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Takayasu's arteritis is a persistent chronic progressive inflammation of the large- and medium-caliber arteries. Controlling cardiovascular variability during anesthesia and overcoming difficulties of cardiovascular monitoring due to the impaired vessels are important in patients with Takayasu's arteritis. Remimazolam is a novel short-acting benzodiazepine with mild effects on hemodynamics. We report the case of a patient with Takayasu's arteritis who underwent oral surgery under general anesthesia. This report highlights the use of remimazolam and remifentanil to reduce hemodynamic perturbations using estimated continuous cardiac output monitoring., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 International Anesthesia Research Society.)

Previously undiagnosed or asymptomatic epiglottic cysts may be coincidentally detected during intubation. This retrospective case series identified undiagnosed epiglottic cysts that were discovered during intubation in 4 patients who underwent oral surgery under general anesthesia at our hospital during a 6-year period. Including 2 additional cases, 1 previously diagnosed and 1 detected during preoperative imaging, epiglottic cysts were observed in 6 of 1112 cases (0.54%) total. Among the undiagnosed epiglottic cyst cases, mild dyspnea on effort or snoring was reported in 2 patients, but all others were asymptomatic. Upon discovering previously undiagnosed epiglottic cysts during intubation, it is essential to proceed cautiously, remain alert for potential airway management difficulties, and avoid injuring or rupturing the cysts. In addition, any available preoperative imaging should be reviewed as information pertinent to the airway and any abnormalities may be useful. This report discusses the anesthetic care of 6 patients with epiglottic cysts that were previously known or initially discovered during intubation., (© 2021 by the American Dental Society of Anesthesiology.)

In budding tunicates, aging accompanies a decrease in the gene expression of mitochondrial transcription factor A ( Tfam ), and the in vivo transfection of Tfam mRNA stimulates the mitochondrial respiratory activity of aged animals. The gene expression of both the transcriptional repressor Yin-Yang-1 ( YY1 ) and corepressor Sirtuin6 ( Sirt6 ) increased during aging, and the cotransfection of synthetic mRNA of YY1 and Sirt6 synergistically downregulated Tfam gene expression. Pulldown assays of proteins indicated that YY1-associated factor 2 (YAF2) was associated with both YY1 and SIRT6. Protein cross-linking confirmed that YAF2 bound YY1 and SIRT6 with a molar ratio of 1:1. YY1 was bound to CCAT- or ACAT-containing oligonucleotides in the 5' flanking region of the Tfam gene. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) showed that SIRT6 specifically induced the histone H3 lysine 9 (H3K9) deacetylation of the Tfam upstream region. YY1 and YAF2 accelerated SIRT6-induced H3K9 deacetylation. YY1 and Sirt6 mRNA transfection attenuated mitochondrial respiratory gene expression and blocked MitoTracker fluorescence. In contrast, the SIRT6 inhibitor and Tfam mRNA antagonized the inhibitory effects of YY1 and Sirt6 , indicating that Tfam acts on mitochondria downstream of YY1 and Sirt6 . We concluded that in the budding tunicate Polyandrocarpa misakiensis, YY1 recruits SIRT6 via YAF2 to the TFAM gene, resulting in aging-related mitochondrial downregulation.

Planula larvae of the scleractinian coral, Acropora tenuis, consist of elongated ectodermal cells and developing inner endodermal cells. To establish in vitro cell lines for future studies of cellular and developmental potential of coral cells, larvae were successfully dissociated into single cells by treating them with a tissue dissociation solution consisting of trypsin, EDTA, and collagenase. Brown-colored cells, translucent cells, and pale blue cells were the major components of dissociated larvae. Brown-colored cells began to proliferate transiently in the culture medium that was devised for the coral, while translucent cells and pale blue cells decreased in number about 1 week after cell dissociation. In addition, when a modular protease, plasmin, was added to the cell culture medium, brown-colored cells extended pseudopodia and assumed amorphous shapes. They then continued to proliferate in clumps for more than 6 months with a doubling time of approximately 4-5 days. From 3 weeks of cell culture onward, brown-colored cells often aggregated and exhibited morphogenesis-like behavior to form flat sheets, and blastula-like clusters or gastrula-like spheres. Single cells or cell-clusters of the cell lines were analyzed by RNA-seq. This analysis showed that genes expressed in these cells in vitro were A. tenuis genes. Furthermore, each cell line expressed a specific set of genes, suggesting that their properties include gastroderm, secretory cells, undifferentiated cells, neuronal cells, and epidermis. All cell properties were maintained stably throughout successive cell cultures. These results confirm the successful establishment of a coral in vitro cell line., (© 2021. The Author(s).)

