Active site rearrangement and structural divergence in prokaryotic respiratory oxidases.

Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the Escherichia coli cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site.

Activation of RHOA-VAV1 signaling in angioimmunoblastic T-cell lymphoma.

Somatic G17V RHOA mutations were found in 50-70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high-throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1-STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), whereas none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in VAV1-STAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1-STAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA-VAV1 axis may provide a new therapeutic target in AITL.

Integrating genomic alterations in diffuse large B-cell lymphoma identifies new relevant pathways and potential therapeutic targets.

Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

Identification of cell-type-specific mutations in nodal T-cell lymphomas.

Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations.

Tissue-specific expression and silencing phenotypes of mitochondrial phosphate carrier paralogues in several insect species.

The mitochondrial phosphate carrier gene (PiC) encodes a membrane protein that mediates the supply of inorganic phosphate from the cytosol into the mitochondrial matrix. This substrate-specific transport system plays an important role in efficient ATP synthesis. Mammals appear to have only one PiC with two alternative splicing variants whose functional differences remain unclear. The present study is the first to characterize the multiple genes that encode PiC in insects. Bombyx mori was found to have two PiC paralogues, one ubiquitous and one testis-specific, the latter seeming to be present only in Lepidoptera. Drosophila melanogaster was found to harbour two PiC paralogues, whereas Liriomyza chinensis, another dipteran, has three PiC paralogues. Two PiCs were found to be present in Plautia stali, and silencing either of these genes affected the normal development of P. stali nymphs, although their expression patterns differed amongst tissues. Schistocerca gregaria and Locusta migratoria have two PiC each, with different expression patterns. Tribolium castaneum was found to have only one PiC, which appears to play an essential role in larval development. Thus, although the inorganic phosphate transport system appears to be conserved across eukaryotes, PiC has become specialized in the different tissues of different insect species.

Impact of renal transplantation on glucose tolerance in Japanese recipients with impaired glucose tolerance.

AIMS: To investigate changes in glucose tolerance, insulin secretion and insulin sensitivity in Japanese recipients before and 1 year after renal transplantation. METHODS: We conducted a study of Japanese recipients without diabetes who underwent renal transplantation at Hokkaido University Hospital. A 75-g oral glucose tolerance test was performed before and 1 year after renal transplantation in these recipients. Insulin sensitivity was estimated using the Matsuda index and homeostasis model assessment of insulin resistance (HOMA-IR). Insulin secretion was evaluated based on the insulin secretion sensitivity index-2 (ISSI-2). RESULTS: Of the 62 renal transplant recipients, 31 were diagnosed as having impaired glucose tolerance before transplantation. Among these 31 recipients, after 1 year, four had developed new-onset diabetes after transplantation, and nine had impaired glucose tolerance. Unexpectedly, 18 changed from impaired to normal glucose tolerance. When these recipients with impaired glucose tolerance were classified into a non-amelioration group and an amelioration group, the ISSI-2 was significantly reduced, with no significant changes in the Matsuda index or HOMA-IR, in the non-amelioration group 1 year after renal transplantation. By contrast, ISSI-2 and Matsuda index values were significantly increased, with no significant changes in HOMA-IR values in the amelioration group. CONCLUSIONS: More than half of Japanese renal transplant recipients with impaired glucose tolerance had normal glucose tolerance 1 year after renal transplantation. These results suggest that an increase in insulin secretion and whole insulin sensitivity was associated with improvement in glucose tolerance in these recipients.

Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis.

There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining immunoglobulin A (IgA) transcytosis across Transwells. IgA transcytosis required induction of polymeric Ig receptor (pIgR) expression, which could be stimulated by a combination of lipopolysaccharide and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that tumor necrosis factor-α, interleukin (IL)-1ß, IL-17, and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that among these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. Interferon-γ, however, did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology.

Epalrestat improves diabetic wound healing via increased expression of nerve growth factor.

