Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part I)

Summary.  Down syndrome (DS) is the most significant genetic disorder with mental retardation and is caused by trisomy 21. The phenotype of DS is thought to result from overexpression of a gene(s) located on the triplicated chromosome (region). An increasing body of evidence that challenge this “gene dosage effect” hypothesis, however, has been reported indicating that this hypothesis still remains to be elucidated. The availability of the complete sequence of genes on chromosome 21 could have an immediate impact on DS research, but no conclusions can be drawn from nucleic acid levels. This made us evaluate protein levels of six proteins, gene products, encoded on chromosome 21 (T-cell lymphoma invasion and metastasis inducing Tiam1 protein, holocarboxylase synthetase, human interferon-regulated resistance GTP-binding protein MxA, Pbx regulating protein 1, autoimmune regulator, and pericentrin) in fetal cortex from DS and controls at 18–19 weeks of gestational age using Western blot technique. None of the investigated proteins showed overexpression in DS compared to controls. Our present data showing unaltered expression of six proteins on chromosome 21 in fetal DS brain suggest that the existence of the trisomic state is not involved in abnormal development of fetal DS brain and that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are in the process of quantifying all gene products of chromosome 21 and our first results do not support the gene dosage hypothesis. Received June 27, 2002 Accepted July 19, 2002 Published online November 14, 2002 Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: AIRE, autoimmune regulator; DS, Down syndrome; HCS, holocarboxylase synthetase; Prep1, Pbx regulating protein 1; Tiam1, T-cell lymphoma invasion and metastasis 1     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part III)

Summary.  Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the gene dosage effect hypothesis has been proposed to explain the impact of extra chromosome 21 on the pathology of DS, a series of evidence that challenge this hypothesis has been reported. The availability of the complete sequences of genes on chromosome 21 serves now as starting point to find functional information of the gene products, but information on gene products is limited so far. We therefore evaluated expression levels of six proteins whose genes are encoded on chromosome 21 (synaptojanin-1, chromosome 21 open reading frame 2, oligomycin sensitivity confering protein, peptide 19, cystatin B and adenosine deaminase RNA-specific 2) in fetal cerebral cortex from DS and controls at 18–19 weeks of gestational age using Western blot analysis. Synaptojanin-1 and C21orf2 were increased in DS, but others were comparable between DS and controls, suggesting that the DS phenotype cannot be simply explained by gene dosage effects. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. These studies are of significance as they show for the first time protein levels that are carrying out specific function in human fetal brain with DS. Received August 12, 2002 Accepted September 12, 2002 Published online January 30, 2003 Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK) Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: ADAR2, adenosine deaminase RNA-specific 2; C21orf2, chromosome 21 open reading frame 2; DS, Down syndrome; NSE, neuron specific enolase; OSCP, oligomycin sensitivity conferring protein; PEP-19, peptide 19     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part II)

Summary.  Down syndrome (DS) is the most common genetic cause of mental retardation. To explain the impact of extra chromosome 21 in the pathology of DS, gene dosage effect hypothesis has been proposed, but several investigators including our group have challenged this hypothesis. Although analysis of the sequence of chromosome 21 has been essentially completed, the molecular and biochemical mechanisms underlying the pathology are still unknown. We therefore investigated expression levels of six proteins encoded on chromosome 21 (HACS1, DYRK1A, αA-crystallin, FTCD, GARS-AIRS-GART, and CBS) in fetal cerebral cortex from DS and controls at 18–19 weeks of gestational age using Western blot analysis. Protein expression of HACS1 was significantly and remarkably decreased in DS, and the expression levels of five proteins were comparable between DS and controls suggesting that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are continuing to quantify proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. Received July 1, 2002 Accepted July 19, 2002 Published online November 14, 2002 Acknowledgement This work was supported, in part (Dr. D. Patterson), by the National Institute of Child Health and Human Development (NICHD; HD17449). Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: DS, Down syndrome; HACS1, hematopoietic adapter containing Src homology 3 domain and sterile α motifs; DYRK1A, dual specificity tyrosine phosphorylated and regulated kinase; αA-crystallin, alpha crystallin subunit A; FTCD, formi-minotransferase cyclodeaminase; GARS-AIRS-GART, glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase; CBS, cystathionine β-synthase; NSE, neuron specific enolase; GFAP, glial fibrillary acidic protein     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: Challenging the gene dosage effect hypothesis (Part IV)

