Beta-catenin mutations are frequent in calcifying odontogenic cysts, but rare in ameloblastomas

We have reported previously that alterations to beta-catenin occur frequently in adamantinomatous craniopharyngioma. Based on its histological resemblance to some odontogenic tumors, we suspected the presence of common genetic alterations among these tumors. To address this issue, 11 cases of calcifying odontogenic cyst (COC) and 20 cases of ameloblastoma were investigated for the presence of beta-catenin mutations and beta-catenin expression. Ten COCs were successfully analyzed by direct sequencing, and nine of them were found to harbor somatic beta-catenin mutations. Immunohistochemically, all of the COCs showed nuclear and cytoplasmic expression of beta-catenin with a heterogeneous pattern. No beta-catenin mutations were found in ameloblastomas, except for one case of the follicular type. All follicular ameloblastomas exhibited moderate nuclear and cytoplasmic accumulation of beta-catenin, in contrast to the predominantly membranous expression seen in the plexiform type. beta-Catenin mutation is considered to be a characteristic genetic feature of COC, and may play a critical role in its histogenesis. Although ameloblastoma closely resembles COC histologically, the two have genetically distinctive features.     

Normal development of the gut-associated lymphoid tissue except Peyer's patch in MyD88-deficient mice

MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88-/-, TLR2-/- and TLR4-/- mice. In MyD88-/- mice, the organogenesis of PPs was not disturbed. However, PPs in 2-week-old MyD88-/- mice were significantly smaller than those in MyD88+/- mice. Also, in 2-week-old TLR4-/-, but not TLR2-/- mice, PPs did not develop rapidly. The development of PPs in MyD88-/- and TLR4-/- mice was completely recovered in 10 weeks. PP cells from MyD88-/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10-week-old MyD88-/- mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.     

Expression of L-type amino acid transporter 1 (LAT1) in neuroendocrine tumors of the lung

Amino acid transport systems play an important role in cellular proliferation. L-type amino acid transporter 1 (LAT1) has been associated with tumor growth, and is highly expressed in the established tumor cell lines and primary human neoplasms. In this study, we investigated the expression of LAT1 to evaluate the malignant potential and prognostic significance in neuroendocrine (NE) tumors of the lung. Twenty-one surgically resected, large cell neuroendocrine carcinomas (LCNEC), 13 small cell lung cancers (SCLC), five atypical carcinoids (AC), and 10 typical carcinoids (TC) were enrolled in the study. LAT1 expression and Ki-67 labeling index of the NE tumors were analyzed by immunohistochemical staining. LAT1 was overexpressed in 52.4% of the LCNEC, in 46.2% of the SCLC, and in 25% of the AC. LAT1 expression in LCNEC was significantly associated with lymph node metastasis and poor outcome. Moreover, a significant correlation was found between LAT1 expression and Ki-67 in both LCNEC and SCLC. Expression of LAT1 tended to increase from low-grade to high-grade NE tumors. The present results suggest that LAT1 may play a significant role in cellular proliferation, lymph node metastasis, and poor outcome in patients with NE tumors of the lung.     

STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo

Alternative splicing of pre-mRNAs greatly contributes to the spatiotemporal diversity of gene expression in metazoans. However, the molecular basis of developmental regulation and the precise sequence of alternative pre-mRNA processing in vivo are poorly understood. In the present study, we focus on the developmental switching of the mutually exclusive alternative splicing of the let-2 gene of Caenorhabditis elegans from the exon 9 form in embryos to the exon 10 form in adults. By visualizing the usage of the let-2 mutually exclusive exons through differential expression of green fluorescent protein (GFP) and red fluorescent protein (RFP), we isolated several switching-defective mutants and identified the alternative splicing defective-2 (asd-2) gene, encoding a novel member of the evolutionarily conserved STAR (signal transduction activators of RNA) family of RNA-binding proteins. Comparison of the amounts of partially spliced let-2 RNAs in synchronized wild-type and asd-2 mutant worms suggested that either of the introns downstream from the let-2 mutually exclusive exons is removed prior to the removal of the upstream ones, and that asd-2 promotes biased excision of intron 10 in the late larval stages. We propose that the developmental switching between alternative sequences of intron removal determines the ratio between the mature let-2 mRNA isoforms.     

