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Knowledge of the histologic type and primary origin of pulmonary tumors is essential when preparing a surgical strategy. Intraoperative diagnosis of hematoxylin and eosin (HE)-stained frozen sections is the gold standard, but reliable pathology requires time-consuming immunohistochemistry (IHC) to distinguish among histological types/organ origins and to analyze molecular status. The aim of this study was to evaluate the clinical reliability of a new rapid-IHC technique for intraoperative diagnosis of pulmonary tumors. In total, 169 patients with undiagnosed pulmonary tumors were enrolled in a multicenter prospective observational study. At three institutes, pulmonary tumor samples were collected through core needle biopsy and/or surgery to determine surgical strategies. Using a new device for rapid IHC, we applied a high-voltage, low-frequency alternating current (AC) field, which mixes the available antibody as the voltage is switched on/off. Rapid IHC can provide tumor histologic type/origin diagnoses within 20 min, as opposed to the 3-6 h required for conventional IHC. No false diagnoses of malignancy were rendered in any of the cases when using simple HE staining. With HE staining alone, the overall definitive diagnosis rate, the rate of defined tumor origin, and the rate of determined histological type were 76.92%, 85.80%, and 90.53%, respectively. When rapid IHC was added, those rates were significantly improved to 88.76%, 94.67%, and 91.72%, respectively. By providing prompt and accurate intraoperative histological/molecular analysis, rapid IHC driven by AC mixing could serve as an effective clinical tool guiding the surgical strategy for undiagnosed pulmonary tumors.

The human epidermis is formed by the proliferation and differentiation of keratinocytes adjacent to the basement membrane. The outermost layer, the stratum corneum, is equipped with a barrier function that prevents water evaporation, and intercellular lipids play an important role in this barrier function. When the barrier is functioning normally, evaporation is prevented; however, when barrier function is impaired, moisture evaporates, resulting in dry and rough skin. Therefore, maintenance of normal barrier function is critical for maintaining normal skin function. Peroxisome proliferator-activated receptor α (PPARα) is mainly not only involved in lipid metabolism in the liver but is also expressed in the epidermis and is involved in inducing keratinocyte differentiation, promoting lipid production, maintaining barrier function and suppressing skin inflammation. Hence, compounds that activate PPARα are expected to control skin function. Therefore, we identified PPARα activators from among extracts of natural resources that have been approved for use in humans and analysed the effects of these extracts on skin function.First, extracts of 474 natural resources were screened using a PPARα activator screening cell line independently constructed in our laboratory. Next, reporter assays were performed using the Gal4-chimera system to evaluate whether these extracts act as ligands for PPARα. We then analysed their effect on primary normal human epidermal keratinocyte cells by using real-time RT-PCR. Finally, we evaluated PPARα activation effect by the combination of these extracts.We identified 36 extracts having the effect of activating PPARα. In particular, #419, a Typha angustifolia spike extract, showed concentration-dependent transcriptional activation through PPARα-LBD and was considered to be likely to contain a compound that is a ligand of PPARα. #419 increased the expression of PPARα target genes and genes related to skin function in primary cultured human epidermal keratinocytes. Finally, the use of #419 in combination with nine extracts increased PPAR activity more than twice as much as #419 alone treatment.These results showed that the reporter cell line could be useful for discovering extracts of natural resources and that the identified Typha angustifolia spike extract could be used in cosmetics that activate PPARα, which expected to improve skin function.L’épiderme humain se forme grâce à la prolifération et à la différenciation des kératinocytes adjacents à la membrane basale. La couche externe, dite « couche cornée », possède une fonction barrière qui empêche l’évaporation de l’eau, dans laquelle les lipides intercellulaires jouent un rôle important. Lorsque la barrière fonctionne normalement, l’évaporation est évitée ; mais lorsqu’elle est altérée, l’évaporation a lieu et la peau, privée d’hydratation, devient sèche et rêche. Par conséquent, il est capital de maintenir cette fonction barrière normale pour que la peau conserve son fonctionnement normal. Le récepteur alpha activé par proliférateurs de peroxysomes (PPARα) intervient surtout non seulement dans le métabolisme lipidique du foie, mais également dans l’épiderme ; il joue en effet un rôle dans l’induction de la différenciation des kératinocytes, la promotion de la production lipidique, le maintien de la fonction barrière et la suppression de l’inflammation de l’épiderme. Par conséquent, les activateurs du PPAR-α devraient être déterminants pour une bonne fonction cutanée. Nous avons donc identifié des activateurs du PPAR-α parmi des extraits de ressources naturelles dont l’utilisation chez l’homme est approuvée, et nous avons analysé les effets de ces extraits sur la fonction cutanée. MÉTHODES: Tout d’abord, des extraits de 474 ressources naturelles ont été sélectionnés à l’aide d’une lignée cellulaire de détection des activateurs du PPAR-α, construite indépendamment dans notre laboratoire. Ensuite, des tests de gènes rapporteurs ont été effectués à l’aide du système Gal4-chimera pour voir si ces extraits jouaient le rôle de ligands pour le PPAR-α. Nous avons ensuite analysé leur effet sur les cellules kératinocytaires épidermiques humaines normales primaires par RT-PCR en temps réel. Enfin, nous avons évalué l’effet d’activation du PPAR-α par l’association de ces extraits. RÉSULTATS: Nous avons identifié 36 extraits ayant pour effet d’activer le PPAR-α. En particulier, le n° 419, un extrait d’épi de Typha angustifolia, a montré une activation transcriptionnelle dépendante de la concentration par le PPAR-α-LBD et a été considéré comme susceptible de contenir un composé qui est un ligand du PPAR-α. Le n° 419 a augmenté l’expression des gènes cibles du PPAR-α et des gènes liés au fonctionnement de la peau dans les kératinocytes épidermiques humains primaires mis en culture. Enfin, l’utilisation du n° 419 en association avec neuf extraits a augmenté de plus du double l’activité du PPAR par rapport au traitement par le n° 419 seul.Ces résultats ont montré que la lignée cellulaire rapporteuse pourrait être utile pour découvrir des extraits de ressources naturelles et que l’extrait d’épi de Typha angustifolia identifié pourrait être utilisé dans des cosmétiques qui activent le PPAR-α, ce qui devrait améliorer la fonction cutanée.

