Activating GNAS and KRAS mutations in gastric foveolar metaplasia, gastric heterotopia, and adenocarcinoma of the duodenum.

BACKGROUND: Heterotopic gastric-type epithelium, including gastric foveolar metaplasia (GFM) and gastric heterotopia (GH), is a common finding in duodenal biopsy specimens; however, there is still controversy regarding their histogenetic backgrounds. METHODS: We analysed a total of 177 duodenal lesions, including 66 GFM lesions, 81 GH lesions, and 30 adenocarcinomas, for the presence of GNAS, KRAS, and BRAF mutations. RESULTS: Activating GNAS mutations were identified in 27 GFM lesions (41%) and 23 GH lesions (28%). The KRAS mutations were found in 17 GFM lesions (26%) and 2 GH lesions (2%). A BRAF mutation was found in only one GFM lesion (2%). These mutations were absent in all 32 normal duodenal mucosa specimens that were examined, suggesting a somatic nature. Among the GFM lesions, GNAS mutations were more common in lesions without active inflammation. Analyses of adenocarcinomas identified GNAS and KRAS mutations in 5 (17%) and 11 lesions (37%), respectively. Immunohistochemically, all the GNAS-mutated adenocarcinomas diffusely expressed MUC5AC, indicating gastric epithelial differentiation. CONCLUSIONS: A significant proportion of GFM and GH harbours GNAS and/or KRAS mutations. The common presence of these mutations in duodenal adenoma and adenocarcinoma with a gastric epithelial phenotype implies that GFM and GH might be precursors of these tumours.

Frequent MED12 mutations in phyllodes tumours of the breast.

BACKGROUND: Phyllodes tumours are rare fibroepithelial tumours of the breast, that include benign, borderline, and malignant lesions. Although the molecular basis of phyllodes tumours largely remains unknown, a recent exome study identified MED12 mutations as a sole recurrent genetic alteration in fibroadenoma, a common benign fibroepithelial tumour that shares some histological features with the phyllodes tumour. METHODS: Forty-six phyllodes tumours and 58 fibroadenomas of the breast were analysed for MED12 mutations by using Sanger sequencing. RESULTS: MED12 mutations were identified in 37 out of the 46 phyllodes tumours (80%). The prevalence of MED12 mutations was similar among benign (15/18, 83%), borderline (12/15, 80%), and malignant tumours (10/13, 77%). MED12 mutations were also identified in 36 of the 58 fibroadenomas (62%). The mutations were frequent among intracanalicular-type (24/32, 75%) and complex-type lesions (4/6, 67%), but were significantly less common among the pericanalicular-type lesions (8/20, 40%). A microdissection-based analysis showed that MED12 mutations were confined to the stromal components in both phyllodes tumours and fibroadenomas. CONCLUSIONS: MED12 mutations were frequent among the phyllodes tumours of the breast, regardless of the tumour grade. Phyllodes tumours and fibroadenomas share, at least in part, a common genetic background.

Frequent GNAS mutations in low-grade appendiceal mucinous neoplasms.

BACKGROUND: The molecular basis for the development of appendiceal mucinous tumours, which can be a cause of pseudomyxoma peritonei, remains largely unknown. METHODS: Thirty-five appendiceal mucinous neoplasms were analysed for GNAS and KRAS mutations. A functional analysis of mutant GNAS was performed using a colorectal cancer cell line. RESULTS: A mutational analysis identified activating GNAS mutations in 16 of 32 low-grade appendiceal mucinous neoplasms (LAMNs) but in none of three mucinous adenocarcinomas (MACs). KRAS mutations were found in 30 LAMNs and in all MACs. We additionally analysed a total of 186 extra-appendiceal mucinous tumours and found that GNAS mutations were highly prevalent in intraductal papillary mucinous tumours of the pancreas (88%) but were rare or absent in mucinous tumours of the colorectum, ovary, lung and breast (0-9%). The prevalence of KRAS mutations was quite variable among the tumours. The introduction of the mutant GNAS into a colorectal cancer cell line markedly induced MUC2 and MUC5AC expression, but did not promote cell growth either in vitro or in vivo. CONCLUSION: Activating GNAS mutations are a frequent and characteristic genetic abnormality of LAMN. Mutant GNAS might play a direct role in the prominent mucin production that is a hallmark of LAMN.