Reporter analyses of Hox1 and Brachyury (Bra) genes have revealed examples of redundant enhancers that provide regulatory robustness. Retinoic acid (RA) activates through an RA-response element the transcription of Hox1 in the nerve cord of the ascidian Ciona intestinalis. We also found a weak RA-independent neural enhancer within the second intron of Hox1. The Hox1 gene in the larvacean Oikopleura dioica is also expressed in the nerve cord. The O. dioica genome, however, does not contain the RA receptor-encoding gene, and the expression of Hox1 has become independent of RA. We have found that the upstream sequence of the O. dioica Hox1 was able to activate reporter gene expression in the nerve cord of the C. intestinalis embryo, suggesting that an RA-independent regulatory system in the nerve cord might be common in larvaceans and ascidians. This RA-independent redundant regulatory system may have facilitated the Oikopleura ancestor losing RA signaling without an apparent impact on Hox1 expression domains. On the other hand, vertebrate Bra is expressed in the ventral mesoderm and notochord, whereas its ascidian ortholog is exclusively expressed in the notochord. Fibroblast growth factor (FGF) induces Bra in the ventral mesoderm in vertebrates, whereas it induces Bra in the notochord in ascidians. Disruption of the FGF signal does not completely silence Bra expression in ascidians, suggesting that FGF-dependent and independent enhancers might comprise a redundant regulatory system in ascidians. The existence of redundant enhancers, therefore, provides regulatory robustness that may facilitate the acquisition of new expression domains.

The accurate monitoring of arterial blood pressure is important for cardiovascular management. However, the frequency characteristics of pressure transducer kits are influenced by the length of the pressure-resistant tube. To date, there have been few studies addressing the frequency characteristics of pressure transducer kits with inserted pressure-resistant extension tubes (pressure-resistant extension tube (ET) circuits). In this study, we examine ET circuits from the viewpoint of the frequency characteristics of pressure transducer kits. DT4812J transducer kits (length 150 cm; Argon Medical Devices, TX, USA) were used. Three original ET circuits were prepared, with the pressure-resistant tube of the DT4812J being extended with a 30-cm length of pressure-resistant tube (180ET circuit), a 60-cm length of pressure-resistant tube (210ET circuit), and a 90-cm length of pressure-resistant tube (240ET circuit). Each of these circuits was evaluated as part of this study. The natural frequency of the original DT4812J circuit was 45.90 Hz while the damping coefficient was 0.160. For the 180 ET circuit, the natural frequency and damping coefficient were 36.4 Hz and 0.162, respectively. For the ET210 circuit, the natural frequency and damping coefficient were 30.3 Hz and 0.175, respectively. For the ET210 circuit, the natural frequency and damping coefficient were 25.3 Hz and 0.180, respectively. As a result of extending the circuit, it was found that the natural frequency decreased drastically, while the damping coefficient increased slightly. When the extension of a pressure transducer kit is required, we should pay careful attention to the major decrease in the natural frequency, which may influence the pressure monitoring.

Respiratory depression can occur during intravenous general anesthesia without tracheal intubation. A new acoustic method for respiratory rate monitoring, RRa ® (Masimo Corp., Tokyo, Japan), has been reported to show good reliability in post-anesthesia care and emergency units. The purpose of this study was to investigate the reliability of the acoustic method for measurement of respiratory rate during intravenous general anesthesia, as compared with capnography. Patients with dental anxiety undergoing dental treatment under intravenous anesthesia without tracheal intubation were enrolled in this study. Respiratory rate was recorded every 30 s using the acoustic method and capnography, and detectability of respiratory rate was investigated for both methods. This study used a cohort study design. In 1953 recorded respiratory rate data points, the number of detected points by the acoustic method (1884, 96.5 %) was significantly higher than that by capnography (1682, 86.1 %) (P < 0.0001). In the intraoperative period, there was a significant difference in the LOA (95 % limits of agreement of correlation between difference and average of the two methods)/ULLOA (under the lower limit of agreement) in terms of use or non-use of a dental air turbine (P < 0.0001). In comparison between capnography, the acoustic method is useful for continuous monitoring of respiratory rate in spontaneously breathing subjects undergoing dental procedures under intravenous general anesthesia. However, the acoustic method might not accurately detect in cases in with dental air turbine.

