Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

Oral polyprenoic acid prevents second primary hepatomas after surgical resection of the original tumor or the percutaneous injection of ethanol.

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In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

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Apolipoprotein E (apoE) is a major apolipoprotein in the brain. The ⑀4 allele of apoE is a major risk factor for Alzheimer disease, and apoE deficiency in mice leads to blood-brain barrier (BBB) leakage. However, the effect of apoE isoforms on BBB properties are as yet unknown. Here, using an in vitro BBB model consisting of brain endothelial cells and pericytes prepared from wild-type (WT) mice, and primary astrocytes prepared from human apoE3-and apoE4-knock-in mice, we show that the barrier function of tight junctions (TJs) was impaired when the BBB was reconstituted with primary astrocytes from apoE4-knock-in mice (apoE4-BBB model). The phosphorylation of occludin at Thr residues and the activation of protein kinase C (PKC) in mBECs were attenuated in the apoE4-BBB model compared with those in the apoE3-BBB model. The differential effects of apoE isoforms on the activation of PKC, the phosphorylation of occludin at Thr residues, and TJ integrity were abolished following the treatment with an anti-low density lipoprotein receptor-related protein 1 (LRP1) antibody or a LRP1 antagonist receptor-associated protein. Consistent with the results of in vitro studies, BBB permeability was higher in apoE4-knock-in mice than in apoE3-knock-in mice. Our studies provide evidence that TJ integrity in BBB is regulated by apoE in an isoform-dependent manner. Apolipoprotein E (apoE)2 is a polymorphic glycoprotein with a molecular mass of 34 kDa. Its three isoforms, apoE2, apoE3, and apoE4, are all products of the same gene, which exists as three alleles (⑀2, ⑀3, and ⑀4) at a single locus (1). Among these three isoforms, apoE4 is a major risk factor for Alzheimer disease (AD) (2, 3). ApoE is expressed in several organs, with the liver showing the highest expression level, followed by the brain. In the brain, apoE is a major apolipoprotein and plays a major role in the transportation of lipids as a lipid acceptor (1). ApoE-containing lipoprotein particles are mainly produced by astrocytes and deliver cholesterol and other essential lipids to neurons through low density lipoprotein (LDL) receptor family members (4 -6). A number of studies revealed that astrocytes are involved in the control of endothelium blood-brain barrier (BBB) properties (7,8) and that apoE deficiency leads to BBB leakage (9 -11).BBB is formed by brain endothelial cells and is essential for the protection of the central nervous system from harmful blood molecules and cells (12). Brain endothelial cells form tight junctions (TJs), which are the fundamental characteristics of BBB (13,14). The assembly of TJs requires at least three types of transmembrane protein, namely, occludin, claudin, and junctional adhesion molecule (15). Protein kinases are localized at TJs or interact directly with TJ proteins (16 -18). Among protein kinases, PKC has been shown to regulate the phosphorylation of occludin at its Thr residues and play a crucial role in the assembly and/or maintenance of TJs (19). Cells surrounding brain capillaries, such as astrocytes and pericytes, con...

In vivo effects of basic fibroblast growth factor (bFGF) on bone formation was examined in rats. Daily systemic injections of 100 micrograms/kg bFGF for 7 days caused a marked stimulation of endosteal bone formation in both cortical and secondary cancellous bone areas. Histological examinations revealed that the sequence of responses to the injections of bFGF consisted of three phases: an early increase in the number of preosteoblastic cells over the osteoblastic cell layer (days 1-3), recruitment of osteoblasts from preosteoblastic cells (days 3-5), and an increase in new bone formation (days 5-7). These histological changes in the endosteum correlated closely with histomorphometrical parameters of bone formation, and the endosteal mineral apposition rate was almost unaffected during the initial 4 days but was markedly enhanced after this period. Immunohistochemical examinations using antitransforming growth factor (TGF)-beta 1 antibody demonstrated that immunostaining of preosteoblastic cells for TGF-beta already increased 1 day after bFGF treatment. Distribution of TGF-beta in osteoblasts and bone matrices began to increase on day 3, and all the osteoblasts and new bone matrices were intensively immuno-stained on day 7. These results demonstrate that systemic injections of bFGF in rats stimulate endosteal bone formation, and that the stimulation of bone formation is preceded by an initial increase in preosteoblastic cells with later recruitment of osteoblasts from these cells. Because the distribution of TGF-beta in the endosteal cells is increased by bFGF, the effect of bFGF may at least in part be mediated by TGF-beta. However, the precise mechanism of action of bFGF on bone formation remains to be clarified.

Effect of recombinant human basic fibroblast growth factor (bFGF) on fracture healing was investigated using a tibial fracture in beagle dogs. Transverse fractures in the middle of the diaphyses were created in the right tibiae and bFGF was injected into the fracture sites at a single dose of 200 g. The time course of changes in callus volume and morphology of the fracture sites were evaluated at weeks 2, 4, 8, 16, and 32 after treatment, and the fracture strength was analyzed at weeks 16 and 32. At week 2, a radiogram of the fracture site showed obvious membranous ossification in the group injected with bFGF. Basic FGF extended the callus area at week 4 and increased the bone mineral content (BMC) in the callus at week 8. bFGF also increased the osteoclast number in the periosteal callus at weeks 2 and 4. In the bFGF group, a maximal increase in the osteoclast index was found at week 4, and an identical increase was recognized in the control group at weeks 8 and 16. These findings strongly suggested that bFGF stimulated not only callus formation but osteoclastic callus resorption. BMC in the bFGF group was followed by a rapid decrease from week 8, while that in the control group was identical from week 4. Fracture strength of the bFGF group showed significant recovery by week 16, and recovery was still evident by week 32. We conclude that bFGF promotes the fracture healing in dogs by the stimulation of bone remodeling. (J Bone Miner Res 1998;13:942-949)