Clinicopathological analysis of early-stage gastric cancers detected after successful eradication of Helicobacter pylori

Background and Aims: The results of a randomized controlled study and meta‐analysis study have recently proved that Helicobacter pylori eradication has a preventive effect against the development of metachronous and primary gastric cancer. However, gastric cancer is sometimes detected after successful eradication. There is a lack of study about gastric cancers in eradicated patients. To clarify the characteristics of gastric cancers detected after H. pylori eradication, we analyzed the clinicopathological features of these cancers. Methods: The subjects were 18 early‐stage gastric cancer specimens resected from 17 patients who had received successful eradication of H. pylori from February 1995 to March 2009. The control group consisted of 36 specimens from noneradicated patients with persistent H. pylori infection who were matched with the subjects in age, sex, and depth of invasion. Clinicopathological features and mucin phenotypes of gastric cancer were clinically and immunohistologically evaluated. Results: The average diameter of gastric cancer was smaller and Ki‐67 index was lower in the eradication group. The morphological distribution of depression types was significantly lower in the control group. Immunohistochemical phenotyping revealed that 72.2% of the lesions in the eradicated group were complete gastric type or gastric predominant mixed type, whereas the percentages of gastric type and intestinal type in the control group were similar. Conclusion: Our findings indicate that the clinicopathological characteristics of gastric cancers detected after H. pylori eradication are different from those of gastric cancers in patients with persistent H. pylori infection. H. pylori eradication may suppress intestinalization during the development of gastric cancer.     

Nuclear protein quality is regulated by the ubiquitin-proteasome system through the activity of Ubc4 and San1 in fission yeast

Eukaryotic cells monitor and maintain protein quality through a set of protein quality control (PQC) systems whose role is to minimize the harmful effects of the accumulation of aberrant proteins. Although these PQC systems have been extensively studied in the cytoplasm, nuclear PQC systems are not well understood. The present work shows the existence of a nuclear PQC system mediated by the ubiquitin-proteasome system in the fission yeast Schizosaccharomyces pombe. Asf1-30, a mutant form of the histone chaperone Asf1, was used as a model substrate for the study of the nuclear PQC. A temperature-sensitive Asf1-30 protein localized to the nucleus was selectively degraded by the ubiquitin-proteasome system. The Asf1-30 mutant protein was highly ubiquitinated at higher temperatures, and it remained stable in an mts2-1 mutant, which lacks proteasome activity. The E2 enzyme Ubc4 was identified among 11 candidate proteins as the ubiquitin-conjugating enzyme in this system, and San1 was selected among 100 candidates as the ubiquitin ligase (E3) targeting Asf1-30 for degradation. San1, but not other nuclear E3s, showed specificity for the mutant nuclear Asf1-30, but did not show activity against wild-type Asf1. These data clearly showed that the aberrant nuclear protein was degraded by a defined set of E1-E2-E3 enzymes through the ubiquitin-proteasome system. The data also show, for the first time, the presence of a nuclear PQC system in fission yeast.     

Molybdophyllysin,a toxic metalloendopeptidase from the tropical toadstool,Chlorophyllum molybdites

A toxic protein, dubbed molybdophyllysin, was isolated from the tropical toadstool Chlorophyllum molybdites by following its lethal effect in mice. Analysis of the protein using SDS–PAGE revealed a single 23-kDa band. Sequence analysis of molybdophyllysin tryptic fragments showed that this protein is highly homologous to metalloendopeptidases (MEPs) obtained from edible mushrooms, such as Grifola frondosa, Pleurotus ostreatus, and Armillaria mellea. These proteins include a HEXXH+D zinc-binding motif known as aspzincin. Accordingly, molybdophyllysin is a member of the deuterolysin family of zinc proteases. Molybdophyllysin retained its proteolytic activity at temperatures up to 60 °C with an optimum pH of 7.0. The activity was inhibited by both 1,10-phenanthroline and N-bromosuccinimide, but molybdophyllysin exhibited strong resistance to SDS.     

Platelet activation receptor CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells

The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2(+/+) platelets, but not by CLEC-2(-/-) platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.     

Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.     

