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Esophageal superficial carcinoma safely can be resected surgically or endoscopically. We evaluated indications for endoscopic mucosal resection (EMR) and optimal treatment modality for superficial carcinoma of the esophagus based on clinical and pathologic analyses. Between January 1, 1984, and September 30, 1999, 113 patients with superficial cancer of the esophagus underwent surgical or endoscopic resection (n = 33 patients, 36 lesions). The two-channel method, esophageal EMR-tube method or EMR cap-fitted panendoscope was used. Mucosal and submucosal cancers were classified to be epithelial layer (m1), proper mucosal layer (m2), muscularis mucosae (m3), upper third of the submucosal level (sm1), middle third of the submucosal layer (sm2), or the lower third of the submucosal level (sm3) cancers, according to criteria of the Japanese Society for Esophageal Disease. Absolute indication for EMR was restricted to m1 or m2 cancers, and relative indications for EMR included m3 or sm1 lesions. In our department, indications for EMR were not related to size or circumference of lesions. Lymph vessel invasion and lymph node metastasis markedly increased in lesions that infiltrated the lamina muscularis mucosa (m3). All lesions resected with use of EMR were 0-II (flat), and the depth of invasion in 10 0-IIa or 0-IIb lesions was m1 or m2. Twenty-one 0-IIc lesions were distributed widely from m1 to sm1. All 0-IIa+IIc lesions were m3 or sm1. Preoperative diagnosis accurately was established preoperatively in 61% of patients. Complications related to EMR were detected in 21% of patients and included perforation, stenosis, and hemorrhage. Ten patients also received radiotherapy, chemotherapy, or esophagectomy with lymph node dissection after use of EMR. No such combination therapy was administered in six patients with m3 lesions, but without lymph vessel invasion. All patients treated with use of EMR, including patients with m3 cancer who did not receive additional treatment, are living without recurrence. Local resection with use of EMR could be regarded to be the preferred treatment of superficial esophageal cancers limited to the lamina propria mucosae. Endoscopic mucosal resection also could be regarded to be the preferred treatment of m3 cancer without lymph vessel invasion. Use of additional therapy, such as radiotherapy, allows the use of EMR for m3 cancer with lymph vessel invasion or sm1 cancers.

A worldwide shortage of donor livers is a limiting factor of the clinical application of hepatocyte transplantation (HTX). To resolve this issue, we focused on a reversible immortalization system that allows temporary expansion of primary hepatocyte populations by transfer of an oncogene that can be subsequently excised. As a preliminary test toward this goal, we examined the efficacy of Cre/loxP site-specific recombination in a transformed human liver cell line, HepG2. The present study utilized retroviral transfer of a prototypical immortalizing gene, simian virus 40 large T antigen (SV40Tag), flanked by a pair of loxP recombination targets and adenovirus-mediated Cre/loxP recombination. Here we report that complete elimination of the retroviral transferred oncogene was achieved by site-specific recombination using a replication-deficient recombinant adenovirus vector producing Cre recombinase (Ad-Cre).

The last-instar larvae of a drosophilid fly, Chymomyza costata enter diapause in response to the dark-phases longer than 9 h (Yoshida, T., Kimura, M.T., 1995. The photoperiodic clock in Chymomyza costata. Journal of Insect Physiology 41, 217-222). In order to switch the developmental programming of the sensitive larvae from continuous development to diapause, after they were transferred from the short (8 h) to the long (14 h) dark-phase, significantly less time (1-2 days) was required when the dark-phase was abruptly and asymmetrically extended into the evening, than when it was extended symmetrically into both morning and evening (2-3 days), or asymmetrically into the morning hours (4-6 days). Diapause was also induced in 40-70% of sensitive larvae that were reared under the gradually shortening light-phase (from 16 h to 2 h, by 1 h in each cycle), despite that the dark-phase remained constant and short (8 h). Larvae developed continuously, however, when reared under the gradually extending light-phase (from 16 h to 24 h) and a constantly short dark-phase. We interpret such results, with the help of the two-oscillator model of circadian rhythmicity (Pittendrigh, C.S., Daan, S., 1976. A functional analysis of circadian pacemakers in nocturnal rodents. V. Pacemaker structure: A clock for all seasons. Journal of Comparative Physiology A 106, 333-355), as indicating that two mutually coupled oscillators (evening and morning) differing in their entrainability may participate in measuring of the dark-phase duration. The levels of dopamine (DA) and serotonin (5-HT) in the larval CNS transiently increased (by up to 20%) after the dusk, while no apparent change was observed during the dawn. The dusk-related increase was observed also after the asymmetric extension of the dark-phase into evening, while the asymmetric extension into morning had no effect on the levels of the DA and 5-HT.