The horizontal transfer of genes between distantly related organisms is undoubtedly a major factor in the evolution of novel traits. Because genes are functionless without expression, horizontally transferred genes must acquire appropriate transcriptional regulations in their recipient organisms, although the evolutionary mechanism is not known well. The defining characteristic of tunicates is the presence of a cellulose containing tunic covering the adult and larval body surface. Cellulose synthase was acquired by horizontal gene transfer from Actinobacteria. We found that acquisition of the binding site of AP-2 transcription factor was essential for tunicate cellulose synthase to gain epidermal-specific expression. Actinobacteria have very GC-rich genomes, regions of which are capable of inducing specific expression in the tunicate epidermis as the AP-2 binds to a GC-rich region. Therefore, the actinobacterial cellulose synthase could have been potentiated to evolve its new function in the ancestor of tunicates with a higher probability than the evolution depending solely on a spontaneous event., (© 2016 The Author(s).)

Blood pressure transducer kits are equipped with two types of Planecta™ ports-the flat-type Planecta™ port (FTP) and the Planecta™ port with a three-way stopcock (PTS). We reported that FTP application decreased the natural frequency of the kits. However, Planecta™ is an invaluable tool as it prevents infection, ensures technical simplicity, and excludes air. Hence, an ideal Planecta™ port that does not decrease the frequency characteristics is required. As a first step in this direction, we aimed to assess the influence of PTSs on the natural frequency of blood transducer kits. A DTXplus transducer kit (DT4812J; Argon Medical Devices, TX, USA) was used along with ≥1 PTSs (JMS, Hiroshima, Japan), and the frequency characteristics were assessed. The natural frequency and damping coefficient of each kit were obtained by using frequency characteristics analysis software, and these parameters were evaluated by plotting them on Gardner's chart. Regardless of whether one or two PTSs were inserted, the natural frequency of the kits only slightly decreased (from 42.5 to 41.1 Hz, when 2 PTSs were used). Thus, the frequency characteristics of the kits with PTSs were adequate for pressure monitoring. The insertion of ≥2 FTPs in pressure transducer kits should be avoided, as they markedly decrease the natural frequency and lead to underdamping. However, the effect of PTS insertion in pressure transducer kits on the frequency characteristics is minimal. Thus, we found that the use of PTS markedly improved the frequency characteristics as compared to the use of FTP.

We report a child with vocal cord adhesion encountered during induction of anesthesia. A 4-month-old girl was scheduled for bilateral lip plasty. She was intubated for one week due to pneumonia at the age of 3 days. Hoarseness and stridor appeared just after extubation. Although laryngo-fiberoptic examination had been tried several times, otorhinologists could not find any abnormality. We once decided to postpone the operation because of severe stridor. However, laryngofiberoptic examination could not reveal any abnormality, and we rescheduled the operation. Tracheal intubation using laryngoscope was not possible due to vocal cord adhesion. Finally, 2.5 mm ID tracheal tube was intubated by using a fiberscope, and lip plasty was performed. The patient stayed in the ICU for 7 days after surgery. Tracheotomy was performed 3 weeks after the operation. We should pay attention to stridor in an infant before general anesthesia, since it suggests severe airway narrowing although laryngo-fiberoptic examination could not find any abnormalities.