Role of aquaporin-5 in gallbladder carcinoma.

BACKGROUND/PURPOSE: Aquaporins (AQPs) are important in controlling bile formation. However, the exact role in human gallbladder carcinogenesis has not yet been defined. METHODS: AQP-5-expressing gallbladder carcinoma (GBC) cell lines (NOZ) were transfected with anti-AQP-5 small interfering RNA (siRNA). Growth, migration, invasion assay, and drug susceptibility tests were performed. Next, microRNA (miRNA) expression was analyzed by miRNA oligo chip (3D-Gene®). AQP-5 and AQP-5-related miRNA target gene expressions were also analyzed using tissue microarray (TMA) in 44 GBC samples. RESULTS: Treatment with AQP-5 siRNA decreased cell proliferation, migration, and invasion. On the other hand, those cells increased IC50 of gemcitabine. By performing miRNA assays, miR-29b, -200a, and -21 were shown to be highly overexpressed in cells treated with AQP-5 siRNA NOZ. When focusing on miR-21, phosphatase and tensin homolog (PTEN) was found to be a target of miR-21. In the TMA, AQP-5/PTEN coexpression was significantly associated with the depth of invasion and MIB-1 index (p = 0.003, 0.010). Survival of patients with a high AQP-5/PTEN coexpression was longer than that of patients with a low coexpression (p = 0.003). CONCLUSIONS: Our result suggested that miR-21 and PTEN may contribute to the role of AQP-5 in GBC. AQP-5 and PTEN cascades are favorable biomarkers of GBC.

Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin.

AIMS/OBJECTIVE: Nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease. Recent studies have demonstrated that podocyte injury is involved in the onset of and progression to renal insufficiency. Here, we describe a novel, highly sensitive ELISA for detecting urinary podocalyxin, a glycoconjugate on the podocyte apical surface that indicates podocyte injury, particularly in the early phase of diabetic nephropathy. METHODS: Urine samples from patients with glomerular diseases (n = 142) and type 2 diabetes (n = 71) were used to quantify urinary podocalyxin by ELISA. Urine samples were obtained from 69 healthy controls for whom laboratory data were within normal values. Podocalyxin was detected in urine by immunofluorescence, immunoelectron microscopy and western blotting. RESULTS: Morphologically, urinary podocalyxin was present as a vesicular structure; western blotting showed it as a positive band at 165-170 kDa. Levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetes. In patients with diabetes, urinary podocalyxin was higher than the cut-off value in 53.8% patients at the normoalbuminuric stage, 64.7% at the microalbuminuric stage and 66.7% at the macroalbuminuric stage. Positive correlations were observed between urinary podocalyxin levels and HbA(1c), urinary ß(2) microglobulin, α(1) microglobulin and urinary N-acetyl-ß-D-glucosaminidase, although urinary podocalyxin levels were not correlated with other laboratory markers such as blood pressure, lipid level, serum creatinine, estimated GFR or proteinuria. CONCLUSIONS/INTERPRETATION: Urinary podocalyxin may be a useful biomarker for detecting early podocyte injury in patients with diabetes.

Tumour necrosis is a postoperative prognostic marker for pancreatic cancer patients with a high interobserver reproducibility in histological evaluation.

BACKGROUND: Tumour necrosis reflects the presence of hypoxia, which can be indicative of an aggressive tumour phenotype. The aim of this study was to investigate whether histological necrosis is a useful predictor of outcome in patients with pancreatic ductal carcinoma (PDC). METHODS: We reviewed histopathological findings in 348 cases of PDC in comparison with clinicopathological information. We counted small necrotic foci (micronecrosis) as necrosis, in addition to massive necrosis that had been only defined as necrosis in previous studies. The reproducibility of identifying histological parameters was tested by asking five independent observers to blindly review 51 examples of PDC. RESULTS: Both micronecrosis and massive necrosis corresponded to hypoxic foci expressing carbonic anhydrase IX detected by immunohistochemistry. Multivariate survival analysis showed that histological necrosis was an independent predictor of poor outcome in terms of both disease-free survival (DFS) and disease-specific survival (DSS) of PDC patients. In addition, metastatic status, and lymphatic, venous, and intrapancreatic neural invasion were independent prognostic factors for shorter DFS and metastatic status, margin status, lymphatic invasion, and intrapancreatic neural invasion were independent prognostic factors for DSS. The interobserver reproducibility of necrosis identification among the five independent observers was 'almost perfect' (κ-value of 0.87). CONCLUSION: Histological necrosis is a simple, accurate, and reproducible predictor of postoperative outcome in PDC patients.