AIMS/INTRODUCTION: Aldose reductase inhibitors (ARIs) are a useful therapy for diabetic neuropathy. Nerve damage is associated with delayed wound healing of skin ulcers in diabetic patients. Therefore, we hypothesized that ARI supplementation would improve diabetic wound healing. MATERIALS AND METHODS: Control and streptozotocin-induced diabetic mice were fed either control diet or diet containing the ARI Epalrestat (40 mg/kg). After 12 weeks, we created skin wounds on the backs of the mice. Wound healing was determined by measuring the reduction in wound area. RESULTS: The wound gap of the diabetic group was significantly larger 9 days after creating the wounds when compared to the other groups (p<0.01). Interestingly, wound healing in the diabetic mice fed Epalrestat was comparable to the non-diabetic mice. To clarify the mechanism(s) behind this improved wound healing, mRNA expression of growth factors reported to be involved in wound healing were examined. Among the growth factors investigated, only the expression of nerve growth factor (NGF) was -significantly decreased (54.0%) in the healing lesions of diabetic mice. Similarly, NGF protein expression was decreased in diabetic mice and recovered in Epalrestat treated diabetic mice. Inhibition of NGF via 2 separate inhibitors (K252a and BSO) reduced the ability of Epalrestat to improve wound healing in diabetic mice. CONCLUSIONS: These findings suggest that Epalrestat is a potential therapy for improving diabetic wound healing and the mechanism involves upregulation of NGF.

[Sarcomatoid carcinoma of the thymus which caused acute epicarditis].

Thymic carcinoma is rare. Particularly sarcomatoid carcinoma of the thymus is a very rare disease it has been reported in only 15 patients to date. The prognosis is very poor and diagnosis and treatment have not yet been established. We report a case of 63-year-old man who was initially diagnosed with acute pericarditis and was finally found to be sarcomatoid carcinoma of the thymus. He underwent surgery and the tumor was completely resected. However, 6 months after surgery, local recurrence was noted. The patient was treated by radiotherapy followed by paclitaxel monotherapy. Partial remission was achieved transiently with paclitaxel, but the tumor again recurred. He died 33 months after surgery. The possibility of diseases like this tumor must be kept in mind for a patient with chest symptoms. Paclitaxel monotherapy is likely to be effective in treating sarcomatoid carcinoma of the thymus.

Transcriptional induction of MMP-10 by TGF-beta, mediated by activation of MEF2A and downregulation of class IIa HDACs.

Transforming growth factor (TGF)-beta regulates the expression of matrix metalloproteinases (MMPs) and components of the extracellular matrix, thereby profoundly affecting the microenvironment of cells including cancerous ones. We studied MMP-10 induction by TGF-beta in mammary epithelial cells and found that the induction was dependent on the myocyte enhancer factor (MEF)-2 transcription factor. TGF-beta upregulated the gene promoter through the MEF2 site, and knockdown of the MEF2A transcription factor negatively affected MMP-10 induction, whereas its overexpression had a positive effect on the induction. In response to TGF-beta, acetylation and concomitant binding of MEF2A to the promoter region increased, thus suggesting a critical role of MEF2A in transactivation of MMP-10 by TGF-beta. Consistent with the fact that class IIa histone deacetylases (HDACs) interact with MEF2 and suppress transcription, knockdown of HDACs increased and their overexpression inhibited MMP-10 expression. Intriguingly, TGF-beta promoted proteasome-dependent degradation of HDACs. Consistent with this, acetylation of core histones was increased around the MEF2 site of the MMP-10 promoter by TGF-beta and alleviated by overexpression of HDACs. Collectively, it is possible that TGF-beta transcriptionally upregulated MMP-10 through activation of MEF2A, concomitant with acetylation of core histones increasing around the promoter, as a consequence of degradation of the class IIa HDACs.

Chromosomal instability by beta-catenin/TCF transcription in APC or beta-catenin mutant cells.

Adenomatous polyposis coli (APC/Apc) gene encodes a key tumor suppressor whose mutations activate beta-catenin/T-cell factor (TCF)-mediated transcription (canonical Wnt signaling). Here, we show that Wnt signaling can cause chromosomal instability (CIN). As an indicator of CIN, we scored anaphase bridge index (ABI) in mouse polyps and ES cells where Wnt signaling was activated by Apc or beta-catenin mutations. We found three to nine times higher ABI than in wild-type controls. Furthermore, karyotype analysis confirmed that the Wnt signal-activated ES cells produced new chromosomal aberrations at higher rates; hence CIN. Consistently, expression of dominant-negative TCFs in these cells reduced their ABI. We also found that Wnt signal activation increased phosphorylation of Cdc2 (Cdk1) that inhibited its activity, and suppressed apoptosis upon exposure of the cells to nocodazole or colcemid. The data suggest that Wnt signaling stimulates the cells to escape from mitotic arrest and apoptosis, resulting in CIN. In human gastric cancer tissues with nuclear beta-catenin, ABI was significantly higher than in those without. These results collectively indicate that beta-catenin/TCF-mediated transcription itself increases CIN through dysregulation of G2/M progression.

MRI appearance of primary giant ovarian leiomyoma in a hysterectomised woman.

Primary leiomyoma is a rare, benign tumour of the ovary. We describe the MRI features of an ovarian leiomyoma identified in a 51-year-old woman after hysterectomy. The tumour appeared as a well-circumscribed low signal intensity mass on T(1) weighted imaging, with mixed signal intensity on T2 weighted imaging. Areas of high signal intensity on T2 weighted imaging corresponded to degeneration of leiomyoma. Dynamic contrast-enhanced imaging revealed early enhancement of the lesion. MRI appearance was thus similar to that of uterine leiomyoma. This case suggests the potential usefulness of dynamic contrast-enhanced MRI for the diagnosis of ovarian leiomyoma.

Accelerated onsets of gastric hamartomas and hepatic adenomas/carcinomas in Lkb1+/-p53-/- compound mutant mice.

Germline mutations in the LKB1 gene are responsible for Peutz-Jeghers syndrome (PJS), which is characterized by gastrointestinal hamartomas and increasing risk of cancer. Mice with Lkb1(+/-) mutation develop gastric hamartomas after >20 weeks of age, and hepatocellular adenomas and carcinomas >30 weeks. It has been reported that, in PJS patients, carcinomas progressed from hamartomas contain p53 mutations, and that LKB1 regulates p53-dependent apoptosis. To investigate the roles of LKB1 and p53 mutations in tumorigenesis, we constructed compound mutant mice of Lkb1 and p53 genes. In the Lkb1(+/-)p53(-/-) mice, formation of gastric hamartomas and hepatic tumors was accelerated. However, histopathology of hamartomas was similar between Lkb1(+/-)p53(-/-) and Lkb1(+/-) mice, and Lkb1 genotype remained heterozygous, suggesting that the p53 mutation affected hamartoma initiation. Contrary to the heterozygous hamartomas in the stomach and duodenum, the hepatic adenomas in Lkb1(+/-)p53(-/-) mice showed loss of Lkb1 heterozygosity (LOH), suggesting that lack of p53 stimulated Lkb1 LOH and tumor initiation in the liver. Taken together, these results indicate that lack of p53 causes earlier onsets of gastric hamartomas and hepatic tumors in Lkb1(+/-)p53(-/-) mice.

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

Simultaneous expression of COX-2 and mPGES-1 in mouse gastrointestinal hamartomas.

Cyclo-oxygenase (COX)-2 is induced in various types of cancer tissues. Here, we demonstrate stromal expression of both COX-2 and microsomal prostaglandin E(2) synthase (mPGES)-1 in gastrointestinal hamartomas developed in Lkb1(+/-), Smad4(+/-) and Cdx2(+/-)mice. These results suggest that PGE(2) produced by COX-2 and mPGES-1 plays an important role in hamartoma development regardless of the mutated genes causing hamartomas.