The patient was a 6-year-old girl with pulmonary atresia, intact ventricular septum and major aortopulmonary collateral artery with tetralogy of Fallot Her Sp(O2) was around 60% under room air, and she could not walk long. She underwent dental treatment under general anesthesia. Invasive monitoring using pulmonary artery catheter should have been avoided, since the risk of monitoring greatly exceeds that of the treatment. The patient entered the operating room with her mother, and anesthesia was induced with intravenous midazolam, propofol and vecuronium. She was intubated orally first and impedance cardiography monitoring was started. FI(O2) was maintained at 0.5-1.0. Increases in airway pressure and Pa(CO2) were appropriately avoided. Dental treatment is important for infants with cardiac disease not only to reduce their pain, but also to reduce the risk of infection. It often requires general anesthesia. We have to conduct it with less invasiveness and less stress.

Dandy-Walker syndrome (DWS) is characterized by perfect or partial defect of the cerebellum vermis and cystic dilatation of the posterior fossa communicating with the fourth ventricle. Common clinical signs are mental retardation, cerebellar ataxia, and those of increased intracranial pressure (ICP). Associated congenital anomalies are craniofacial, cardiac, renal, and skeletal abnormalities. We experienced a case of intravenous sedation and six times of "the same day" general anesthesia for a school-aged boy (10-13 years old) with DWS and hypodentinogenesis. The patient underwent an examination and dental treatments. We had to pay attention to airway management tracheal tube selection and control of ICP. In addition, we should prevent tooth injuries through mishaps during tracheal intubations, since all-tooth-hypoplasia with fragile dental crowns was strongly suggested in this case. Detailed postoperative care is also required for general anesthesia afflicted with DWS.

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aicardi syndrome is a rare hereditary disorder that develops in only girls with the trilogy of nutatory epilepsy, callosal agenesis and chorioretinopathy. We experienced general anesthesia twice for a patient with Aicardi syndrome in addition to heavy mental retardation. She underwent surgical correction for cleft lip and palate at 6 months of age and at 2 years of age, respectively. Anesthesia was induced slowly with inhalation of nitrous oxide, oxygen and sevoflurare. After securing an intravenous route, midazolam, thiopental and vecuronium were administered and intubated orally. Anesthesia was maintained with isoflurane safely. Patients with Aicardi syndrome have a high risk of aspiration pneumonia caused by underdeveloped swallowing ability due to callosal agenesis. We should, therefore, pay attention to prevention of seizure and aspiration pneumonia during the perioperative period.

Pressure-transducer kits have frequency characteristics such as natural frequency and damping coefficient, which affect the monitoring accuracy. The aim of the present study was to investigate the effect of planecta ports and a damping device (ROSE™, Argon Medical Devices, TX, USA) on the frequency characteristics of pressure-transducer kits. The FloTrac sensor kit (Edwards Lifesciences, CA, USA) and the DTXplus transducer kit (Argon Medical Devices) were prepared with planecta ports, and their frequency characteristics were tested with or without ROSE™. The natural frequency and damping coefficient of each kit were obtained using frequency characteristics analysis software and evaluated by plotting them on the Gardner's chart. By inserting a planecta port, the natural frequency markedly decreased in both the FloTrac sensor kit (from 40 to 22 Hz) and the DTXplus transducer kit (from 35 to 22 Hz). In both kits with one planecta port, the damping coefficient markedly increased by insertion of ROSE™ from 0.2 to 0.5, optimising frequency characteristics. In both kits with two planecta ports, however, the natural frequency decreased from 22 to 12 Hz. The damping coefficient increased from 0.2 to 0.8 by insertion of ROSE™; however, optimisation was not achieved even by ROSE™ insertion. Planecta ports decrease the natural frequency of the kit. ROSE™ is useful to optimise the frequency characteristics in the kits without or with one planecta port. However, optimisation is difficult with two or more planecta ports, even with the ROSE™ device.