Architectures of class-defining and specific domains of glutamyl-tRNA synthetase.

The crystal structure of a class I aminoacyl-transfer RNA synthetase, glutamyl-tRNA synthetase (GluRS) from Thermus thermophilus, was solved and refined at 2.5 A resolution. The amino-terminal half of GluRS shows a geometrical similarity with that of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of the same subclass in class I, comprising the class I-specific Rossmann fold domain and the intervening subclass-specific alpha/beta domain. These domains were found to have two GluRS-specific, secondary-structure insertions, which then participated in the specific recognition of the D and acceptor stems of tRNA(Glu) as indicated by mutagenesis analyses based on the docking properties of GluRS and tRNA. In striking contrast to the beta-barrel structure of the GlnRS carboxyl-terminal half, the GluRS carboxyl-terminal half displayed an all-alpha-helix architecture, an alpha-helix cage, and mutagenesis analyses indicated that it had a role in the anticodon recognition.

Histological diversity in basaloid squamous cell carcinoma of the esophagus.

Basaloid squamous cell carcinoma of the esophagus (BSCCE) is a distinct variant of esophageal cancer. This study investigated histopathological variations of BSCCE. Thirty-eight surgical and two endoscopically resected specimens of BSCCE were examined. Histological features were classified into five components: solid nest (SN), microcyst and/or trabecular nest (MT), ductal differentiation (DD), cribriform pattern (CP), and an invasive squamous cell carcinoma (SCC) component. The immunohistochemical phenotypes of each component were examined using antibodies against cytokeratin (CK) 7, CK14, and alpha smooth muscle actin (SMA). SN, MT, DD, CP, and SCC were present in 95.0, 97.5, 27.5, 32.5, and 82.5% of the cases, respectively, and combinations of SN & MT, SN & DD, SN, MT & DD, SN, MT & CP, and SN, MT, DD & CP were found in 50.0, 2.5, 10.0, 17.5, and 15.0%, respectively. All the intraepithelial lesions observed in 18 (45.0%) cases were SCC. Immunoreactivity for CK7, CK14, and SMA was seen in 10.5, 86.8, and 18.4% of SN; 30.8, 97.4, and 38.5% of MT; 54.5, 100.0, and 54.5% of DD; 7.7, 76.9, and 23.1% of CP; and 6.1, 97.0, and 0.0% of SCC, respectively. CK14 immunoreactivity was seen in the periphery of most of the SN component. CK7, CK14, and SMA immunoreactivity was seen in the inner layer, all layers, and the outer layer of DD, respectively. MT and CP showed partial peripheral positivity for CK14 and SMA in microcystic, trabecular, and cribriform-like pseudoglandular structures. BSCCE demonstrates various histopathological and immunohistochemical features including a ductal and cribriform growth pattern.

In situ synthesis of Au-shelled Ag nanoparticles on PDMS for flexible, long-life, and broad spectrum-sensitive SERS substrates.

A simple and fast one-step fabrication method of silver nanoparticles (AgNPs) on a polydimethylsiloxane (PDMS) film and their improvement as highly sensitive surface enhanced Raman scattering (SERS) substrates via atomically thin Au coatings is demonstrated. The thin Au layer provides oxidation resistivity while maintaining the broad spectral range SERS sensitivity of Ag nanoparticles.

Assessment of the impact of scheduled postmarketing safety summary analyses on regulatory actions.

In addition to standard postmarketing drug safety monitoring, Section 915 of the Food and Drug Administration Amendments Act of 2007 (FDAAA) requires the US Food and Drug Administration (FDA) to conduct a summary analysis of adverse event reports to identify risks of a drug or biologic product 18 months after product approval, or after 10,000 patients have used the product, whichever is later. We assessed the extent to which these analyses identified new safety signals and resultant safety-related label changes. Among 458 newly approved products, 300 were the subjects of a scheduled analysis; a new safety signal that resulted in a safety-related label change was found for 11 of these products. Less than 2% of 713 safety-related label changes were based on the scheduled analyses. Our study suggests that the safety summary analyses provide only marginal value over other pharmacovigilance activities.

