Purine and pyrimidine receptors

Adenosine 5′-triphosphate (ATP), in addition to its intracellular roles, acts as an extracellular signalling molecule via a rich array of receptors, which have been cloned and characterised. P1 receptors are selective for adenosine, a breakdown product of ATP, produced after degradation by ectonucleotidases. Four subtypes have been identified, A₁, A_2A, A_2B and A₃ receptors. P2 receptors are activated by purines and some subtypes also by pyrimidines. P2X receptors are ligand-gated ion channel receptors and seven subunits have been identified, which form both homomultimers and heteromultimers. P2Y receptors are G protein-coupled receptors, and eight subtypes have been cloned and characterised to date. Received 22 November 2006; received after revision 11 January 2007; accepted 27 February 2007     

Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca²⁺ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca²⁺ concentrations. Contrarily, mutation of the homologous position in the Ca²⁺-activated Cl^? channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca²⁺-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.     

Tetraspanin TSPAN12 regulates tumor growth and metastasis and inhibits β-catenin degradation

Ablation of tetraspanin protein TSPAN12 from human MDA-MB-231 cells significantly decreased primary tumor xenograft growth, while increasing tumor apoptosis. Furthermore, TSPAN12 removal markedly enhanced tumor-endothelial interactions and increased metastasis to mouse lungs. TSPAN12 removal from human MDA-MB-231 cells also caused diminished association between FZD4 (a key canonical Wnt pathway receptor) and its co-receptor LRP5. The result likely explains substantially enhanced proteosomal degradation of β-catenin, a key effecter of canonical Wnt signaling. Consistent with disrupted canonical Wnt signaling, TSPAN12 ablation altered expression of LRP5, Naked 1 and 2, DVL2, DVL3, Axin 1, and GSKβ3 proteins. TSPAN12 ablation also altered expression of several genes regulated by β-catenin (e.g. CCNA1, CCNE2, WISP1, ID4, SFN, ME1) that may help to explain altered tumor growth and metastasis. In conclusion, these results provide the first evidence for TSPAN12 playing a role in supporting primary tumor growth and suppressing metastasis. TSPAN12 appears to function by stabilizing FZD4–LRP5 association, in support of canonical Wnt-pathway signaling, leading to enhanced β-catenin expression and function.     

Structure and evolution of vertebrate aldehyde oxidases: from gene duplication to gene suppression

Aldehyde oxidases (AOXs) and xanthine dehydrogenases (XDHs) belong to the family of molybdo-flavoenzymes. Although AOXs are not identifiable in fungi, these enzymes are represented in certain protists and the majority of plants and vertebrates. The physiological functions and substrates of AOXs are unknown. Nevertheless, AOXs are major drug metabolizing enzymes, oxidizing a wide range of aromatic aldehydes and heterocyclic compounds of medical/toxicological importance. Using genome sequencing data, we predict the structures of AOX genes and pseudogenes, reconstructing their evolution. Fishes are the most primitive organisms with an AOX gene (AOXα), originating from the duplication of an ancestral XDH. Further evolution of fishes resulted in the duplication of AOXα into AOXβ and successive pseudogenization of AOXα. AOXβ is maintained in amphibians and it is the likely precursors of reptilian, avian, and mammalian AOX1. Amphibian AOXγ is a duplication of AOXβ and the likely ancestor of reptilian and avian AOX2, which, in turn, gave rise to mammalian AOX3L1. Subsequent gene duplications generated the two mammalian genes, AOX3 and AOX4. The evolution of mammalian AOX genes is dominated by pseudogenization and deletion events. Our analysis is relevant from a structural point of view, as it provides information on the residues characterizing the three domains of each mammalian AOX isoenzyme. We cloned the cDNAs encoding the AOX proteins of guinea pig and cynomolgus monkeys, two unique species as to the evolution of this enzyme family. We identify chimeric RNAs from the human AOX3 and AOX3L1 pseudogenes with potential to encode a novel microRNA.     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B₃ to bovine serum albumin. Aflatoxin B₃ was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B₁ (AFB₁) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B₁, B₂, G₁, and G₂ when tritiated AFB₁ was used as the marker ligand. The concentrations causing 50% inhibition of binding of³H-AFB₁ to the antibodies by unlabeled aflatoxins B₁, B₂, G₁, G₂ and B₃ were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B₁ and G₁, two of the most important toxic metabolites produced byAspergillus flavus andA. parasiticus.     

