The error-prone DNA polymerase ι provides quantitative resistance to lung tumorigenesis and mutagenesis in mice.

Opposite undamaged nucleotide T, DNA polymerase ι (Polι) preferentially incorporates G rather than A, violating the Watson-Crick rule. Although the actual biological role of Polι remains enigmatic, we have identified its coding gene as a candidate for pulmonary adenoma resistance 2 (Par2), a mouse quantitative trait locus modulating chemically induced lung tumor susceptibility. Notably, the most tumor-sensitive Par2 allele possessed by the 129X1/SvJ mouse is associated with a loss-of-function mutation in Polι. To determine whether the nonfunctional Polι is responsible for the 129X1/SvJ-specific Par2 phenotype, we knocked out Polι in a C57BL/6J mouse carrying a less tumor-sensitive Par2 allele. Disruption of the C57BL/6J Polι conferred 129X1/SvJ-like sensitivity on the C57BL/6J Par2 locus and increased the in vivo mutation frequency in the lung, providing definitive proof that Polι causes the Par2 effect and inhibits tumorigenesis and mutagenesis, despite its extreme replication infidelity.

Peroral video cholangioscopy to evaluate indeterminate bile duct lesions and preoperative mucosal cancerous extension: a prospective multicenter study.

BACKGROUND AND STUDY AIMS: Despite the development of peroral video cholangioscopy (PVCS), no prospective multicenter studies have been undertaken to investigate the diagnostic accuracy of PVCS in biliary tract diseases. Therefore, the aim of this study was to clarify the accuracy of PVCS in evaluating biliary tract lesions. PATIENTS AND METHODS: This study was a prospective multicenter study at five tertiary referral centers in Japan and included 87 eligible patients with biliary tract diseases who underwent PVCS. The study evaluated the ability of PVCS to diagnose indeterminate biliary tract diseases, detect mucosal cancerous extension preoperatively in extrahepatic bile duct cancers, and predict adverse events. RESULTS: The use of PVCS appearance alone correctly distinguished benign from malignant indeterminate biliary lesions in 92.1 % of patients whereas biopsy alone was accurate in 85.7 %. In extrahepatic bile duct cancer, mucosal cancer extended histologically at least 20 mm in 34.7 % (17/49) of patients. The accuracy rate of PVCS to evaluate the presence or absence of mucosal cancerous extension by endoscopic retrograde cholangiography (ERC) alone, ERC with PVCS, and ERC with PVCS + biopsy were 73.5 %, 83.7 %, and 92.9 %, respectively. Adverse events were seen in 6.9 % of PVCS patients, but no serious complications were observed. CONCLUSION: PVCS enhanced the accurate diagnosis of biliary tract lesions by providing excellent resolution in combination with biopsy.

Oncogenic and cell survival properties of the retinoic acid metabolizing enzyme, CYP26A1.

Vitamin A deficiency (VAD) is associated with increased susceptibility to carcinogenesis in animal models and elevated risk for a number of human cancers. Here, we found that CYP26A1, the gene encoding a cytochrome P450 enzyme specifically involved in metabolic inactivation of retinoic acid (RA), the most active vitamin A derivative, is highly expressed in 42% (27/65) of primary breast cancers. We also showed that enhanced expression of CYP26A1 suppresses cellular responses to anoikis and consequently promotes anchorage-independent growth. This transformed phenotype was sufficient to markedly increase tumorigenic and metastatic potential. Suppression of CYP26A1 significantly reversed the CYP26A1-mediated oncogenic characteristics, suggesting a direct link between intracellular RA status and tumorigenicity. Our observations provide strong evidence for oncogenic and cell survival properties of CYP26A1 in carcinogenesis, and suggest mechanisms whereby VAD might promote cancer development.

Ca(2+)-dependent Ca(2+) clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals.

Ca(2+) clearance in frog motor nerve terminals was studied by fluorometry of Ca(2+) indicators. Rises in intracellular Ca(2+) ([Ca(2+)](i)) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca(2+) transient) reached a peak or plateau within 6-20 stimuli and decayed at least in three phases with the time constants of 82-87 ms (81-85%), a few seconds (11-12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca(2+) pumps at the cell membrane by external Li(+) and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li(+) alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca(2+) transients. Blocking Ca(2+) pumps at Ca(2+) stores by thapsigargin did not affect 100 stimuli-induced Ca(2+) transients but increased the initial decay time constant of 200 stimuli-induced Ca(2+) transients with no change in other parameters. Inhibiting mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca(2+) transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca(2+) transients against [Ca(2+)](i) yielded the supralinear [Ca(2+)](i) dependence of Ca(2+) efflux out of the cytosol. Blocking Ca(2+) extrusion or mitochondrial Ca(2+) uptake significantly reduced this [Ca(2+)](i)-dependent Ca(2+) efflux. Thus Ca(2+)-dependent mitochondrial Ca(2+) uptake and plasmalemmal Ca(2+) extrusion clear out a small Ca(2+) load in frog motor nerve terminals, while thapsigargin-sensitive Ca(2+) pump boosts the clearance of a heavy Ca(2+) load. Furthermore, the activity of plasmalemmal Ca(2+) pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.