Isolation and characterization of a novel growth hormone cDNA from chum salmon (Oncorhynchus keta).

A cDNA clone which codes for a novel growth hormone has been isolated from the library of chum salmon pituitaries. The clone encodes a polypeptide of 210 amino-acid residues including 22 amino-acid residues of signal peptide, which is identical in length with known chum salmon growth hormone. In the coding region, there are 30 base substitutions, some of which result in 12 amino-acid substitutions. There are 8 base changes in the 5' untranslated region, and large insertions/deletions are in the 3' non-coding region. These results clearly indicate that there are at least two species of mRNAs for growth hormone in chum salmon pituitary.

Major identity determinants in the "augmented D helix" of tRNA(Glu) from Escherichia coli.

By a kinetic analysis of 59 variant transcripts of Escherichia coli tRNA(Glu) with glutamyl-tRNA synthetase (GluRS), the U11.A24 base-pair, the U13.G22..A46 base-triple, and the lack of residue 47 (delta47) were found to serve as major determinants for tRNA(Glu) identity. This is the first system for which major identity determinants are reported to be clustered in the "augmented D helix", consisting of the D stem with some neighboring residues and the variable loop. Other identity determinants are U34, U35, C36 and A37 in the anticodon loop, and G1.C72 and U2.A71 in the acceptor stem. Phosphate-group protection by GluRS from ethylnitrosourea was observed most strongly for the minor groove side of D-stem helix, indicating that GluRs tightly binds to the D stem for recognition, on the minor groove side, of the potent identity-determinant groups of the U11.A24 and U13.G22 base-pairs. A46 is not involved in direct recognition by GluRS; the U13.G22..A46 base-triple is required probably for formation of the structural features that are recognized by GluRS. In this context, the essential role of characteristic delta47 in tRNA(Glu) identity may be to maintain the U13.G22..A46 base-triple.

Molecular cloning and expression analyses of mouse betaklotho, which encodes a novel Klotho family protein.

We report here the identification of mouse betaklotho (betakl), which encodes a type I membrane protein with high resemblance to Klotho (KL). Both betaKL and KL consist of two internal repeats with homology to family 1 glycosidases, while these essential glutamates for the enzymatic activities were not conserved. The identical pattern of substitution and variation in the substituted amino acids between these two proteins indicate that they likely to form a unique family within the glycosidase family 1 superfamily. During mouse embryonic development, strong betakl expression was detected in the yolk sac, gut, brown and white adipose tissues, liver and pancreas, and in the adult, predominantly in the liver and pancreas. Despite the high structural similarity between betaKL and KL, their expression profiles were considerably different and betakl expression was not induced in kl-deficient mouse mutants.

Pleiotropic effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors: candidate mechanisms for anti-lipid deposition in blood vessels.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) are considered first-line therapeutic agents for the prevention of coronary heart disease and atherosclerotic disorders related to hypercholesterolemia. Statins inhibit lipid deposition in the aortic endothelium. Although it has been accepted that the statins are potent inhibitors of cholesterol biosynthesis in the liver and that they lower circulating cholesterol levels, several cholesterol-independent (pleiotropic) effects have been reported. The cholesterol-independent effects of statins involve normalization of the nitric oxide (NO)-NO synthase system, anti-inflammatory effects through the inhibition of cytokine/chemokine production, inhibition of vascular smooth muscle cell proliferation and migration, and inhibition of platelet thrombus formation/reduction of the thrombotic response. Some pleiotropic effects of statins may depend on the inhibition of the biosynthesis of farnesyl- and geranylgeranyl-nonsterol compounds from mevalonate in the cells. The Rho/Rho kinase pathway and the phospatidylinositol-3 kinase/Akt pathway mediate the pleiotropic effects of statins. As variations occur in absorption, metabolism, and excretion mechanisms due to the characteristics of specific statins including their hydrophilicity and lipophilicity, there are differences in the transfer mechanisms of statins into tissues. However, the pleiotropic effects occur regardless of statin hydrophilicity and lipophilicity. This review summarizes the pleiotropic effects of statins on lipid deposition in blood vessels.

