiated cells expressed mRNA encoding the steroidogenic enzymes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, CYP17A1, and CYP19, and secreted progesterone was detected in the cell medium. However, expression of human chorionic gonadotropin was also detected, suggesting the differentiated cells were trophoblast like. We next tried a multistep approach. As a first step, human ES/iPS cells were induced to differentiate into the mesodermal lineage. After 7 d of differentiation induced by 6-bromoindirubin-3'-oxime (a glycogen synthase kinase-3β inhibitor), the human ES/iPS cells had differentiated into fetal liver kinase-1- and platelet derived growth factor receptor-α-expressing mesodermal lineage cells. As a second step, plasmid DNA encoding steroidogenic factor-1, a master regulator of steroidogenesis, was introduced into these mesodermal cells. The forced expression of steroidogenic factor-1 and subsequent addition of 8-bromoadenosine 3',5'-cyclic monophosphate induced the mesodermal cells to differentiate into the steroidogenic cell lineage, and expression of CYP21A2 and CYP11B1, in addition to steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, CYP11A1, and CYP17A1, was detected. Moreover, secreted cortisol was detected in the medium, but human chorionic gonadotropin was not. These findings indicate that the steroid-producing cells obtained through the described multistep method are not trophoblast like; instead, they exhibit characteristics of adrenal cortical cells.
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