Summary.  Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the molecular mechanisms of the various phenotypes of DS could be due to overexpression of gene(s) on chromosome 21, several groups have challenged this gene dosage effect hypothesis. The near completion of the sequencing of human chromosome 21 provides unprecedented opportunities to understand the molecular pathology of DS, however, functional information on gene products is limited so far. We therefore evaluated the levels of six proteins whose genes are encoded on chromosome 21 (trefoil factor 1, trefoil factor 2, trefoil factor 3, coxsackie virus and adenovirus receptor, carbonyl reductase 1 and interferon-α receptor) in fetal cerebral cortex from DS and controls at the early second trimester using Western blot analysis. None of the investigated proteins showed overexpression in DS compared to controls suggesting that these proteins are not involved in abnormal development of fetal DS brain and that DS phenotype can not be simply explained by the gene dosage effect hypothesis. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 and these studies may provide a better understanding of genotype-phenotype correlation in DS. Received November 28, 2002 Accepted March 10, 2003 Acknowledgements's of Hospital of Philadelphia, PA, (USA) and Biogen, Inc. (anti-IFNAR-1 antibody; Cambridge, USA) for kindly providing the antibodies and comments. Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: AD, Alzheimer's disease; CAR, coxsackievirus and adenovirus receptor; CBR1, carbonyl reductase 1; CNS, central nervous system; DS, Down syndrome; IFNs, interferons; IFNAR-1, interferon-α receptor; NSE, neuron specific enolase; TFF, trefoil factor     

Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures1

Diadenosine 5′,5′”-P¹,P⁴-tetraphosphate (Ap₄A) cleaving enzymes are assumed to regulate intracellular levels of Ap₄A, a compound known to affect cell proliferation and stress responses. From plants an Ap₄A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap₄A hydrolase in 4-day-old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein-5-isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap₄A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6-diamidino-2-phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap₄A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap₄A in triggering DNA synthesis and cell proliferation.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Modeling of α(1–4) chain arrangements in α(1–4)(1–6) glucans: The action and outcome of β-amylase and Pseudomonas stutzeri amylase on an α(1–4)(1–6) glucan model

Simulated enzymic debranching of a β-limit dextrin model, prepared from a computed construct made by random extension and branching, and given the CCL value of w-maize amylopectin (and equal amounts of external chains with ECL values of 2 and 3) has been related to experimental chromatograms of the debranched β-limit dextrin of the amylopectin. The profile was similar to those from gel chromatograms and IEC-PAD chromatography.The equivalent lengths in glucosyl units of grid-links (g-links) of internal and external chains in constructs were calculated from the ICL and ECL values of amylopectin and models produced from the constructs with the appropriate lengths for internal and external chains. These derived models were subjected to simulated hydrolysis by Pseudomonas stutzeri amylase and the products compared with those of the experimental distribution from w-maize amylopectin. With the model the amounts of maltotetraose and maltodextrins released were similar to the experimental values but the distribution of branched maltodextrins was quite different. Unlike w-maize amylopectin – a polymer with the cluster structure – which has given a profile of molecular sizes of maltodextrins with low amounts of single and small numbers of internal chains and with a peak at a M_W of about 14,000 (13 chains), in the model the proportion of maltodextrin with one internal chain was high and as d.p. increased the amounts decreased exponentially. This would be expected if the distribution of internal chains in the core was random. It is suggested that in the core of a model prepared from a construct made with alternating probabilities of extension – one in which this probability is high relative to branching, and a second in which it is low – may give clusters of branched maltodextrins with short internal chains which are joined by longer chains; more closely approximating the distribution of internal chains of different lengths in amylopectin.An arrangement for amylopectin molecules in the starch granule has been proposed. In this, they have a wafer-like, discoidal shape, composed of the amorphous zone overlain with the double helical, crystalline region. The flat macromolecules are concentrically layered with the former on the inside and the latter oriented to the outside of the granule.     