Background Fibrous bands (FBs) are one of the histological features in tumors which can be confirmed by hematoxylin and eosin (H&E)‐stained slides. FBs have been reported to correlate with malignancy in various tumors. This study aimed to investigate whether the presence of FBs is associated with malignancy in thymoma. Methods A total of 123 consecutive patients with thymoma who underwent microscopically complete resections from January 2000 to December 2018 were enrolled into this study. H&E‐stained slides of all thymoma patients were re‐examined. Study patients were classified into two groups: with FBs (n = 36) and without FBs (n = 87). Clinicopathological characteristics, overall survival (OS), and recurrence‐free survival (RFS) were compared between the two groups. Furthermore, multivariate analyses were performed to identify whether the presence of FBs was associated with higher Masaoka stage and poor prognosis in patients with thymoma. Results The Masaoka stage was found to be higher and recurrence more likely in thymoma patients with FBs than in those without. RFS was significantly poorer in thymoma patients with FBs than in those without, although no significant difference was observed in OS between them. The presence of FBs was significantly associated with higher Masaoka stage in the multivariate analysis using logistic regression. Additionally, the presence of FBs was an independent prognostic factor for poor RFS in multivariate analysis using Cox's proportional hazards model. Conclusions The presence of FBs in patients with thymoma was associated with higher Masaoka stage, higher recurrence rate, and poorer RFS. Key points Significant findings of the study Fibrous bands (FBs) are bands of fibrosis dividing tumors into different‐sized irregular islands. The presence of FBs is associated with higher Masaoka stage and poor recurrence‐free survival in patients with thymoma. What this study adds The presence of fibrous bands might be associated with the malignant behavior of thymoma. Confirming the presence or absence of FBs may result in personalized medication for patients with thymoma., • Fibrous bands (FBs) are bands of fibrosis dividing tumors into different‐sized irregular islands. • The presence of FBs is associated with the malignant behavior of thymomas. • Confirmation of FBs might lead to individualized medications in patients with thymoma.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis and closely related to exposure to asbestos. MPM is a heterogeneous tumor with three main histological subtypes, epithelioid, sarcomatoid, and biphasic types, among which sarcomatoid type shows the poorest prognosis. The Ror-family of receptor tyrosine kinases, Ror1 and Ror2, is expressed in various types of tumor cells at higher levels and affects their aggressiveness. However, it is currently unknown whether they are expressed in and involved in aggressiveness of MPM. Here, we show that Ror1 and Ror2 are expressed in clinical specimens and cell lines of MPM with different histological features. Studies using MPM cell lines indicate that expression of Ror2 is associated tightly with high invasiveness of MPM cells, whereas Ror1 can contribute to their invasion in the absence of Ror2. However, both Ror1 and Ror2 promote proliferation of MPM cells. We also show that promoted invasion and proliferation of MPM cells by Ror signaling can be mediated by the Rho-family of small GTPases, Rac1, and Cdc42. These findings elucidate the critical role of Ror signaling in promoting invasion and proliferation of MPM cells.