Clinicopathological characterization of so-called “cholangiocarcinoma with intraductal papillary growth” with respect to “intraductal papillary neoplasm of bile duct (IPNB)”

Cholangiocarcinoma (CC) of the biliary tract occasionally presents a predominant intraductal papillary growth in the bile ducts, called as biliary tract carcinoma (BTC) of papillary growth (PG) and intrahepatic CC (ICC) of intraductal growth (IG) type. Recently, intraductal papillary neoplasm of bile duct (IPNB) has been proposed as a pre-invasive biliary neoplasm. This study was performed to characterize pathologically BTC of PG type and ICC of IG type with respect to IPNB. It was found that 126 of such 154 CCs (81.8%) fulfilled the criteria of IPNB, while the remaining 28 cases showed different histologies, such as tubular adenocarcinoma and carcinosarcoma. These IPNBs occurred in old aged patients with a male predominance, and the left lobe was rather frequently affected in the liver. A majority of these cases were high grade IPNB (43 cases) and invasive IPNB (77 cases), while low grade IPNB was rare (6 cases). Pancreatobiliary type was predominant (48 cases) followed by gastric (30 cases), intestinal (29 cases) and oncocytic (19 cases) types. Mucus hypersecretion was found in 45 cases, and this was frequent in IPNB at the intrahepatic large bile duct and hilar bile ducts but rare at the extrahepatic bile ducts. Interestingly, 36 cases of high grade and invasive IPNBs contained foci of moderately differentiated adenocacinoma within the intraductal papillary tumor. In conclusion, a majority of ICC of IG type and BTC of PG type could be regarded as a IPNB lineage, and clinically detectable IPNBs were already a malignant papillary lesion.     

Gastrointestinal stromal tumor arising from anorectum: correlation of imprint cytology and radiologic imaging

The diagnosis of gastrointestinal stromal tumor (GIST) is generally established on histopathologic examination of surgical specimens. Gastrointestinal stromal tumor comprises a heterogenous group of neoplasms of the gastrointestinal tract previously referred to as leiomyomas, leiomyosarcomas, or schwannomas. Gastrointestinal stromal tumor arising from anorectum is a rare instance. We report a case of GIST for the correlation of imaging and cytologic features with immunocytochemical staining. A computed tomography and magnetic resonance imaging confirmed a 2-cm tumor growing into the rectal lumen. The central portion of the tumor showed T1-weighted imaging of low signal and suspected central necrosis by the T2-weighted imaging of high signal. Imprint cytology from excised tumors showed isolated or loosely aggregated spindle cells with scanty and fibrillary cytoplasmic processes, nuclear pleomorphism, fine granular chromatin, and irregular nuclear margins. Epithelioid tumor cells showed grooves with abundant cytoplasm and several round nucleoli. Both c-kit and CD34 antigen were positive with strong and diffuse stainability in smears as well as paraffin sections by immunoperoxidase staining. We suggest that the combined use of imaging diagnosis and cytology with immunocytochemical staining are useful initial diagnosis of GIST.     

Hyperpolarization-activated cyclic nucleotide-gated channels and T-type calcium channels confer automaticity of embryonic stem cell-derived cardiomyocytes

