Establishment and characterization of human pancreatic adenocarcinoma cell line in tissue culture and the nude mouse

We established a human pancreatic carcinoma cell line, designated SPH, from cancerous ascites of a 57-year-old male patient with ductal adenocarcinoma of the pancreas. The cells have been cultured for 32 months with RPMI-1640 medium supplemental with 10% fetal calf serum. The population doubling time of this cell line was about 35 h, and the modal number of chromosomes was 85 at passage 20. The cells produced CA19-9, SPan-1, and DUPAN-2 in the conditioned medium and formed tumors in nude mice, the histology of which was similar to that of the primary tumor. Based on these findings, this cell line is considered to be a very useful model for studying many aspects of primary and metastatic pancreatic cancer cell biology.     

Predominant expression of lysosomal N-acylethanolamine-hydrolyzing acid amidase in macrophages revealed by immunochemical studies

Bioactive N-acylethanolamines, including anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory substance), and N-oleoylethanolamine (an anorexic substance) are enzymatically hydrolyzed to fatty acids and ethanolamine. Fatty acid amide hydrolase plays a major role in this reaction. In addition, we cloned cDNA of an isozyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)" [K. Tsuboi, Y.-X. Sun, Y. Okamoto, N. Araki, T. Tonai, N. Ueda, Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase, J. Biol. Chem. 280 (2005) 11082-11092]. Previous biochemical analyses suggested the expression of NAAA in macrophage cells and various rat tissues including lung and brain. To clarify the physiological significance of NAAA, here we immunochemically studied NAAA for the first time. We developed an antibody specific for rat NAAA, and by Western blotting revealed that NAAA is glycosylated and subjected to specific proteolysis. In alveolar macrophages isolated from rat lung, NAAA was immunocytochemically localized in lysosomes. In the whole lung tissue, only alveolar macrophages were immunostained for NAAA. Conformably, the mRNA and protein levels and activity of NAAA in alveolar macrophages were much higher than those in the whole lung tissue. In brain, intraventricular macrophages were positively stained with anti-NAAA antibody, while microglia appeared to be negative. These results strongly suggested the importance of macrophages as an expression site of NAAA in rat tissues.     

Role and interaction of connective tissue growth factor with transforming growth factor-β in persistent fibrosis: A mouse fibrosis model

Skin fibrotic disorders are understood to develop under the influence of some growth factors, such as transforming growth factor-beta (TGF-β), basic fibroblast growth factor (bFGF), or connective tissue growth factor (CTGF). To establish an appropriate animal model of skin fibrosis by exogenous application of growth factors, we investigated the in vivo effects of growth factors by injecting TGF-β, CTGF, and bFGF into the subcutaneous tissue of newborn mice. A single application of TGF-β or bFGF resulted in the formation of transient granulated tissue that disappeared despite 7 days of consecutive injections. A single CTGF injection also caused slight granulation. However, injecting TGF-β plus CTGF produced long-term fibrotic tissue, which persisted for at least 14 days. Also, fibrotic tissue was observed when CTGF was injected from 4 to 7 days after TGF-β injections for the first 1–3 days. In situ hybridization analysis revealed the expression of CTGF mRNA in the fibroblasts at least in a few fibrotic conditions. These findings suggest that either CTGF mRNA or an application of exogenous CTGF protein is required for the development of persistent fibrosis. From our study, it appears that interaction of several growth factors is required for persistent fibrotic tissue formation, with TGF-β causing the induction and CTGF needed for maintenance of skin fibrosis. The animal model on skin fibrosis by exogenous application of growth factors developed in this study may prove useful for future studies on fibrotic disorders. J. Cell. Physiol. 181:153–159, 1999. © 1999 Wiley-Liss, Inc.     

Nucleotide sequence of BamHI family satellite DNA and its unit length polymorphism in bluegill sunfish Lepomis macrochirus

We have isolated and sequenced a member of tandem repetitive DNA containing BamHI site (BamHI family satellite DNA) from bluegill sunfish Lepomis macrochirus. PCR amplification with specific primers was performed to define the size of unit length repeat of the BamHI family satellite DNA, revealing that there were two distinct size of DNA fragments (0.9 kb and 1.3 kb) in the PCR products. The longer fragment (1.3 kb) consisted of internal sub-duplication of shorter fragment (0.9 kb). We have compared the size of PCR products among four fish populations, and found that both fragments co-existed in one population whereas the longer fragment was dominant in other three populations. The results may reflect ongoing homogenization of satellite DNA type over a short evolutionary time scale.     

Recovery of greenery resources in Hiroshima City after World War II

With a population of 1.12 million (in 2005) and an area of 741 km², Hiroshima City is regarded as a metropolis in Japan. On August 6, 1945, Hiroshima became the first city in the world to have an atomic bomb dropped on it, and the delta area was completely destroyed. Almost all the vegetation was burnt and disappeared from the area. This paper summarizes the content and achievements of every campaign conducted throughout the period from the dropping of the bomb in 1945 until 2005. The achievements of the first to third greenery campaigns have been analyzed individually from the viewpoint of the social environmental management system (SEMS). The first campaign corresponds to the system-making stage of the SEMS, the second is the system-working stage, and the last was categorized as the self-management stage. Compared with the brown issue (abiotic environmental problem), the time needed to achieve the goals of each greenery campaign was much longer, because of a variety of struggles caused by urban development in the limited space of the delta area. A case study was conducted on the small parks in the deltaic urban zone in the master plan of greenery. Unexpectedly, new parks have fewer plants and simple structural diversity. Problems relating to the greenery policy and the future direction of biodiversity conservation in urban areas are also discussed in the post-third greenery campaign.     

Decreased Plasma Histidine Level Predicts Risk of Relapse in Patients with Ulcerative Colitis in Remission

Ulcerative colitis (UC) is characterized by chronic intestinal inflammation. Patients with UC have repeated remission and relapse. Clinical biomarkers that can predict relapse in UC patients in remission have not been identified. To facilitate the prediction of relapse of UC, we investigated the potential of novel multivariate indexes using statistical modeling of plasma free amino acid (PFAA) concentrations. We measured fasting PFAA concentrations in 369 UC patients in clinical remission, and 355 were observed prospectively for up to 1 year. Relapse rate within 1 year was 23% (82 of 355 patients). The age- and gender-adjusted hazard ratio for the lowest quartile compared with the highest quartile of plasma histidine concentration was 2.55 (95% confidence interval: 1.41–4.62; p = 0.0020 (log-rank), p for trend = 0.0005). We demonstrated that plasma amino acid profiles in UC patients in clinical remission can predict the risk of relapse within 1 year. Decreased histidine level in PFAAs was associated with increased risk of relapse. Metabolomics could be promising for the establishment of a non-invasive predictive marker in inflammatory bowel disease.     

Identification of residues required for stalled-ribosome rescue in the codon-independent release factor YaeJ

The YaeJ protein is a codon-independent release factor with peptidyl-tRNA hydrolysis (PTH) activity, and functions as a stalled-ribosome rescue factor in Escherichia coli. To identify residues required for YaeJ function, we performed mutational analysis for in vitro PTH activity towards rescue of ribosomes stalled on a non-stop mRNA, and for ribosome-binding efficiency. We focused on residues conserved among bacterial YaeJ proteins. Additionally, we determined the solution structure of the GGQ domain of YaeJ from E. coli using nuclear magnetic resonance spectroscopy. YaeJ and a human homolog, ICT1, had similar levels of PTH activity, despite various differences in sequence and structure. While no YaeJ-specific residues important for PTH activity occur in the structured GGQ domain, Arg118, Leu119, Lys122, Lys129 and Arg132 in the following C-terminal extension were required for PTH activity. All of these residues are completely conserved among bacteria. The equivalent residues were also found in the C-terminal extension of ICT1, allowing an appropriate sequence alignment between YaeJ and ICT1 proteins from various species. Single amino acid substitutions for each of these residues significantly decreased ribosome-binding efficiency. These biochemical findings provide clues to understanding how YaeJ enters the A-site of stalled ribosomes.     

Clinical Significance of Soluble CD26 in Malignant Pleural Mesothelioma

There is no established single diagnostic marker for malignant pleural mesothelioma (MPM). CD26 is a 110 kDa, multifunctional, membrane-bound glycoprotein that has dipeptidyl peptidase IV (DPPIV) enzyme activity. The aim of this study was to evaluate the clinical significance of soluble CD26 (sCD26) in patients with MPM. The study included 80 MPM patients, 79 subjects with past asbestos exposure (SPE), and 134 patients with other benign pleural diseases (OPD) that were included as a control group. sCD26 levels and DPPIV activity in serum and/or pleural fluid were determined using an ELISA kit. Serum sCD26 levels and DPPIV enzyme activity in patients with MPM were significantly decreased compared with those in the SPE group (P = 0.000). The level of serum sCD26 was significantly decreased in patients with advanced stages of MPM compared with those with earlier stages (P = 0.047). The median OS of patients with MPM who had higher DPPIV enzyme activity was significantly longer than that of those with lower DPPIV enzyme activity (P = 0.032). The sCD26 levels in the pleural fluid of MPM patients with an epithelioid subtype were significantly increased compared with the OPD cohort (P = 0.012). Moreover, DPPIV enzyme activity in the pleural fluid of patients with MPM with an epithelioid subtype were significantly increased compared with those in the OPD cohort (P = 0.009). Patients with MPM who had lower specific DPPIV activity, determined as DPPIV/sCD26, showed significantly prolonged survival compared with those with higher specific DPPIV activity (P = 0.028). Serum sCD26 and DPPIV enzyme activity appear to be useful biomarkers for differentiating patients with MPM from SPE. The sCD26 levels or DPPIV enzyme activity in pleural fluid appear to be biomarkers in patients with an epithelioid subtype of MPM. DPPIV activity in serum or pleural fluid appears to be predictive for the prognosis of patients with MPM.