Background: The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly.

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Bone morphogenetic protein (BMP) was extracted from human dentin matrix with 4 mol/L guanidine-HCl and was purified by liquid chromatography. SDS-PAGE and IEF showed that the purified BMP was homogeneous and induced new bone formation in situ after three weeks when implanted into muscle pouches in Wistar rats. The molecular weight of BMP was estimated to be about 20.0 kDa by SDS-PAGE, and the pI value was 8.8 by IEF. Amino acid analysis suggested that BMP is a protein containing 191 amino acids. A partial amino acid sequence was obtained from the final purified BMP. Dentin-matrix-derived BMP is probably not identical to, but is similar to, bone-matrix-derived BMP, though both types of BMP have the same action in vivo.

Pre-mRNA 59 spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a highthroughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained ;250,000 high-quality reads corresponding to 8790 genes, ;58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of ''infrequently trans-spliced'' genes, accounting for ;28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing ;80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis-and trans-splicing and indicates that the prevalence of 63-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca 2+ homeostasis, and actin cytoskeleton.[Supplemental material is available online at http://www.genome.org. The sequence data from this study have been submitted to the NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession no. SRX006190.]A striking evolutionary variation in eukaryotic gene expression mechanisms is the presence or absence in diverse organismal groups of a form of RNA splicing-pre-mRNA spliced leader (SL) trans-splicing (Davis 1996;Nilsen 2001;Hastings 2005). SL transsplicing is closely related to conventional RNA splicing, or cissplicing. The same nucleotide sequence features define donor and acceptor sites, and both processes occur in spliceosomes and involve formation of a 59,39,29 branchpoint upstream of the acceptor site (Agabian 1990;Nilsen 1993). Whereas cis-splicing joins paired donor and acceptor sites within a single RNA molecule, in SL transsplicing the donor exon is the 59-segment of a specialized small 59-capped RNA molecule-the SL RNA-and the target is an unpaired acceptor site near the 59-end of a pre-mRNA molecule.Transfer of the SL RNA 59-segment, the SL sequence, to the pre-mRNA molecule occurs with loss of the pre-mRNA's initial 59-segment ups...

Extrinsic fibroblast growth factor (FGF) signal and intrinsic factors that determine the response of the signal-receiving blastomeres to FGF regulate mesoderm patterning in embryos of the ascidian Halocynthia roretzi. To investigate how cells integrate information from extrinsic and intrinsic inputs, we examined Brachyury (Hr-Bra) promoter activity in the early embryo. Hr-Bra, which encodes a key transcription factor for notochord development, is expressed exclusively in notochord precursors in a manner dependent on the FGF-MEK-MAPK-Ets signaling pathway and on the intrinsic factors Zic and FoxA. Reporter gene expression driven by the 900-bp upstream region of the Hr-Bra promoter was detected as early as the 110-cell stage in notochord precursors by in situ hybridization with a LacZ probe. Deletion analysis combined with MEK inhibitor treatment demonstrated that the -598/-499 region carries FGF-responsiveness. Electrophoretic mobility shift assay identified three Ets-binding sites in this region that were required for promoter activity. Further deletion analysis conducted by injecting eggs with reporter constructs at higher concentration suggested that the -398/-289 region also has enhancer activity, although ectopic reporter expression was detected in nerve cord and endoderm precursors. The -398/-289 region has a Zic-binding site that was also essential for the enhancer activity. These results indicate that Ets- and Zic-binding sites are critical for the initiation of Hr-Bra expression. In conclusion, information from both extrinsic and intrinsic factors is integrated at the level of enhancer of the target gene by direct binding of the transcription factors to the enhancer region.

The outcome of pulmonary metastasectomy of hepatocellular carcinoma (HCC) was appraised in this study. Twenty patients with pulmonary metastasis from HCCs undergoing pulmonary resection between 1990 and 2003 were included in this study. They had undergone curative treatment for the primary lesion and were candidates for a pulmonary metastasectomy for complete resection. Among the 20 patients, 13 died: 5 from hepatic failure, 5 from respiratory failure, and 2 from brain metastasis due to recurrence of the HCC. One patient died from cardiac failure without HCC recurrence. At the latest observation, three of the seven survivors were doing well without HCC recurrence, and others survived with recurrence. The overall survival rates after the initial lung surgery were 45.3% at 1 year and 23.8% at 3 years, respectively. The survival rates without recurrence were 32.4% at 1 year and 21.6% at 3 years, respectively. A Kaplan-Meier analysis showed that multiple lung surgeries and a negative histologic finding of the liver cut surface were favorable characteristics for survival without recurrence. In conclusion, the selected patients were Candidates for pulmonary metastasectomy after a curative hepatectomy for HCC and could benefit from the complete resection. Also, repeated pulmonary resections through thoracoscopy could result in the long-term survival of patients with pulmonary recurrence of HCC.

Background: The neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, laninamivir and peramivir are available in Japan. However, the selective use of NAIs for treating outpatients with influenza has not been clearly defined. Methods: We assigned 191 patients with influenza to 4 groups, each treated with a different NAI, and then compared how long it took to alleviate fever and other symptoms and to eliminate the virus. Results: Alleviation of fever occurred significantly sooner with peramivir than with either zanamivir (p = 0.0002) or oseltamivir (p = 0.0059), but was not significantly different from that with laninamivir (p = 0.0457; p < 0.0083). Other symptoms were also alleviated sooner by peramivir than by the other 3 NAIs. Conclusions: The ability of each NAI to alleviate influenza symptoms and fever varied. The appropriate use of NAIs requires further study.

The ascidian embryo, a model for the primitive mode of chordate development, rapidly forms a dorsal nervous system which consists of a small number of neurons. Here, we have characterized the transcriptional regulation of an ascidian synaptotagmin (syt) gene to explore the molecular mechanisms underlying development of synaptic transmission. In situ hybridization showed that syt is expressed in all neurons described in previous studies and transiently in the embryonic epidermis. Neuronal expression of syt requires induction from the vegetal side of the embryo, whereas epidermal expression occurs autonomously in isolated ectodermal blastomeres. Introduction of green fluorescent protein reporter gene constructs into the ascidian embryos indicates that a genomic fragment of the 3.4-kb 5' upstream region contains promoter elements of syt gene. Deletion analysis of the promoter suggests that syt expression in neurons and in the embryonic epidermis depends on distinct cis-regulatory regions.