Regeneration of cardiac pacemakers is an important target of cardiac regeneration. Previously, we developed a novel embryonic stem (ES) cell differentiation system that could trace cardiovascular differentiation processes at the cellular level. In the present study, we examine expressions and functions of ion channels in ES cell-derived cardiomyocytes during their differentiation and identify ion channels that confer their automaticity. ES cell-derived Flk1(+) mesoderm cells give rise to spontaneously beating cardiomyocytes on OP9 stroma cells. Spontaneously beating colonies observed at day 9.5 of Flk1(+) cell culture (Flk-d9.5) were significantly decreased at Flk-d23.5. Expressions of ion channels in pacemaker cells hyperpolarization-activated cyclic nucleotide-gated (HCN)1 and -4 and voltage-gated calcium channel (Cav)3.1 and -3.2 were significantly decreased in purified cardiomyocytes at Flk-d23.5 compared with at Flk-d9.5, whereas expression of an atrial and ventricular ion channel, inward rectifier potassium channel (Kir)2.1, did not change. Blockade of HCNs and Cav ion channels significantly inhibited beating rates of cardiomyocyte colonies. Electrophysiological studies demonstrated that spontaneously beating cardiomyocytes at Flk-d9.5 showed almost similar features to those of the native mouse sinoatrial node except for relatively deep maximal diastolic potential and faster maximal upstroke velocity. Although approximately 60% of myocytes at Flk-d23.5 revealed almost the same properties as those at Flk-d9.5, approximately 40% of myocytes showed loss of HCN and decreased Cav3 currents and ceased spontaneous beating, with no remarkable increase of Kir2.1. Thus, HCN and Cav3 ion channels should be responsible for the maintenance of automaticity in ES cell-derived cardiomyocytes. Controlled regulation of these ion channels should be required to generate complete biological pacemakers.     

Adhesion activity of fetal gonadal cells to EGF and discoidin domains of milk fat globule-EGF factor 8 (MFG-E8), a secreted integrin-binding protein which is transiently expressed in mouse early gonadogenesis

MFG-E8, a secreted integrin-binding protein, consists of two EGF domains containing a RGD motif and two discoidin domains. In mouse embryogenesis, MFG-E8 is highly expressed in gonadal stromal cells near mesonephros at 11.5–12.5 dpc, but its function in gonadogenesis has not been characterized. To clarify a possible role of MFG-E8 in developing gonads, we analyzed the adhesion activity of 10.5–15.5 dpc gonadal cells to recombinant proteins of EGF or discoidin domains of MFG-E8. In EGF-coated wells, the gonadal cells at 11.5–12.5 dpc revealed a significantly higher adhesion activity as compared to those at 10.5 and 15.5 dpc, while discoidin domains showed a constant number of the adhered cells throughout these stages. To identify the adhesive cells of 11.5-dpc gonads, immunohistochemistry with anti-SF1/Ad4Bp antibody (a specific marker for supporting, steroidogenic, and coelomic epithelial cells) and staining for alkaline phosphatase (a germ cell marker) were carried out. As a result, EGF domains, as well as discoidin domains, were capable of binding to all three groups of SF1/Ad4Bp-positive and negative somatic cells, and germ cells of 11.5-dpc gonads. These findings therefore suggest that MFG-E8 mediates the cell-to-cell interaction among several somatic cell types and germ cells in mouse early gonadogenesis.     

Contribution of sarcoplasmic reticulum Ca²+ release and Ca²+ transporters on sarcolemmal channels to Ca²+ transient in fetal mouse heart

Sarcoplasmic reticulum (SR) Ca release has been shown not to be the predominant mechanism responsible for excitation-contraction (E-C) coupling in fetal myocytes. However, most of the studies have been conducted either on primary cultures or acutely isolated cells, in which an apparent reduction of ryanodine receptor density have been reported. We aimed to elucidate the contribution of SR Ca release and Ca transporters on sarcolemmal channels to Ca transients in fetal mouse whole hearts. On embryonic day 13.5, ryanodine significantly reduced the amplitude of the Ca transient to 27.2 ± 4.4% of the control, and both nickel and SEA0400 significantly prolonged the time to peak from 84 ± 2 ms to 140 ± 5 ms and 129 ± 6 ms, respectively, whereas nifedipine did not alter it. Therefore, at early fetal stages, SR Ca release should be an important component of E-C coupling, and T-type Ca channel and reverse mode sodium-calcium exchanger (NCX)-mediated SR Ca release could be the predominant contributors. Using embryonic mouse cultured cardiomyocytes, we showed that both nifedipine and nickel inhibited the ability of NCX to extrude Ca from the cytosol. From these results, we propose a novel idea concerning E-C coupling in immature heart.