Selection of cDNAs encoding putative type II membrane proteins on the cell surface from a human full-length cDNA bank.

We have developed a simple method to test whether a hydrophobic segment near the N-terminus of a protein functions as a type II signal anchor (SA) in which the N-terminus faces the cytoplasm. A cDNA fragment containing the putative SA sequence of a target clone was fused in-frame to the 5' end of a cDNA fragment encoding the protease domain of urokinase-type plasminogen activator (u-PA). The resulting fused gene was expressed in COS7 cells. Fibrinolytic activity on the cell surface was measured by placing a fibrin sheet in contact with the transfected COS7 cells after removing the medium. When the cDNA fragment encoded a SA, the fibrin sheet was lysed by the u-PA expressed on the cell surface. The fibrinolytic activity was not detected in the culture medium, suggesting that the u-PA remains on the cell surface anchored via the SA in the membrane without being cleaved by signal peptidase. This fibrin sheet method was successfully applied to select five novel cDNA clones encoding putative type II membrane proteins from a human full-length cDNA bank.

Molecular cloning of eel growth hormone cDNA and its expression in Escherichia coli.

cDNA clones coding for growth hormone (eGH) of Japanese eel (Anguilla japonica) have been isolated from a cDNA library prepared from pituitary gland poly(A)+ RNA. The nucleotide sequence of the eGH cDNA was determined. It codes for the prehormone of 209 amino acids (aa) including a putative signal peptide of 19 aa. The deduced amino acid sequence was identical with that determined for eGH protein. The primary structure of eGH was compared with those of other species growth hormones (chum salmon, chicken, rat, and human). Mature eGH was expressed in Escherichia coli harboring a plasmid in which the eGH cDNA was under control of the phage lambda pL promoter. Recombinant eGH polypeptide was immunoreactive to rabbit antiserum against natural eGH. Furthermore, eGH derivative with amino-terminal deletion (delta 1-3 eGH) was produced in E. coli reaching up to 5% of total cellular proteins.

Construction of a human full-length cDNA bank.

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKA1, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.

A three-dimensional structure model of the complex of glutamyl-tRNA synthetase and its cognate tRNA.

A docking model of glutamyl-tRNA synthetase (GluRS) and tRNAGlu was constructed, on the basis of the distinguished similarity between the X-ray crystallographic three-dimensional structures of the N-terminal halves of the Thermus thermophilus GluRS in the free state and the Escherichia coli glutaminyl-tRNA synthetase in a complex with tRNAGln. The modeled structure is energetically favorable and is also well consistent with the results of site-directed mutagenesis studies. The model indicates that the GluRS-specific insertions 2 and 3 fit and bind to the acceptor stem and the D arm, respectively, of the cognate tRNA without affecting other contacts. In particular, insertion 3 strongly interacts with the two D-stem base pairs that are essential for the tRNA-GluRS recognition.

Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein.

We previously established a novel mouse model for human aging and identified the genetic foundation responsible for it. A defect in expression of a novel gene, termed klotho (kl), leads to a syndrome resembling human aging in mice. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. In this report, we present the entire mouse kl gene organization. The mouse kl gene spans about 50 kilobases and consists of five exons. The promoter region lacks a TATA-box and contains four potential binding sites for SP1. We further show that two kl gene transcripts encoding membrane or secreted protein are generated through alternative transcriptional termination. These findings provide fundamental information for further study of the kl gene which may regulate aging in vivo.

Outbreak of herpangina associated with Coxsackievirus B3 infection.

An outbreak of acute febrile illness was observed in summer, 1987, in a welfare home in which 31 healthy infants were accommodated. Within a 5-day period 25 infants (81%) acquired a febrile illness. Coxsackievirus B3 was isolated from 16 (64%) of 25 throat swabs. In the patients in whom viral culture was negative or not performed, 6 were serologically identified as having a coxsackievirus B3 infection. Among 22 patients identified as having a coxsackievirus B3 infection 7 had typical herpangina and the others had pharyngitis with or without a few small vesicles. Serum alpha-interferon was detected in all but 2 cases (one with proved infection and another with indefinite infection). Herpangina can be associated with coxsackievirus B3 as well as with the more frequently associated coxsackievirus Group A; this explosive type of outbreak might be transmitted by a small particle aerosol.