Delayed eruption of premolars with periodontitis of primary predecessors and a cystic lesion: a case report.

Apical periodontitis after pulp therapy in a primary tooth can cause delayed eruption of the permanent successor. A case of bilateral delayed eruption of mandibular premolars is presented. The patient. a 13-year-old girl, was referred by her dentist. Oral findings showed that the right first and left second primary molars were retained. Other premolars had erupted. An orthopantomogram revealed apical periodontitis, affecting both retained primary molars. The right first mandibular premolar was impacted against the alveolar bone and root of the second premolar, and there was a large cystic lesion in close association with the left second mandibular premolar. Both primary molars were extracted, and the cystic lesion was treated by marsupialization. Fenestration and traction were performed on the right first premolar. Correct tooth alignment was achieved with orthodontic appliances. If the problem had been detected earlier, treatment of the premolars might have been easier. Clinical and radiological follow-up, therefore, of primary teeth that have undergone pulp therapy procedures should be performed until eruption of succedaneous teeth.

Analysis of Ca(2+) currents in spermatocytes from mice lacking Ca(v)2.3 (alpha(1E)) Ca(2+) channel.

In mammalian male germ-line cells, low-voltage-activated (LVA) Ca(2+) current has been identified and its electrophysiological properties have been studied. To investigate whether alpha(1)2.3 (alpha(1E)) subunit of the voltage-dependent Ca(2+) channel codes for the LVA current, whole-cell patch clamp and following reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed in pachytene spermatocytes from Ca(v)2.3+/+ and Ca(v)2.3-/- mice. Whole-cell current in acutely dissociated pachytene spermatocytes from Ca(v)2.3+/+ and Ca(v)2.3-/- mice displayed a typical profile of LVA Ca(2+) currents and kinetics with no significant differences. Single-cell RT-PCR revealed the expression of Cacna1g in the pachytene spermatocytes from Ca(v)2.3+/+ and Ca(v)2.3-/- mice in which LVA Ca(2+) currents were actually recorded. These results suggest that the Ca(v)2.3 channel makes no detectable contribution to the LVA Ca(2+) current in the pachytene spermatocyte. Instead, Ca(v)3 family such as Ca(v)3.1 may be the likely candidates responsible for the LVA currents in pachytene spermatocytes.

Requirement of c-jun N-terminal kinase for apoptotic cell death induced by farnesyltransferase inhibitor, farnesylamine, in human pancreatic cancer cells.

Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs and are now in clinical trial. We have previously shown that farnesylamine, synthetic isoprenoid-linked with "amine" which acts as a potent FTI, induces apoptosis in human pancreatic cancer cells through the ras signaling cascade. Since the effect of FTI is usually "cytostatic" rather than "cytotoxic", we speculated another apoptotic machinery of farnesylamine in addition to the effect of FTI. Farnesylamine induced sustained activation of c-jun N-terminal kinase (JNK), which was not caused by other FTI, FTI-277. Blockage of JNK activity by dominant-negative mutant abrogated the DNA laddering and significantly reduced "cytotoxic" effect of farnesylamine. Strikingly similar effect on JNK activation and apoptosis was induced by structurally related long-chain fatty amine (LFA), oleylamine, but not by farnesol, an isoprenoid analogue of farnesylamine without "amine." Taken together, apoptosis induction through JNK activation by farnesylamine based on the LFA structure rather than an effect of FTI.

Adenocarcinoma arising in gastric heterotopic pancreas: clinicopathological and immunohistochemical study with genetic analysis of a case.

Heterotopic pancreas in the stomach is a relatively common congenital condition, but the risk of malignant transformation is extremely low. In this study, we describe a case of adenocarcinoma arising from a gastric heterotopic pancreas and we consider its morphological and immunohistochemical features and genetic analysis, in order to examine its histogenesis. This unusual sequela was seen in a 57-year-old woman. Image studies showed a protruding lesion with a central ulcer located in the lesser curvature from the angle to the body of the stomach. A biopsy specimen confirmed this lesion as adenocarcinoma before total gastrectomy. The tumor showed mixed patterns of solid neoplastic-cell proliferation and moderately differentiated glandular structures, and also showed transitional lesions to obvious malignancy, that is, dysplasia, or adenocarcinoma in situ. Neoplastic cells had positive immunoreactivity for carbohydrate antigen (CA) 19-9, mucin (MUC) 1, and insulin, and the mutant allele-specific amplification method revealed a point mutation at K-ras codon 12 (GGT [Gly]-->GAT [Asp]), which is the most common mutational change observed in patients with pancreatic carcinoma. The features of the present case provide clear evidence that this tumor originated from heterotopic pancreatic tissue rather than from gastric epithelium.

Syntheses of glycine and L-serine by their interconversion in the posterior silkgland of the silkworm, Bombyx mori.