Two novel mutations in the EYS gene are possible major causes of autosomal recessive retinitis pigmentosa in the Japanese population

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease including autosomal recessive (ar), autosomal dominant (ad), and X-linked inheritance. Recently, arRP has been associated with mutations in EYS (Eyes shut homolog), which is a major causative gene for this disease. This study was conducted to determine the spectrum and frequency of EYS mutations in 100 Japanese arRP patients. To determine the prevalence of EYS mutations, all EYS exons were screened for mutations by polymerase chain reaction amplification, and sequence analysis was performed. We detected 67 sequence alterations in EYS, of which 21 were novel. Of these, 7 were very likely pathogenic mutations, 6 were possible pathogenic mutations, and 54 were predicted non-pathogenic sequence alterations. The minimum observed prevalence of distinct EYS mutations in our study was 18% (18/100, comprising 9 patients with 2 very likely pathogenic mutations and the remaining 9 with only one such mutation). Among these mutations, 2 novel truncating mutations, c.4957_4958insA (p.S1653KfsX2) and c.8868C>A (p.Y2956X), were identified in 16 patients and accounted for 57.1% (20/35 alleles) of the mutated alleles. Although these 2 truncating mutations were not detected in Japanese patients with adRP or Leber's congenital amaurosis, we detected them in Korean arRP patients. Similar to Japanese arRP results, the c.4957_4958insA mutation was more frequently detected than the c.8868C>A mutation. The 18% estimated prevalence of very likely pathogenic mutations in our study suggests a major involvement of EYS in the pathogenesis of arRP in the Japanese population. Mutation spectrum of EYS in 100 Japanese patients, including 13 distinct very likely and possible pathogenic mutations, was largely different from the previously reported spectrum in patients from non-Asian populations. Screening for c.4957_4958insA and c.8868C>A mutations in the EYS gene may therefore be very effective for the genetic testing and counseling of RP patients in Japan.     

Oct-3/4 promotes migration and invasion of glioblastoma cells

As a result of increased glioblastoma migration and invasion into normal brain parenchyma, treatment of local tumor recurrence following initial treatment in glioblastoma patients remains challenging. Recent studies have demonstrated increased Oct-3/4 expression, a self-renewal regulator in stem cells, in glioblastomas. However, little is known regarding the influence of Oct-3/4 in glioblastoma cell invasiveness. The present study established Oct-3/4-overexpressing glioblastoma cells, which were prepared from human glioblastoma patients, to assess migration, invasion, and mRNA expression profiles of integrins and matrix metalloproteinases (MMPs). Compared with control cells, Oct-3/4 expressing-glioblastoma cells exhibited increased migration and invasion in wound healing and Matrigel invasion assays. Oct-3/4 overexpression resulted in upregulated FAK and c-Src expression, which mediate integrin signals. Vinculin accumulated along the leading edges of Oct-3/4 expressing-glioblastoma cells and associated with membrane ruffles during cell migration. Oct-3/4 expressing-cells exhibited increased MMP-13 mRNA expression and MMP-13 knockdown by shRNA suppressed cell invasion into Matrigel and organotypic brain slices. These results suggested that Oct-3/4 enhanced degradation of surrounding extracellular matrix by increasing MMP-13 expression and altering integrin signaling. Therefore, Oct-3/4 might contribute to tumor promoting activity in glioblastomas.     

Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin

Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities. A large crystal was prepared at pD 7.4 for neutron protein crystallography. Neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. The neutron structure solved at 2.0 Å resolution revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is composed of Thr75, Trp79, His88, Ser112, Pro113, Thr118-B and four water molecules, and is involved in both monomer–monomer and dimer–dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. In addition, the comparison with X-ray structure at pH 4.0 indicated that the protonation occurred to Asp74, His88 and Glu89 at pH 4.0. Our neutron model provides insights into the molecular stability of TTR related to the hydrogen-bond network, the pH sensitivity and the CH···O weak hydrogen bond.     

Enhanced formation of aerenchyma and induction of a barrier to radial oxygen loss in adventitious roots of Zea nicaraguensis contribute to its waterlogging tolerance as compared with maize (Zea mays ssp. mays)

Enhancement of oxygen transport from shoot to root tip by the formation of aerenchyma and also a barrier to radial oxygen loss (ROL) in roots is common in waterlogging‐tolerant plants. Zea nicaraguensis (teosinte), a wild relative of maize (Zea mays ssp. mays), grows in waterlogged soils. We investigated the formation of aerenchyma and ROL barrier induction in roots of Z. nicaraguensis, in comparison with roots of maize (inbred line Mi29), in a pot soil system and in hydroponics. Furthermore, depositions of suberin in the exodermis/hypodermis and lignin in the epidermis of adventitious roots of Z. nicaraguensis and maize grown in aerated or stagnant deoxygenated nutrient solution were studied. Growth of maize was more adversely affected by low oxygen in the root zone (waterlogged soil or stagnant deoxygenated nutrient solution) compared with Z. nicaraguensis. In stagnant deoxygenated solution, Z. nicaraguensis was superior to maize in transporting oxygen from shoot base to root tip due to formation of larger aerenchyma and a stronger barrier to ROL in adventitious roots. The relationships between the ROL barrier formation and suberin and lignin depositions in roots are discussed. The ROL barrier, in addition to aerenchyma, would contribute to the waterlogging tolerance of Z. nicaraguensis.