Claudin‐4 (CLDN‐4), a tight‐junction protein, is overexpressed in various malignant tumors, including gastric, colorectal, pancreatic, and breast cancers. However, CLDN‐4 is also expressed in normal tissues, including the liver, pancreas, kidney, and small intestine. Whether CLDN‐4 is an effective and safe target for cancer therapy has been unclear owing to the lack of a binder with both CLDN‐4 specificity and cross‐reactivity to human and murine cells. In this study, we successfully generated a rat anti‐CLDN‐4 monoclonal antibody (5D12) that was specific to, and cross‐reactive with, human and mouse CLDN‐4. 5D12 recognized the second extracellular domain of human CLDN‐4 in a conformation‐dependent manner. A human–rat chimeric IgG1 of 5D12 (xi‐5D12) activated the Fcγ IIIa receptor, indicating the activation of antibody‐dependent cellular cytotoxicity in CLDN‐4‐expressing cells. Moreover, xi‐5D12 significantly suppressed tumor growth in mice bearing human colorectal and gastric tumors without apparent adverse effects, such as weight loss or liver and kidney damage. These results suggest that CLDN‐4 is a potent target for cancer therapy and that an anti‐CLDN‐4 antibody is a promising candidate anticancer agent.

Liver X receptor alpha (LXRalpha) is a member of the nuclear receptor superfamily that is activated by oxysterols, and plays a pivotal role in regulating the metabolism, transport and uptake of cholesterol. Here, we demonstrate that LXRalpha also regulates the low-density lipoprotein receptor (LDLR) gene, which mediates the endocytic uptake of LDL cholesterol in the liver. An LXR agonist induced the expression of LDLR in cultured hepatoblastoma cells. Moreover, the LDLR promoter contained an LXR response element that was recognized by LXRalpha/RXRalpha (retinoid X receptor alpha) heterodimers in hepatoblastoma cells. These results suggest a novel pathway whereby LXRalpha might modulate cholesterol metabolism.

We have prepared a model receptor containing a Lys cluster (320-340) in the collagen-like domain of the bovine macrophage scavenger receptor, and have shown that it has a similar binding specificity to the native scavenger receptor. The native scavenger receptor is reported to bind the quadruplex structure of nucleotides. In this study, we analyzed the model receptor binding of nucleotides with various structures, random, parallel or antiparallel quadruplex and aggregate forms. This was carried out by direct binding assays using labeled oligonucleotides or surface plasmon resonance, and by an inhibition assay using Chinese hamster ovary (CHO) cells expressing the scavenger receptor. The results showed that the nucleotides forming the quadruplex structure did not exhibit any binding. Only the aggregate forms of the nucleotide could bind to the model receptor. They also inhibited the degradation of acetylated low density lipoprotein by CHO cells expressing the native scavenger receptor, whereas nucleotides that did not bind to the model receptor had no effect on cellular acetylated low density lipoprotein degradation. Our results suggest that the quadruplex structure is not essential but may be required for the formation of the nucleotide aggregates, which can interact with the scavenger receptor.