ING1 and ING2: multifaceted tumor suppressor genes

Inhibitor of Growth 1 (ING1) was identified and characterized as a “candidate” tumor suppressor gene in 1996. Subsequently, four more genes, also characterized as “candidate” tumor suppressor genes, were identified by homology search: ING2, ING3, ING4, and ING5. The ING proteins are characterized by a high homology in their C-terminal domain, which contains a Nuclear Localization Sequence and a Plant HomeoDomain (PHD), which has a high affinity to Histone 3 tri-methylated on lysine 4 (H3K4Me3). The ING proteins have been involved in the control of cell growth, senescence, apoptosis, chromatin remodeling, and DNA repair. Within the ING family, ING1 and ING2 form a subgroup since they are evolutionarily and functionally close. In yeast, only one gene, Pho23, is related to ING1 and ING2 and possesses also a PHD. Recently, the ING1 and ING2 tumor suppressor status has been fully established since several studies have described the loss of ING1 and ING2 protein expression in human tumors and both ING1 and ING2 knockout mice were reported to have spontaneously developed tumors, B cell lymphomas, and soft tissue sarcomas, respectively. In this review, we will describe for the first time what is known about the ING1 and ING2 genes, proteins, their regulations in both human and mice, and their status in human tumors. Furthermore, we explore the current knowledge about identified functions involving ING1 and ING2 in tumor suppression pathways especially in the control of cell cycle and in genome stability.     

Interaction of DISC1 with the PTB domain of Tensin2

The gene for Disrupted-in-Schizophrenia 1 (DISC1) is amongst the most significant risk genes for schizophrenia. The DISC1 protein is an intracellular scaffolding molecule thought to act an important hub for protein interactions involved in signalling for neural cell differentiation and function. Tensin2 is an intracellular actin-binding protein that bridges the intracellular portion of transmembrane receptors to the cytoskeleton, thereby regulating signalling for cell shape and motility. In this study, we probed in molecular detail a novel interaction between DISC1 and Tensin2. Western blot and confocal microscopic analyses revealed widespread expression of both DISC1 and Tensin2 proteins throughout the mouse brain. Furthermore, we have developed novel anti-DISC1 antibodies that verified the predominant expression of a 105-kDa isoform of DISC1 in the rodent brain as well as in human cells. In the mouse brain, both proteins showed region-specific expression patterns, including strong expression in the pyramidal cell layer of the hippocampus and dentate gyrus. DISC1–Tensin2 colocalisation was most clearly observed in the Purkinje cells of the mouse cerebellum. Biochemical coimmunoprecipitation experiments revealed an interaction between endogenous DISC1 and Tensin2 proteins in the mouse brain. Further pulldown studies in human cells using Myc-tagged Tensin2 constructs revealed that DISC1 specifically interacts with the C-terminal PTB domain of Tensin2 in a phosphorylation-independent manner. This new knowledge on the DISC1–Tensin2 interaction, as part of the wider DISC1 interactome, should further elucidate the signalling pathways that are perturbed in schizophrenia and other mental disorders.     

Über den Vitamin-B12-Gehalt von Organen verschieden ernährter Ratten

Summary Adult, male rats bred for over 10 generations on a soy meal-corn-diet had vitamin B₁₂ values of liver and kidney about 10 times lower than the controls. If the deficient ration was supplemented with 5µg/kg of B₁₂, these values were still about 1/5 of the controls. 3 weeks on the deficient diet lowered the B₁₂ levels in the livers and kidneys of previously undepleted rats to about 1/2, and a similar diet containing 0.1% of iodized casein lowered these levels to about 1/3 of the normal values but did not lower the B₁₂-concentration of organs of already deficient rats.Rats bred on the deficient diet and receiving for 1 month a supplement of 30µg/kg of vitamin B₁₂ or the stock diet with a similar B₁₂-content, had normal B₁₂-levels in livers and kidneys.     

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 – 30 detectable by the 3F4 antibody against human PrP109 – 112. We recently identified a new PK-resistant PrP species, designated PrP^*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 – 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP^*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP^*20 but also a small amount of 3F4-detected PK-resistant PrP27 – 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP^*20 to the occurrence of the 3F4-detected PrP27 – 30 was observed. Our study suggests that an increase in the level of PrP^*20 characterizes the early stages of prion diseases. Received 17 October 2007; received after revision 5 December 2007; accepted 14 December 2007     

Biomedicine and diseases: the Klippel-Trenaunay syndrome, vascular anomalies and vascular morphogenesis

Vascular morphogenesis is a vital process for embryonic development, normal physiologic conditions (e.g. wound healing) and pathological processes (e.g. atherosclerosis, cancer). Genetic studies of vascular anomalies have led to identification of critical genes involved in vascular morphogenesis. A susceptibility gene, VG5Q (formally named AGGF1), was cloned for Klippel-Trenaunay syndrome (KTS). AGGF1 encodes a potent angiogenic factor, and KTS-associated mutations enhance angiogenic activity of AGGF1, defining ‘increased angiogenesis’ as one molecular mechanism for the pathogenesis of KTS. Similar studies have identified other genes involved in vascular anomalies as important genes for vascular morphogenesis, including TIE2, VEGFR-3, RASA1, KRIT1, MGC4607, PDCD10, glomulin, FOXC2, NEMO, SOX18, ENG, ACVRLK1, MADH4, NDP, TIMP3, Notch3, COL3A1 and PTEN. Future studies of vascular anomaly genes will provide insights into the molecular mechanisms for vascular morphogenesis, and may lead to the development of therapeutic strategies for treating these and other angiogenesis-related diseases, including coronary artery disease and cancer.Received 24 November 2004; received after revision 21 January 2005; accepted 2 March 2005     

Cisplatin-induced genes as potential markers for thyroid cancer

Despite the uncontested role of p53 in cycle arrest/cell death after cisplatin treatment, to date the question whether wild-type p53 confers a resistant or sensitive status on the cell is still a matter of debate. Isogenic and isophenotypic human thyroid papillary carcinoma cell line variants for p53 differently expressed cycle genes after cisplatin treatment. Seven genes (CDC6-related protein, CCNC, GAS1, TFDP2, MAPK10/JNK3, WEE1, RPA1) selected after expression on an Atlas human cell cycle array were analyzed by quantitative real-time PCR. While cisplatin treatment increased their expression in p53 wild-type cells it decreased it in cells with inactivated p53 and had no or less effect on cells with mutated p53. These results show that in a well-defined system, different alterations of p53 can lead to a different regulation of genes and hence to either resistance or sensitivity to cisplatin. Moreover for the first time, MAPK10/JNK3 was identified in human thyroid cells and tissue. Four of the genes (CDC6-related protein, CCNC, GAS1 and TFDP2) were decreased in human papillary carcinoma tissues. Relevance of these genes (especially a decrease in GAS1 in thyroid papillary carcinoma) in various malignant pathologies has already been shown. These genes may be explored as new markers in advanced thyroid cancer such as metastatic and anaplastic forms displaying p53 alterations.Received 26 July 2004; received after revision 14 September 2004; accepted 26 October 2004     

Ca<Superscript>2+</Superscript> signals,cell membrane disintegration,and activation of TMEM16F during necroptosis

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca²⁺. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT₂₉, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca²⁺, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca²⁺, which was paralleled by the activation of outwardly rectifying Cl^? currents, which were typical for TMEM16F/ANO6. Ca²⁺ oscillations were due to Ca²⁺ release from endoplasmic reticulum, and were independent of extracellular Ca²⁺. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl^? currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.     

Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.     

Defining G protein-coupled receptor peptide ligand expressomes and signalomes in human and mouse islets

Introduction

Islets synthesise and secrete numerous peptides, some of which are known to be important regulators of islet function and glucose homeostasis. In this study, we quantified mRNAs encoding all peptide ligands of islet G protein-coupled receptors (GPCRs) in isolated human and mouse islets and carried out in vitro islet hormone secretion studies to provide functional confirmation for the species-specific role of peptide YY (PYY) in mouse islets.

Materials and methods

GPCR peptide ligand mRNAs in human and mouse islets were quantified by quantitative real-time PCR relative to the reference genes ACTB, GAPDH, PPIA, TBP and TFRC. The pathways connecting GPCR peptide ligands with their receptors were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases. Distribution of PYY protein in mouse and human islets was determined by immunohistochemistry. Insulin, glucagon and somatostatin secretion from islets was measured by radioimmunoassay.

Results

We have quantified GPCR peptide ligand mRNA expression in human and mouse islets and created specific signalomes mapping the pathways by which islet peptide ligands regulate human and mouse GPCR signalling. We also identified species-specific islet expression of several GPCR ligands. In particular, PYY mRNA levels were ~ 40,000-fold higher in mouse than human islets, suggesting a more important role of locally secreted Pyy in mouse islets. This was confirmed by IHC and functional experiments measuring insulin, glucagon and somatostatin secretion.