Developmental profiles of dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) in the larval CNS of Chymomyza costata were measured by HPLC using electrochemical detection. Larvae of two strains, wild-type (W) and nondiapause mutant (M), were maintained either under long-day (LD, inducing pupariation) or short-day (SD, inducing diapause in W-strain) photoperiods. The levels of DA ranged from 10 fmol/CNS (early 3rd instar larvae) to 60 fmol/CNS (150-day-old diapausing larvae); the range for 5-HT was from 10 fmol/CNS to 75 fmol/CNS in the same larvae. During the 3rd larval instar, which is the decisive stage for photoperiodic induction of diapause, no differences were found in DA developmental profiles between different strains or conditions. Some differences were found in 5-HT developmental profiles, but only after the end of sensitive stage, and were therefore regarded as insignificant for regulation of developmental mode. Similarly, no clear correlations between the developmental profiles of DA and 5-HT and the course of developmental changes during the maintenance and termination of a few-month-long larval diapause were observed. Furthermore, the DA and 5-HT levels in the CNS were pharmacologically manipulated by feeding the larvae with either precursors or enzyme inhibitors of DA and 5-HT biosynthesis. Although retardations of growth and development were observed, the treated larvae retained full capacity for the photoperiodic response, irrespective of the level of DA or 5-HT in their CNS. Larvae with their 5-HT depleted to trace levels survived and were capable of diapause induction, maintenance, and termination. Depletion of DA to trace levels resulted in 100% mortality. Collectively, the present study indicates that 5-HT in the CNS is dispensable for the photoperiodic response in C. costata. More information is needed to elucidate the potential role of DA.

Parasitoid wasps never kill their hosts before the wasp larvae emerge from the host. However, almost 100% of the host armyworm larvae Pseudaletia separata die within 2-3 days by parasitization with the wasp Cotesia kariyai or by injection of polydnavirus, the wasp symbiont virus, when they are simultaneously infected by the pathogenic bacterium Serratia marcescens. The present study was conducted to elucidate the crucial factor causing this larval mortality. An insecticidal protein has been shown to exist in the hemolymph of dying host larvae; it has been purified by procedures consisting of reverse-phase column extraction, gel filtration and ion-exchange column chromatography. The purified protein showed a strong insecticidal effect with a median lethal dosage (LD50) of 13 pmol/larva and was estimated to have a molecular mass of 57 kDa. The amino acid sequence of the insecticidal protein was partially characterized and used for isolation and sequencing of the genomic DNA. The deduced amino acid sequence for this protein revealed striking similarity with the metalloprotease of S. marcescens enterobacter.

Experiments were conducted to examine the relationship between an onset of diapause and dopamine (DA) content in the cabbage armyworm Mamestra brassicae during pupation. The DA levels were significantly higher in haemolymph, integument and brain-central nervous system of diapause-destined pupae than in non-diapause-destined pupae. The elevated level of the integumental DA content was demonstrated to be due to an increase in dopa decarboxylase activity in diapausing pupal integuments through an enhancement of transcript levels of this enzyme. Elevation of the DA level accomplished by feeding l-DOPA to last instar larvae induced a diapause-like state in more than 50% of the pupae under long daylengths.

Type 1 diabetes mellitus is an autoimmune disease, which results in the permanent destruction of β-cells of the pancreatic islets of Langerhans. While exogenous insulin therapy has dramatically improved the quality of life, chronic diabetic complications develop in a substantial proportion of subjects and these complications generally progress and worsen over time. Although intensive insulin therapy has proven effective to delay and sometimes prevent the progression of complications such as nephropathy, neuropathy or retinopathy, it is difficult to achieve and maintain long term in most subjects. Reasons for this difficulty include compliance issues and the increased risk of severe hypoglycemic episodes, which are generally associated with intensification of exogenous insulin therapy. Clinical studies have shown that transplantation of pancreas or purified pancreatic islets can support glucose homeostasis in type 1 diabetic patients. Islet transplantation carries the special advantages of being less invasive and resulting in fewer complications compared with the traditional pancreas or pancreas-kidney transplantation. However, islet transplantation efforts have limitations including the short supply of donor pancreata, the paucity of experienced islet isolation teams, side effects of immunosuppressants and poor long-term results. The purpose of this article is to review recent progress in clinical islet transplantation for the treatment of diabetes.

Nat. Biotechnol. 24, 1412–1419 (2006); published online 5 November 2006; corrected after print 7 February 2007. In the version of the article initially published, the fifth author's name is misspelled. The correct spelling is Debiao Zhao. The error has been corrected in the HTML and PDF versions of the article.