It was observed by solution-state 13C NMR spectroscopy that a great portion of the 13C of [1-13C]L-serine fed to the 5th instar larvae of the silkworm, Bombyx mori was incorporated into C1 of glycine in silk fibroin. [1-13C]Glycine was detected along with [1-13C]serine in fibroin of the posterior silkgland cultured in a medium containing [1-13C]serine. This formation of [1-13C]glycine was inhibited by addition of aminopterin to the culture medium. These findings suggest that an active conversion from serine to glycine, which needs tetrahydrofolate, occurs in the posterior silkgland for fibroin synthesis. Moreover, the solid-state 13C CP/MAS spectrum of the fibroin prepared from cocoons spun by larvae fed with [13C]formate revealed that serine C3 was labelled specifically with 13C, suggesting that the reverse conversion from glycine to serine took place in the silkworm. The posterior silkgland has the ability to synthesize not only fibroin but also its major materials, glycine and serine.

Occludin and claudin-1 concentrate in the midbody of immortalized mouse hepatocytes during cell division.

It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. In late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis.

Retinoid X receptor alpha and retinoic acid receptor gamma mediate expression of genes encoding tight-junction proteins and barrier function in F9 cells during visceral endodermal differentiation.

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9:rtTA:Cre-ER(T) L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et al., 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs. We also compared the RA-induced expression of TJ components and barrier function in RXRalpha(-/-)-RARgamma(-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs.

Unique cellular features in atypical adenomatous hyperplasia of the lung: ultrastructural evidence of its cytodifferentiation.

Atypical adenomatous hyperplasia (AAH) of the lung could be a good material to understand the histogenesis of peripherally occurring, well-differentiated adenocarcinoma. However, its true biological significance remains to be clarified. The authors present the histomorphological studies of this lesion and compare the ultrastructure with that of nonmucinous bronchioloalveolar carcinoma (BAC) to define characteristic features of AAH. Light microscopy showed the well-preserved pulmonary architecture, proliferated neoplastic cells without marked cellular atypia, and no transitional area to obvious adenocarcinoma. Intranuclear inclusion was present in a large number of neoplastic cells. Electron microscopy revealed that cuboidal or low columnar neoplastic cells proliferated actively but were not crowded on slightly thickened fibrous alveolar septa with both Clara-like granules and small lamellar bodies in the cytoplasm resembling that of Clara cell and type 2 pneumocyte. Some of the nuclei had characteristic invaginations of its nuclear membrane. Although the findings appear to be nonspecific for AAH, the authors emphasize that AAH is an alveolar intraepithelial neoplasia that represents a very early stage in the continuous developmental spectrum of adenomatous neoplasia in the bronchioloalveolar region corresponding to dysplasia or intraepithelial neoplasia in other organs, and will give the significance to speculate its histogenesis.

Ca2+ dynamics at the frog motor nerve terminal.

Rises in free [Ca2+]i in response to various tetanic stimuli (Ca2+ transient) in frog motor nerve terminals were measured by recording fluorescence changes of Ca2+ indicators and analyzed in relation to short-term synaptic plasticity. Ca2+ transients reached a plateau after 10-20 impulses at 100 Hz and decayed in a three-exponential manner, in which the fast component was predominant. The plateau and fast component of the Ca2+ transient were elevated infralinearly with an increase in tetanus frequency. Computer simulation showed that the Ca2+ transients estimated from fluorescence changes faithfully reflect the true changes in [Ca2+]i except for the initial 20 ms. A slow Ca2+ chelator, EGTA, loaded into the nerve terminal, decreased the magnitude of both the fast and slow components of facilitation of transmitter release and the time constant of the former. A fast Ca2+ chelator, BAPTA, decreased the magnitude of fast facilitation but slightly increased its time constant. These results suggest that Ca2+ transients in the frog motor nerve terminals are primarily caused by Ca2+ entry and are dissipated by three components, in which the rate of the fast component is equivalent to that of free Ca2+ diffusion. The residual Ca2+ in the nerve terminals after stimulation accounts for the fast component of facilitation.

Expression of GFRalpha-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB.

It is well known that the blood-brain barrier (BBB) matures at approximately 2 wk after birth in the rat. Recently, we showed that glial cell line-derived neurotrophic factor (GDNF) enhances the barrier function of porcine endothelial cells forming the BBB in culture. In the present study, we examined the relation between permeability of the BBB, using Evans blue as a tracer, and expression of the GDNF family receptor (GFRalpha-1) during postnatal development of the BBB. Morphometric analysis showed that exudation of Evans blue from capillaries of the cerebral cortex progressively decreased until postnatal day 21. Inversely, immunohistochemical examinations showed expression of GFRalpha-1 in the capillaries at postnatal day 3 and expression that reached the same levels as observed in adult rats by postnatal day 10. However, c-ret, which is thought to mediate a signal evoked by binding of GDNF to GFRalpha-1, was not expressed in the capillaries of the brain cortex in 3-mo-old rats. On the other hand, the tight junction proteins occludin and ZO-1 appeared to be fully expressed at birth. The reciprocal relation between GFRalpha-1 expression and the permeability of the BBB strongly suggests active participation of GDNF in postnatal development of the BBB, although the mechanism(s) involved is still veiled.