Discussion

The detailed human and mouse islet GPCR peptide ligand atlases will allow accurate translation of mouse islet functional studies for the identification of GPCR/peptide signalling pathways relevant for human physiology, which may lead to novel treatment modalities of diabetes and metabolic disease.

     

Combinatorial activity of Six1-2-4 genes in cephalic neural crest cells controls craniofacial and brain development

The combinatorial expression of Hox genes is an evolutionarily ancient program underlying body axis patterning in all Bilateria. In the head, the neural crest (NC)––a vertebrate innovation that contributes to evolutionarily novel skeletal and neural features––develops as a structure free of Hox-gene expression. The activation of Hoxa2 in the Hox-free facial NC (FNC) leads to severe craniofacial and brain defects. Here, we show that this condition unveils the requirement of three Six genes, Six1, Six2, and Six4, for brain development and morphogenesis of the maxillo-mandibular and nasofrontal skeleton. Inactivation of each of these Six genes in FNC generates diverse brain defects, ranging from plexus agenesis to mild or severe holoprosencephaly, and entails facial hypoplasia or truncation of the craniofacial skeleton. The triple silencing of these genes reveals their complementary role in face and brain morphogenesis. Furthermore, we show that the perturbation of the intrinsic genetic FNC program, by either Hoxa2 expression or Six gene inactivation, affects Bmp signaling through the downregulation of Bmp antagonists in the FNC cells. When upregulated in the FNC, Bmp antagonists suppress the adverse skeletal and cerebral effects of Hoxa2 expression. These results demonstrate that the combinatorial expression of Six1, Six2, and Six4 is required for the molecular programs governing craniofacial and cerebral development. These genes are crucial for the signaling system of FNC origin, which regulates normal growth and patterning of the cephalic neuroepithelium. Our results strongly suggest that several congenital craniofacial and cerebral malformations could be attributed to Six genes’ misregulation.     

Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rarα, and rarβ) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgfβ2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rarβ, meis2, hoxb5, tgfβ2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgfβ2, and id2 spatial distribution; to increase rarβ, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal–distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.     

The structures of minor congeners of detoxin complex,the selective antagonist of blasticidin S1

Summary The structures of the minor congeners of detoxin complex, viz., detoxins E₁, C₁, C₂, C₃, B₁, B₃ and A₁ have been established on the basis of spectral and degradative evidence.Acknowledgment. This work was supported by a Grant-in-Aid for Special Project Research (510208) from the Ministry of Education, Science and Culture, Japan. We are grateful to Kaken Chemical Co. Ltd for the supply of the detoxin complex. This is Part V of studies of Detoxin Complex, the Selective Antagonists of Blasticidin S'. For Part IV, see Ogita et al.⁸.     

Neural stem cells in Parkinson’s disease: a role for neurogenesis defects in onset and progression

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, leading to a variety of motor and non-motor symptoms. Interestingly, non-motor symptoms often appear a decade or more before the first signs of motor symptoms. Some of these non-motor symptoms are remarkably similar to those observed in cases of impaired neurogenesis and several PD-related genes have been shown to play a role in embryonic or adult neurogenesis. Indeed, animal models deficient in Nurr1, Pitx3, SNCA and PINK1 display deregulated embryonic neurogenesis and LRRK2 and VPS35 have been implicated in neuronal development-related processes such as Wnt/β-catenin signaling and neurite outgrowth. Moreover, adult neurogenesis is affected in both PD patients and PD animal models and is regulated by dopamine and dopaminergic (DA) receptors, by chronic neuroinflammation, such as that observed in PD, and by differential expression of wild-type or mutant forms of PD-related genes. Indeed, an increasing number of in vivo studies demonstrate a role for SNCA and LRRK2 in adult neurogenesis and in the generation and maintenance of DA neurons. Finally, the roles of PD-related genes, SNCA, LRRK2, VPS35, Parkin, PINK1 and DJ-1 have been studied in NSCs, progenitor cells and induced pluripotent stem cells, demonstrating a role for some of these genes in stem/progenitor cell proliferation and maintenance. Together, these studies strongly suggest a link between deregulated neurogenesis and the onset and progression of PD and present strong evidence that, in addition to a neurodegenerative disorder, PD can also be regarded as a developmental disorder.