1α,25(OH)2-Vitamin D3 signaling in chick enterocytes: Enhancement of tyrosine phosphorylation and rapid stimulation of mitogen-activated protein (MAP) kinase

The steroid hormone 1α,25(OH)₂–vitamin D₃ (1α,25(OH)₂D₃) generates biological responses in intestinal and other cells via both genomic and rapid, nongenomic signal transduction pathways. We examined the hypothesis that 1α,25(OH)₂D₃ action in chick enterocytes may be linked to pathways involving tyrosine phosphorylation. Brief exposure of isolated chick enterocytes to 1α,25(OH)₂D₃ demonstrated increased tyrosine phosphorylation of several cellular proteins (antiphosphotyrosine immunoblots of whole cell lysates) with prominent bands at 42–44, 55–60, and 105–120 Kda. The 42–44 Kda bands comigrated with mitogen-activated protein (MAP) kinase (immunoblotting with anti-MAP kinase antibody) The response occurred within 30 s, peaked at 1 min, and was dose-dependent (0.01–10 nM), with maximal stimulation at 1 nM (three- to fivefold). This effect was specific for 1α,25(OH)₂D₃ since its metabolic precursors 25(OH)D₃and vitamin D₃ did not increase MAP kinase tyrosine phosphorylation. The tyrosine kinase inhibitor, genistein, blocked 1α,25(OH)₂D₃-induced tyrosine phosphorylation of MAP kinase, while staurosporine, a PKC inhibitor, attenuated the hormone's effects by 30%. We have evaluated the ability of 1α,25(OH)₂D₃ analogs, which have complete flexibility around the 6,7 carbon-carbon bond (6F) or which are locked in either the 6-s-cis (6C) or the 6-s-trans(6T) shape(s), to activate MAP kinase. Thus, two 6F and one 6C analog stimulated while one 6T analog did not stimulate MAP kinase tyrosine phosphorylation. In addition, 1β,25(OH)₂D₃, a known antagonist of 1α,25(OH)₂D₃-mediated rapid responses, blocked the hormone effects on MAP kinase. We conclude that 1α,25(OH)₂D₃ and analogs which can achieve the 6-s-cis shape (6F and 6C) can increase tyrosine phosphorylation and activation of MAP kinase in chick enterocytes. J. Cell. Biochem. 69:470–482, 1998. © 1998 Wiley-Liss, Inc.     

Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo

Aiming at the development of new drugs for the treatment of prostate cancer, the effects of steroidal compounds and one non-steroidal substance on androgen biosynthesis were evaluated in vitro and in vivo. Sa 40 [17-(5-pyrimidyl)androsta-5,16-diene-3beta-ol], its 3-acetyl derivate Sa 41 and BW 19 [3,4-dihydro-2-(4-imidazolylmethyl)-6-methoxy-1-methyl-naphthalene] are compounds from our group, which have been developed as inhibitors of CYP 17 (17alpha-hydroxylase-C17, 20-lyase, the key enzyme in androgen biosynthesis). They have been compared with CB 7598 [abiraterone: 17-(3-pyridyl)androsta-5,16-diene-3beta-ol], its 3-acetyl compound CB 7630 and ketoconazole, compounds which already have been used clinically. The most potent compound toward human CYP 17 (testicular microsomes) was Sa 40 (IC(50) value of 24 nM), followed by Sa 41, CB 7598, BW 19, CB 7630 and ketoconazole. Sa 40 shows a type II difference spectrum and a non-competitive type of inhibition (K(i) value of 16 nM). No recovery of enzyme activity was observed after preincubation of CYP 17 with Sa 40 and subsequent charcoal treatment. In Escherichia coli cells coexpressing human CYP 17 and NADPH-P450 reductase, Sa 40 was more active than CB 7598 and BW 19, whereas the acetyl compounds were not active. The latter three compounds were equally active towards rat CYP 17. Male Sprague-Dawley (SD) rats were administered daily for 14 days BW 19 and the acetyl derivatives Sa 41 and CB 7630 as prodrugs (0.1 mmol/kg intraperitoneally). The test compounds strongly reduced plasma testosterone concentration, as well as prostate and seminal vesicles weights. They showed moderate inhibitory effects on the weights of levator ani, bulbocavernosus and testes, whereas they led to an increase in adrenal and pituitary weights. The only exception was BW 19 which did not change pituitary weights. Based on its superiority on the human enzyme, it was concluded that Sa 40 in its 3beta-acetate form (Sa 41) could be a promising candidate for clinical evaluation.     