Chondroitin sulfate (CS) is a carbohydrate component of proteoglycans. Several types of sulfotransferases determine the pattern of CS sulfation, and thus regulate the biological functions of proteoglycans. The protochordate ascidians are the closest relatives of vertebrates, but the functions of their sulfotransferases have not been investigated. Here, we show that two chondroitin 4-O-sulfotransferases (C4STs) play important roles in the embryonic morphogenesis of the ascidian Ciona intestinalis. Ci-C4ST-like1 is predominantly expressed in the epidermis and muscle. Epidermal and muscle cells became spherical upon the injection of a Ci-C4ST-like1-specific morpholino oligo (MO), thus suggesting weakened cell adhesion. Co-injection of a Ci-C4ST-like1-expressing transgene rescued the phenotype, suggesting that the effects of the MO were specific. Ci-C4ST-like3 was expressed in the central nervous system, muscle, and mesenchyme. A specific MO appeared to affect cell adhesion in the epidermis and muscle. Convergent extension movement of notochordal cells was also impaired. Forced expression of Ci-C4ST-like3 restored normal morphogenesis, suggesting that the effects of the MO were specific. The present study suggests that Ci-C4ST-like1 and Ci-C4ST-like3 are required for cell adhesion mainly in the epidermis and muscle., (© 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.)

Osteogenesis imperfecta (OI) is a rare hereditary disorder characterized by an excessive tendency to bone fractures and retarded growth. We report an anesthetic management of the patient with OI who has the history of vertebral bone fracture by coughing. A 44-year-old female underwent mandibular resection and reconstruction with a metal instrument due to ossifying fibroma 35 years ago. Since then, she had undergone mandibular resection and shaving the instrument several times because of recurrence of the tumor and/or fracture of the instrument. This time, some parts of the instrument were removed under general anesthesia since it had exposed from the skin. Difficulty in mask ventilation and intubation was predicted due to the defect of mandible and some muscles supporting the tongue and the pharynx. Awake fiber-optic nasotracheal intubation, therefore, was performed in consideration of airway obstruction. Dexmedetomidine was administered to reduce the risk of bone fracture in addition to low doses of midazolam and fentanyl. Considering incomplete respiration after extubation, the tracheal tube was extubated after inserting the tube exchanger into the trachea through the tube. The tube exchanger was pulled out after confirming spontaneous respiration and upper airway patency. The patient was cooperative, and respiratory and hemodynamic conditions were stable throughout.

In ascidian embryos, ectodermal tissues derive from blastomeres in the animal hemisphere. The animal hemisphere-specific gene expression is observed as early as the 16-cell stage. Here, we characterized animal hemisphere-specific enhancers of three genes, Ci-ephrin-Ad, Ci-TGFβ-NA1 and Ci-Fz4. Deletion analyses identified minimal essential elements. Although these elements contained multiple GATA sequences, electrophoretic mobility shift assays revealed that only some of them were strong binding sites for the transcription factor Ci-GATAa. On the other hand, the motif-searching software MEME identified an octamer, GA (T/G) AAGGG, shared by these enhancers. In Ci-ephrin-Ad and Ci-TGFβ-NA1, the octamer was GATAAGGG, which strongly bound Ci-GATAa. The 397-bp upstream region of Ci-ephrin-Ad contained two strong Ci-GATAa-binding sites, one of which was the octamer motif. Mutation in the octamer motif, but not the other Ci-GATAa-binding site, severely affected the enhancer activity. The 204-bp upstream region of Ci-TGFβ-NA1 contained four strong Ci-GATAa-binding sites, including the octamer motif. Mutation only in the octamer motif, leaving the other three Ci-GATAa-binding sites intact, abolished the enhancer activity. These results suggest a crucial role for the octamer motif., (© 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.)

The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci-Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci-Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid-response element (RARE). Mutation of the RARE in the Ci-Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci-Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA-dependent and one RA-independent., (© 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.)

The budding ascidian Polyandrocarpa misakiensis proliferates asexually by budding. The atrial epithelium is a multipotent but differentiated tissue, which transdifferentiates into various tissues and organs after the bud separates from the parental body. We isolated cDNA clones homologous to the myc proto-oncogene from P. misakiensis. The cDNA, named Pm-myc, encoded a polypeptide of 639 amino acid residues, containing Myc-specific functional motifs, Myc box I and Myc box II, and the basic helix-loop-helix domain. Expression of Pm-myc was observed in the atrial epithelium in the organ-forming region of the developing bud, where the epithelial cells dedifferentiate and re-enter the cell cycle. The expression was also observed in fibroblast-like cells, which are known to participate in the organogenesis together with the epithelial cells. Unexpectedly, the atrial epithelium expressed Pm-myc more than one day before the dedifferentiation. The organogenesis was disturbed by Pm-myc-specific double-stranded RNA. In situ hybridization revealed that Pm-myc-positive fibroblast-like cells disappeared around the organ primordium of the dsRNA-treated bud. The results suggest that the mesenchymal-epithelial transition of fibroblast-like cells is important for the organogenesis in this budding ascidian species., (© 2011 The Authors. Development, Growth & Differentiation © 2011 Japanese Society of Developmental Biologists.)