BETA2/NeuroD protein is important for regulating insulin gene transcription and for the terminal differentiation of islet cells, including insulin- and glucagon-producing cells. We reported that BETA2/NeuroD protein can permeate several cell types, including pancreatic islets, because of an arginine- and lysine-rich protein transduction domain (PTD) sequence in its structure. Here we provide genetic and biochemical evidence that cell membrane heparan sulfate proteoglycans are involved in extracellular BETA2/NeuroD internalization. We tested whether soluble glycosaminoglycans (GAGs) could inhibit BETA2/NeuroD internalization. Heparin almost completely prevented BETA2/NeuroD entry, whereas chondroitin sulfate A, B, and C caused only limited inhibition. Moreover, treatment with heparinase III impaired BETA2/NeuroD internalization, whereas treatment with chondroitinase ABC, or with chondroitinase AC, was completely ineffective in inhibiting BETA2/NeuroD internalization. We also examined various mutant cell lines originating from CHOK1 cells and defective in GAG biosynthesis. The observation using mutant cell lines supports the notion that the selective sulfation of heparan sulfate is an important determinant for NeuroD/heparan sulfate recognition. These data indicate that cell surface heparan sulfate proteoglycans are required for BETA2/NeuroD internalization and that BETA2/NeuroD protein transduction could be a safe and valuable strategy for enhancing insulin gene transcription without requiring gene transfer technology.

Construction of a safe and functional bioartificial pancreas (BAP) that provides an adequate environment for islet cells may be an important approach to treating diabetic patients. Various types of BAP devices have been developed, but most of them involve extravascular implantation of islets in microcapsules or diffusion chambers. These devices have poor diffusive exchange between the islets and blood, and often rupture. To overcome these problems, we developed a new type of BAP composed of polyethylene-vinyl alcohol (EVAL) hollow fibers that are permeable to glucose and insulin and a poly-amino-urethane-coated, non-woven polytetrafluoroethylene (PTFE) fabric that allows cell adhesion. Porcine islets attached to the surface of the PTFE fabric, but not to the surface of the EVAL hollow fibers, allowing nutrient and oxygen exchange between blood flowing inside the fibers and cells outside. We inoculated this BAP with porcine islets and connected it to the circulation of totally pancreatectomized diabetic pigs. We found that blood glucose levels were reduced to a normal range and general health was improved, resulting in longer survival times. In addition, regulation of insulin secretion from the BAP was properly controlled in response to glucose both in vitro and in vivo. These results indicate that our newly developed BAP may be a potential therapy for the treatment of diabetes in humans.

Background. Temporary support of a damaged liver by a bioartificial liver (BAL) devise is a promising approach for the treatment of acute liver failure. Although human primary hepatocytes are an ideal source of hepatic function in BAL, shortage of human livers available for hepatocyte isolation is the limiting factor for the use of this modality. A clonal human hepatocyte cell line that can grow economically in culture and exhibit liver-specific functions should be an attractive solution to this problem. Methods. To test this alternative, primary human fetal hepatocytes were immortalized using Simian virus 40 large T antigen. To investigate the potential of the immortalized cells for BAL, we transplanted the cells into the spleen of adult rats and performed a 90% hepatectomy 12 hr later. Results. One of the cloned human liver cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplanting of 20×10 6 OUMS-29 cells protected the animals from hyperammonemia and the associated hepatic encephalopathy. Survival was significantly prolonged in 90% of hepatectomized rats receiving OUMS-29 cells. Conclusions. A highly differentiated immortalized human hepatocyte cell line, OUMS-29, was able to provide metabolic support during acute liver failure induced by 90% hepatectomy in rats. Essentially unlimited availability of OUMS-29 cells may be clinically useful for BAL treatment.

Neural stem cells (NSCs) have the ability to self-renew and give rise to neurons and glial cells (astrocytes and oligodendrocytes) in the mammalian central nervous system. This multipotency is acquired by NSCs during development and is maintained throughout life. Proliferation, fate specification, and maturation of NSCs are regulated by both cell intrinsic and extrinsic factors. Epigenetic modification is a representative intrinsic factor, being involved in many biological aspects of central nervous system development and adult neurogenesis through the regulation of NSC dynamics. In this review, we summarize recent progress in the epigenetic regulation of NSC behavior in the embryonic and adult brain, with particular reference to DNA methylation, histone modification, and noncoding RNAs.

Hepatocyte transplantation (HTX) could be an attractive treatment for patients with liver failure and liver-based metabolic disease. Human primary hepatocytes are ideal in this modality, but the shortage of human livers available for hepatocyte isolation severely limits the use of this form of therapy. A tightly regulated human hepatocyte cell line that grows economically in culture and exhibits differentiated liver functions would be an attractive alternative to the primary human hepatocytes. To test the feasibility, human hepatocytes were immortalized by a retroviral vector expressing simian virus 40 large T antigen and herpes simplex virus-thymidine kinase. A highly differentiated immortal hepatocyte line NKNT-3 was established. NKNT-3 cells grew in chemically defined serum-free medium, retained highly differentiated liver functions, and were sensitivity to ganciclovir as a prodrug. Essentially unlimited availability of NKNT-3 cells may be clinically useful for HTX and bioartificial liver.