Induction of SCEs in CHL cells by dichlorobiphenyl derivative water pollutants, 2-phenylbenzotriazole (PBTA) congeners and river water concentrates

We recently identified dichlorobiphenyl (DCB) derivatives and 2-phenylbenzotriazole (PBTA) congeners as major mutagenic constituents of the waters of the Waka River and the Yodo River system in Japan, respectively. In this study we examined sister chromatid exchange (SCE) induction by two dichlorobiphenyl derivatives, 3,3′-dichlorobenzidine (DCB, 4,4′-diamino-3,3′-dichlorobiphenyl) and 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl (5-nitro-DCB); three PBTA congeners, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1), 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), and 2-[2-(acetylamino)amino]-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6); and water concentrates from the Waka River in Chinese hamster lung (CHL) cells. Concentration-dependent induction of SCE was found for all DCBs and PBTAs examined in the presence of S9 mix, and statistically significant increases of SCEs were detected at 2 μg per ml of medium or higher concentrations. SCE induction of MeIQx was examined to compare genotoxic activities of these water pollutants. According to the results, a ranking of the SCE-inducing potency of these compounds is the following: 5-nitro-DCB ≈ MeIQx > PBTA6 > PBTA-1 ≈ PBTA-2 > DCB.Water samples collected at a site at the Waka River showed concentration-related increases in SCEs at 6.25–18.75 ml-equivalent of river water per ml of medium with S9 mix. The concentrations of 5-nitro-DCB and DCB in the river water samples were from 2.5 to 19.4 ng/l and from 4100 to 18,900 ng/l, respectively. However, these chemicals showed only small contribution to SCE induction by the Waka River water.     

Effects of Moritella viscosa antigens on pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) cell line (SHK-1)

Moritella viscosa is the causative agent of winter ulcer disease in salmonids reared in North-Atlantic countries. In this study the effects of selected M. viscosa antigens on cytotoxicity and pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) macrophage-like cell line (SHK-1) were examined. SHK-1 cells were stimulated with live and heat-killed bacterial cells, extracellular products (ECP) and an extracellular vibriolysin, termed MvP1. Following incubation, cytotoxicity and expression levels of interleukin-1β (IL-1β) and interleukin-8 (IL-8) were examined at different time points. Both live M. viscosa cells and ECP were cytotoxic, but neither heat-killed cells, nor the MvP1 peptidase caused cell death. Expression levels of both IL-1β and IL-8 increased significantly after stimulation with live cells, but heat-killed cells only caused increased IL-8 expression. ECP did not affect IL-1β expression, but did stimulate IL-8 expression. The isolated MvP1 peptidase stimulated both IL-1β and IL-8 expression at the highest concentration tested. This study reveals a difference in the induction of pro-inflammatory gene expression in salmon SHK-1 cells between live and heat-killed M. viscosa cells, and also that an unknown secreted factor is the main stimulant of IL-β and IL-8 expression.     