The protochordate ascidian Polyandrocarpa misakiensis has a striking ability to regenerate. When the posterior half of the adult body is amputated, the anterior half completely loses the esophagus, stomach and intestine. These organs are reconstituted in a week. Histological observation revealed that the regeneration involves transdifferentiation of the atrial epithelium near the cut surface. The morphological features of the gut primordium were similar to those observed in the developing bud of this species. Inhibitors of the synthesis of retinoic acid (RA) suppressed the formation of the gut. 13-cis RA rescued the regenerates from the inhibitor-induced hypoplasia. These results suggest that RA is required for the regeneration of the gut. A gene encoding the RA receptor (Pm-RAR) and its target gene, TRAMP, were expressed in and around the regenerating gut. Pm-RAR-specific and TRAMP-specific double-stranded RNA molecules inhibited the regeneration of the gut, indicating that the RA signal is mediated at least in part by Pm-RAR and TRAMP. These results suggested that RA triggers the transdifferentiation of the atrial epithelium into the gut in regenerating animals, as it does during asexual reproduction.

Retinoic acid regulates the spatial pattern of gene expression mainly in the epidermis in the protochordate ascidian, Ciona intestinalis. Our previous study characterized the enhancer element responsible for the activation of the retinoic acid receptor (RAR) in the dorsal midline epidermis (DME). In the present study, detailed analysis identified two small sequence elements required for the transcriptional activation in the DME. Deletion of either one of these sequences resulted in suppression of the DME enhancer, suggesting that both sites are necessary. The nucleotide sequences of these two sites were similar to the consensus recognition sequences for the Msx and Sox transcription factors, respectively. These transcription factors are expressed in the DME lineage blastomeres (b7.9 and b7.10 blastomere pairs of bilaterally symmetrical embryos of this species) at the 64-cell stage. Electrophoretic mobility shift assays revealed that recombinant Msxb and SoxB1 proteins specifically bound to these sequences. These results suggest that Msxb and SoxB1 synergistically activate the enhancer in the DME.

The acetylcholinesterase-encoding gene in the ascidian Ciona intestinalis (Ci-AChE) is expressed in tail muscle cells from the gastrula stage. When the embryo was continuously treated with aphidicolin from the 32-cell stage, Ci-AChE was not expressed even when control embryos reached the tailbud stage. This result suggests that Ci-AChE acquires the competence to be transcribed after passing through a certain number of DNA replication cycles. A lacZ reporter gene containing the 5' flanking region of Ci-AChE was expressed in the tail muscle cells. Aphidicolin treatment from the 32-cell stage affected, but did not completely suppress, the expression of lacZ. A bisulfite sequencing analysis was carried out to examine the methylation status of four regions within the 5' flanking sequence and the first exon. However, all of these regions remained unmethylated from the 16-cell to 110-cell stages. The results suggested that the DNA of the Ci-AChE locus is not responsible for counting the rounds of replication. We examined the expression of the C. intestinalis MyoD (Ci-MyoD), a transcription factor that activates Ci-AChE. Aphidicolin treatment from the 32-cell stage suppressed the expression of Ci-MyoD, even when control embryos reached the gastrula stage. These results suggest that a lack of Ci-MyoD is critical to the suppression of Ci-AChE in aphidicolin-treated embryos.