In the rat platelet factor 4 (PF4) promoter, Ets motifs and GATA motifs are located at positions -880, -75 and -135, -30, respectively, and their motifs are found in the promoter region of most megakaryocyte protein genes. In order to investigate how the Ets and GATA motifs affect PF4 promoter activity, we constructed Ets and/or GATA motif mutant genes. A single disruption of either -75Ets, -135GATA, or -30GATA significantly reduced PF4 promoter activity, and double disruptions involving these motifs completely abolished it. Furthermore, gel-retardation assays revealed that Ets-1 and GATA-1 proteins from HEL and MEG-01 cells bound to the Ets motifs and GATA motifs, respectively. Co-transfection experiments showed that the overexpression of Ets-1 and/or GATA-1 enhanced the expression of the PF4 promoter reporter gene. These effects of Ets-1 and GATA-1 on PF4 promoter activity are additive. When HEL cells were treated with dimethylsulfoxide in order to induce differentiation into megakaryocytes, the mRNA level of ets-1 increased 10-fold, which might be directly correlated with the significant increase in PF4 mRNA level induced by dimethylsulfoxide. All these results strongly suggest that both Ets-1 and GATA-1 play key roles in the positive regulation of PF4 gene expression.

Scavenger receptors bind modified low-density lipoproteins (LDL) on a collagen-like domain which possesses a lysine cluster at the car☐y end. We previously constructed a receptor model peptide containing the lysine cluster. In the present study, we evaluated the ligand specificity of the receptor model peptide. It selectively bound modified-LDLs, and not LDL. The binding of acetylated-LDL (Ac-LDL) was inhibited by dextran sulfate, fucoidan, and sulfatides in a manner similar to that of the natural receptor. Both polyguanylic and polyinosinic acids inhibit the Ac-LDL binding whereas polycytidylic acid did not. These results indicate that the lysine cluster in the collagen-like domain has important roles in both ligand binding and ligand specificity.

A novel cationic lipid with a symmetrical and biodegradable structure was synthesized, and the transfection efficiency and the cytotoxicity of the cationic liposomes composed of the cationic lipid and dioleoylphosphatidylethanolamine (DOPE) were studied.

Bicyclic nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneuridine and -cytidine, were incorporated into oligonucleotides, and the binding efficiency of the modified oligonucleotides to the complementary DNA and RNA as well as the CD spectra of the modified DNA-DNA and modified DNA-RNA duplexes were studied.

3-(4-Alkoxyphenyl)propanoic acid derivatives were prepared as candidate peroxisome proliferator-activated receptor (PPAR) alpha/delta/gamma pan agonists, based on our previous SAR studies directed toward the development of subtype-selective PPAR agonists. Those studies indicated that the steric bulkiness of substituents introduced at the distal benzene ring had an important influence on PPAR activity. The finding that a 4-adamantyl derivative exhibited not only PPARalpha/delta activity but also significant PPARgamma activity prompted us to search for structurally novel phenylpropanoic acid derivatives with more potent adipocyte differentiation activity than the well-known PPARgamma agonist, rosiglitazone, as well as well-balanced PPARalpha and PPARdelta agonistic activities. A representative phenylpropanoic acid derivative (12) bearing a 4-adamantylphenyl substituent proved to be a well-balanced PPAR-pan agonist with activities to regulate the expression of genes involved in lipid and glucose homeostasis, and should be useful as a candidate drug for the treatment of altered PPAR function.

A novel low Mr GTP-binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8-amino acid sequence specific for c25KG, a GTP-binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000–17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr of 25068. The protein is designated as ram (ras-related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP-binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H-ras, smg25A and c25KG proteins, respectively. The 3.5-kb ram mRNA was detected abundantly in spleen cells.

Megakaryocytes are the hematopoietic precursors of platelets, which play an essential role in thrombosis and hemostasis. Platelet factor 4 (PF4) is expressed exclusively in megakaryocytes and platelets and serves as a lineage-specific marker of megakaryocytic differentiation. We previously characterized a number of upstream enhancer and repressor elements and demonstrated that GATA-1 and ETS-1 are important for PF4 gene expression. Recently, we have determined the novel regulatory element termed "TME" in the PF4 promoter and identified a group of binding proteins from megakaryocytic HEL cells. Here we review the function of these proteins in PF4 gene expression and discuss megakaryocyte-specific gene expression and megakaryocytepoiesis.

The single-stranged region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNA Met f was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNA Met f , which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine.