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.

[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.

Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors.

Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable, long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation.

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.

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.

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.

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.

Hormonal control of substrate cycling in humans.

Recent studies have established the existence of substrate cycles in humans, but factors regulating the rate of cycling have not been identified. We have therefore investigated the acute response of glucose/glucose-6P-glucose (glucose) and triglyceride/fatty acid (TG/FA) substrate cycling to the infusion of epinephrine (0.03 microgram/kg.min) and glucagon. The response to a high dose glucagon infusion (2 micrograms/kg.min) was tested, as well as the response to a low dose infusion (5 ng/kg.min), with and without the simultaneous infusion of somatostatin (0.1 microgram/kg.min) and insulin (0.1 mU/kg.min). Additionally, the response to chronic prednisone (50 mg/d) was evaluated, both alone and during glucagon (low dose) and epinephrine infusion. Finally, the response to hyperglycemia, with insulin and glucagon held constant by somatostatin infusion and constant replacement of glucagon and insulin at basal rates, was investigated. Glucose cycling was calculated as the difference between the rate of appearance (Ra) of glucose as determined using 2-d1- and 6,6-d2-glucose as tracers. TG/FA cycling was calculated by first determining the Ra glycerol with d5-glycerol and the Ra FFA with [1-13C]palmitate, then subtracting Ra FFA from three times Ra glycerol. The results indicate that glucagon stimulates glucose cycling, and this stimulatory effect is augmented when the insulin response to glucagon infusion is blocked. Glucagon had minimal effect on TG/FA cycling. In contrast, epinephrine stimulated TG/FA cycling, but affected glucose cycling minimally. Prednisone had no direct effect on either glucose or TG/FA cycling, but blunted the stimulatory effect of glucagon on glucose cycling. Hyperglycemia, per se, had no direct effect on glucose or TG/FA cycling. Calculations revealed that stimulation of TG/FA cycling theoretically amplified the sensitivity of control of fatty acid flux, but no such amplification was evident as a result of the stimulation of glucose cycling by glucagon.

Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas.

Cholestatic liver injury appears to result from the induction of hepatocyte apoptosis by toxic bile salts such as glycochenodeoxycholate (GCDC). Previous studies from this laboratory indicate that cathepsin B is a downstream effector protease during the hepatocyte apoptotic process. Because caspases can initiate apoptosis, the present studies were undertaken to determine the role of caspases in cathepsin B activation. Immunoblotting of GCDC-treated McNtcp.24 hepatoma cells demonstrated cleavage of poly(ADP-ribose) polymerase and lamin B1 to fragments that indicate activation of effector caspases. Transfection with CrmA, an inhibitor of caspase 8, prevented GCDC-induced cathepsin B activation and apoptosis. Consistent with these results, an increase in caspase 8-like activity was observed in GCDC-treated cells. Examination of the mechanism of GCDC-induced caspase 8 activation revealed that dominant-negative FADD inhibited apoptosis and that hepatocytes isolated from Fas-deficient lymphoproliferative mice were resistant to GCDC-induced apoptosis. After GCDC treatment, immunoprecipitation experiments demonstrated Fas oligomerization, and confocal microscopy demonstrated DeltaFADD-GFP (Fas-associated death domain-green fluorescent protein, aggregation in the absence of detectable Fas ligand mRNA. Collectively, these data suggest that GCDC-induced hepatocyte apoptosis involves ligand-independent oligomerization of Fas, recruitment of FADD, activation of caspase 8, and subsequent activation of effector proteases, including downstream caspases and cathepsin B.

Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c.

TNF-alpha-induced apoptosis is thought to involve mediators from acidic vesicles. Cathepsin B (cat B), a lysosomal cysteine protease, has recently been implicated in apoptosis. To determine whether cat B contributes to TNF-alpha-induced apoptosis, we exposed mouse hepatocytes to the cytokine in vitro and in vivo. Isolated hepatocytes treated with TNF-alpha in the presence of the transcription inhibitor actinomycin D (AcD) accumulated cat B in their cytosol. Further experiments using cell-free systems indicated that caspase-8 caused release of active cat B from purified lysosomes and that cat B, in turn, increased cytosol-induced release of cytochrome c from mitochondria. Consistent with these observations, the ability of TNF-alpha/AcD to induce mitochondrial release of cytochrome c, caspase activation, and apoptosis of isolated hepatocytes was markedly diminished in cells from CatB(-/-) mice. Deletion of the CatB gene resulted in diminished liver injury and enhanced survival after treatment in vivo with TNF-alpha and an adenovirus construct expressing the IkappaB superrepressor. Collectively, these observations suggest that caspase-mediated release of cat B from lysosomes enhances mitochondrial release of cytochrome c and subsequent caspase activation in TNF-alpha-treated hepatocytes.

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.

Consistent disruption of the AML1 gene occurs within a single intron in the t(8;21) chromosomal translocation.

The AML1 gene on chromosome 21 was rearranged by the t(8;21) chromosomal translocation in acute myeloid leukemia (AML). Southern blot analysis of 21 AML patients with t(8;21), including three with complex translocations, t(8;V;21), demonstrated that all the breakpoints occurred at random within a single intron between two coding exons of AML1. Clustering of the breakpoints in the restricted intron suggests the formation of a unique fusion gene between the AML1 gene and a presumable counterpart gene on chromosome 8. Nucleotide sequencing of the breakpoint region revealed that the translocation event was accompanied by deletion of a short stretch of nucleotides.

Gastric and duodenal polyps in Smad4 (Dpc4) knockout mice.

The SMAD4 (DPC4) gene was initially isolated as a candidate tumor suppressor from the convergent site of homozygous deletions on 18q in a panel of pancreatic carcinoma cell lines. It encodes a common cytoplasmic signaling molecule shared by the transforming growth factor-beta, activin, and bone morphogenic pathways. We recently inactivated its mouse homologue Smad4 and demonstrated its role in the malignant progression of benign adenomas to invasive adenocarcinomas by analyzing mice with Apc and Smad4 compound mutations. Although simple Smad4 homozygotes were embryonically lethal, the heterozygotes were fertile and appeared normal up to the age of 1 year. Upon further investigation, however, they have developed inflammatory polyps in the glandular stomach and duodenum. By PCR genotyping and immunohistochemical staining, the wild-type Smad4 allele has been lost in the polyp epithelial cells, ie., loss of heterozygosity. On the other hand, we have not found any mutations in such genes as K-Ras, H-Ras, N-Ras, p53, or PTEN. Histologically, the polyps are similar to human juvenile polyps showing moderate stromal cell proliferation and infiltrations by eosinophils and plasma cells. In addition, foci of adenocarcinoma with signet ring cells are also found. These results are consistent with a recent report that germ-line SMAD4 mutations are found in a subset of familial juvenile polyposis.

Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis.

The mechanism of hepatocarcinogenesis in hepatitis C virus (HCV) infection is still undefined. One possibility is the involvement of oxidative stress, which can produce genetic mutations as well as gross chromosomal alterations and contribute to cancer development. We recently showed that after a long period, the core protein of HCV induces hepatocellular carcinoma (HCC) in transgenic mice with marked hepatic steatosis but without inflammation, indicating a direct involvement of HCV in hepatocarcinogenesis. To elucidate the biochemical events before the development of HCC, we examined several parameters of oxidative stress and redox homeostasis in a mouse model of HCV-associated HCC. For young mice ages 3-12 months, there was no significant difference in the levels of hydroperoxides of phosphatidylcholine (PCOOH) and phosphatidylethanolamine in liver tissue homogenates between transgenic and nontransgenic control mice. In contrast, the PCOOH level was increased by 180% in old core gene transgenic mice > 16 months old. Concurrently, there was a significant increase in the catalase activity, and there were decreases in the levels of total and reduced glutathione in the same mice. A direct in situ determination by chemiluminescence revealed an increase in hydroperoxide products by 170% even in young transgenic mice, suggesting that hydroperoxides were overproduced but immediately removed by an activated scavenger system in young mice. Electron microscopy revealed lipofuscin granules, secondary lysosomes carrying various cytoplasmic organelles, and disruption of the double membrane structure of mitochondria, and PCR analysis disclosed a deletion in mitochondrial DNA. Interestingly, alcohol caused a marked increase in the PCOOH level in transgenic mice, suggesting synergism between alcohol and HCV in hepatocarcinogenesis. The HCV core protein thus alters the oxidant/antioxidant state in the liver in the absence of inflammation and may thereby contribute to or facilitate, at least in part, the development of HCC in HCV infection.

Transcription of the RelB gene is regulated by NF-kappaB.

RelA and RelB are two members of the NF-kappaB family that differ structurally and functionally. While RelA is regulated through its cytosolic localization by inhibitor proteins or IkappaB and not through transcriptional mechanisms, the regulation of RelB is poorly understood. In this study we demonstrate that stimuli (TNF or LPS) lead within minutes to the nuclear translocation of RelA, but require hours to result in the nuclear translocation of RelB. The delayed nuclear translocation of RelB correlates with increases in its protein synthesis which are secondary to increases in RelB gene transcription. RelA is alone sufficient to induce RelB gene transcription and to mediate the stimuli-driven increase in RelB transcription. Cloning and characterization of the RelB 5' untranslated gene region indicates that RelB transcription is dependent on a TATA-less promoter containing two NF-kappaB binding sites. One of the NF-kappaB sites is primarily involved in the binding of p50 while the other one in the binding and transactivation by RelA and also RelB. Lastly, it is observed that p21, a protein involved in cell cycle control and oncogenesis known to be regulated by NF-kappaB, is upregulated at the transcriptional level by RelB. Thus, RelB is regulated at least at the level of transcription in a RelA and RelB dependent manner and may exert an important role in p21 regulation.

Structure-activity relationships of some complex I inhibitors.

A wide variety of complex I inhibitors act at or close to the ubiquinone reduction site. Identification of the structural factors required for exhibiting inhibitory actions on the basis of structure-activity relationships is useful to elucidate the manner in which inhibitors interact with the enzyme. This review summarizes studies on the structure-activity relationship of rotenoids, piericidins, capsaicins, pyridinium-type inhibitors and modern synthetic agrochemicals acting at mitochondrial complex I.

Quantitative analyses of the uncoupling activity of substituted phenols with mitochondria from flight muscles of house flies.

Uncoupling activity with flight-muscle mitochondria from house flies was measured for a series of weakly acidic uncouplers (substituted phenols) and compared with the protonophoric potency across lecithin liposomal membranes. The activity was linearly related to the protonophoric potency when such factors as the stability of anionic species in the membrane phase and the difference in the pH conditions of the extramembranous aqueous phase were taken into account. Relationships of the flight-muscle activity with activities measured previously with rat-liver mitochondria and spinach chloroplasts were linear. Our findings were further evidence for the shuttle-type mechanism of the uncoupling action of weakly acidic uncouplers.