Hox genes play important roles in the specification of spatial identity during development of vertebrate embryos. Retinoic acid regulates the transcription of Hox genes in vertebrates. We identified an epidermal enhancer in the 5' flanking region of an ortholog of Hox1 (Ci-Hox1) in the ascidian Ciona intestinalis. This enhancer element drives the transcription of a lacZ reporter gene in the epidermis in the posterior trunk and the anterior tail region of tailbud-stage embryos. Inhibition of retinoic acid synthesis resulted in inactivation of the expression of the reporter gene. The enhancer contains a putative retinoic acid response element. When this element was mutagenized, the expression of the reporter gene disappeared from the epidermis. This sequence was also required for the response to exogenously administered retinoic acid. A heterodimeric nuclear receptor, consisting of the retinoic acid receptor and retinoid X receptor, bound to this sequence. These results indicate that retinoic acid directly activates the epidermal enhancer of Ci-Hox1. This is the first demonstration that retinoic acid is necessary for endogenous gene expression in ascidian embryos.

We carried out RNA interference by expressing short hairpin RNA (shRNA) in the Ciona intestinalis embryo. For this purpose, we identified a gene encoding U6 small nuclear RNA (snRNA) in the C. intestinalis genome. The 1-kb sequence upstream of the U6 snRNA gene was sufficient for directing transcription of short RNA as revealed by Northern blot hybridization. An shRNA-expressing plasmid vector was constructed, in which shRNA-encoding oligonucleotides are inserted downstream of the U6 promoter. An shRNA that contained a sequence homologous to the C. intestinalis tyrosinase gene (Ci-tyrosinase) suppressed melanization of pigment cells in the brain of morphologically normal tailbud embryos. An shRNA that perfectly matched the translated sequence of enhanced green fluorescent protein (EGFP) (a mutant type of Aequorea victoria green fluorescent protein) suppressed the expression of the coelectroporated EGFP transgene. These results suggest that the expression of shRNA interferes with functions of both endogenous and exogenous genes. The shRNA-expressing plasmid constructed in the present study provides an easy and inexpensive alternative for the functional analysis of genes in ascidian embryos.

The cellular and molecular features of multipotent epithelial cells during regeneration and asexual reproduction in colonial tunicates are described in the present study. The epicardium has been regarded as the endodermal tissue-forming epithelium in the order Enterogona, because only body fragments having the epicardium exhibit the regenerative potential. Epicardial cells in Polycitor proliferus have two peculiar features; they always accompany coelomic undifferentiated cells, and they contain various kinds of organelles in the cytoplasm. During strobilation a large amount of organelles are discarded in the lumen, and then, each tissue-forming cell takes an undifferentiated configuration. Septum cells in the stolon are also multipotent in Enterogona. Free cells with a similar configuration to the septum inhabit the hemocoel. They may provide a pool for epithelial septum cells. At the distal tip of the stolon, septum cells are columnar in shape and apparently undifferentiated. They are the precursor of the stolonial bud. In Pleurogona, the atrial epithelium of endodermal origin is multipotent. In Polyandrocarpa misakiensis, it consists of pigmented squamous cells. The cells have ultrastructurally fine granules in the cytoplasm. During budding, coelomic cells with similar morphology become associated with the atrial epithelium. Then, cells of organ placodes undergo dedifferentiation, enter a cell division cycle, and commence morphogenesis. Retinoic acid-related molecules are involved in this dedifferentiation process of multipotent cells. We conclude that in colonial tunicates two systems support the flexibility of tissue remodeling during regeneration and asexual reproduction; dedifferentiation of epithelial cells and epithelial transformation of coelomic free cells.

Overexpression of a lefty orthologue, Ci-lefty, caused a failure of neural tube closure in the protochordate ascidian Ciona intestinalis. The body bent dorsally, and anterior-posterior elongation was inhibited. A similar phenotype was observed in embryos treated with SB431542, an inhibitor of Nodal receptors, suggesting that Ci-Lefty antagonized Nodal signaling as reported in other deuterostome species. Overexpression of Ci-nodal also resulted in a similar phenotype, suggesting that a correct quantity and/or a spatial restriction of Nodal signaling are important for the neural tube to form. In addition to known Ci-Nodal target genes, orthologues of Zic (Ci-ZicL) and cdx (Ci-cdx) were activated by Ci-Nodal. Expression of a dominant negative Ci-cdx caused defects in neural tube formation similar to those obtained on treatment with SB431542 or overexpression of Ci-lefty. A regulatory cascade composed of Ci-Nodal, Ci-ZicL, and Ci-Cdx may play an important role in neural tube formation in the Ciona embryo.

We identified 5' upstream enhancers of two Ci-ZicL genes and characterized one of them in detail. Although the genes are tandemly repeated in the genome, the transcription of each seemed to be individually regulated. The 259-bp 5' flanking sequence contained essential elements for driving a correct spatiotemporal expression. This enhancer can be divided into two distinct modules. The A module was located between nucleotide positions -259 and -205 upstream of the putative transcription start site, and was necessary for activation in A6.2 and A6.4 blastomeres at the 32-cell stage. The BM module lay between nucleotide positions -205 and -89 and was responsible for activation in B6.2 and B6.4 blastomeres at the 32-cell stage and in A-line presumptive notochord, nerve cord, and muscle lineage cells at later stages. Two putative Fox-binding sites, one located within and the other downstream of the BM module, were necessary for the latter activity. Mutation at a potential Ets-binding site, located downstream of the BM module, caused ectopic activation of the reporter gene in a-line presumptive ectoderm cells. This suggests that repression in the a-line blastomeres is necessary for correct transcriptional control of the Ci-ZicL gene.

A new series of 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives were found to show potent and selective inhibition of platelet-dervied growth factor (PDGF) receptor phosphorylation. In this exploration of the structure-activity relationships (SARs) of the prototype inhibitor KN1022, the 4-nitrophenylurea moiety was probed. We found that 4-substitution on the phenyl ring was optimal and the introduction of more than two substituents on the phenyl ring decreased activities. Bulky substituents on the phenyl ring enhanced activities. Thiourea analogues were also prepared, and the SARs were found to be slightly different from those of the urea derivatives. Through this research, we obtained some potent KN1022 derivatives such as 4-(4-methylphenoxy)phenyl (36, IC(50) 0.02 micromol/L), 4-tert-butylphenyl (16, IC(50) 0.03 micromol/L), and 4-phenoxyphenyl (21, IC(50) 0.08 micromol/L) analogues, which had almost a 10-fold increase in activity against KN1022. These potent compounds retained their high selectivity against the PDGF receptor family similar to KN1022. We also observed that these compounds could inhibit the PDGF-BB-induced proliferation of porcine vascular smooth muscle cells without cell toxicity almost at the same IC(50) values observed for PDGF receptor phosphorylation. To evaluate the biological effects in vivo, we selected some analogues on the basis of the measurement of the plasma drug concentration after oral administration to rats. Oral administration of the 4-chlorophenyl (6), 4-bromophenyl (9), or 4-isopropoxyphenyl (20) analogue to Sprague-Dawley rats (30 mg/kg, twice daily) resulted in significant inhibition (24-38%) of neointima formation in the carotid artery of the balloon catheter deendothelialized vessel in the rats. Therefore, 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives, which are potent inhibitors of PDGFR phosphorylation, may be expected to represent a new therapeutic approach for the treatment of various aspects of atherosclerosis and other cellular proliferative disorders.

Two closely related cDNA fragments, named pTC14-1 and pTC14-2, encoding C-type lectins were cloned from the budding ascidian Polyandrocarpa misakiensis by means of the polymerase chain reaction. The amino acid sequence deduced from pTC14-1 was identical to that of a 14-kDa calcium-dependent galactose-binding lectin, TC-14, that had been purified from this species. Between the two clones, nucleotide sequence similarity was 90%, whilst that of the deduced amino acid sequences was 82%. The cDNA inserts of these clones hybridized weakly with each other. Antisense RNA probes prepared from these clones gave intense hybridization signals on Northern blots of the W strain, but very weak signals on those of the other strains. Therefore, both clones were suggested to originate from the W strain, but from two separate genes, since the base substitution was scattered throughout the entire translated region. The amount of TC14-1 mRNA increased during bud development, and peaked at 36 h after separation of the bud from the parental body wall. At this stage, extracellular matrix containing TC-14 lectin developed in the mesenchymal space around the morphogenetic region of the bud. There was much less TC14-2, than TC14-1 mRNA at every stage of bud development. TC14-1 and TC14-2 mRNAs were detected on Northern blots of RNAs from adults and growing buds, suggesting that these genes can be used as the earliest markers of budding in this species.