Effect of chronic l-DOPA treatment on 5-HT1A receptors in parkinsonian monkey brain

After chronic use of l-3,4-dihydroxyphenylalanine (l-DOPA), most Parkinson’s disease (PD) patients suffer from its side effects, especially motor complications called l-DOPA-induced dyskinesia (LID). 5-HT_1A agonists were tested to treat LID but many were reported to worsen parkinsonism. In this study, we evaluated changes in concentration of serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) and of 5-HT_1A receptors in control monkeys, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys, dyskinetic MPTP monkeys treated chronically with l-DOPA, low dyskinetic MPTP monkeys treated with l-DOPA and drugs of various pharmacological activities: Ro 61-8048 (an inhibitor of kynurenine hydroxylase) or docosahexaenoic acid (DHA) and dyskinetic MPTP monkeys treated with l-DOPA + naltrexone (an opioid receptor antagonist). Striatal serotonin concentrations were reduced in MPTP monkeys compared to controls. Higher striatal 5-HIAA/serotonin concentration ratios in l-DOPA-treated monkeys compared to untreated monkeys suggest an intense activity of serotonin axon terminals but this value was similar in dyskinetic and nondyskinetic animals treated with or without adjunct treatment with l-DOPA. As measured by autoradiography with [³H]8-hydroxy-2-(di-n-propyl) aminotetralin (8-OH-DPAT), a decrease of 5-HT_1A receptor specific binding was observed in the posterior/dorsal region of the anterior cingulate gyrus and posterior/ventral area of the superior frontal gyrus of MPTP monkeys compared to controls. An increase of 5-HT_1A receptor specific binding was observed in the hippocampus of MPTP monkeys treated with l-DOPA regardless to their adjunct treatment. Cortical 5-HT_1A receptor specific binding was increased in the l-DOPA-treated MPTP monkeys alone or with DHA or naltrexone and this increase was prevented in low dyskinetic MPTP monkeys treated with l-DOPA and Ro 61-8048. These results highlight the importance of 5-HT_1A receptor alterations in treatment of PD with l-DOPA.     

Cell selection and inheritance of imidazolinone resistance in sugarbeet (Beta vulgaris)

 Sugarbeets are sensitive to imidazolinone herbicide residues applied to rotational crops. Two imidazolinone-resistance (IMI-R) sugarbeet traits were developed by somatic cell selection to overcome rotation restrictions for sugarbeets where imidazolinones have been applied. Sir-13 is an IMI-R/SU-S (sulfonylurea-sensitive) variant selected from an imidazolinone-sensitive (IMI-S) sugarbeet clone, REL-1. A second variant, 93R30B, resistant to imidazolinone as well as to sulfonylurea herbicides (IMI-R/SU-R), was selected from a plant homozygous for a previously described sulfonylurea-specific resistance trait, Sur (IMI-S/SU-R). The IMI-R alleles (Sir-13 and 93R30B) were found to be corresponding allelic variants at the same ALS locus and both were tightly associated with the Sur allele. Each resistant allele is dominant to the sensitive wild-type allele; however, incomplete dominance is shown among resistance alleles. Diploid sugarbeet contains a single ALS gene copy, limiting the ability to stack these resistance traits in the same plant by traditional breeding. Received: 1 May 1997 / Accepted: 30 June 1997     

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3′,5′-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I₂, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B₄ synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B₄ itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B₄ is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B₄ synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B₄ can limit their own activity in neutrophils.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

Crystal Structure of Human RNA Helicase A (DHX9): Structural Basis for Unselective Nucleotide Base Binding in a DEAD-Box Variant Protein

RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long α-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5′-triphosphate, cytidine 5′-triphosphate, and uridine 5′-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.     

Involvement of Ceramide in the Mechanism of Cr(VI)-induced Apoptosis of CHO Cells

Mitochondria reduce Cr(VI) to Cr(V) with concomitant generation of reactive oxygen species, thereby exhibiting cytotoxic effects leading to apoptosis in various types of cells. To clarify the mechanism by which Cr(VI) induces apoptosis, we examined the effect of Cr(VI) on Chinese hamster ovary (CHO) cells. Cr(VI) increased cellular levels of ceramide by activating acid sphingomyelinase (ASMase) and inhibiting the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt). Cr(VI) also induced cyclosporin A- and trifluoperazine-sensitive depolarization of mitochondria and activated caspase-3, 8 and 9, thereby causing fragmentation of cellular DNA. The presence of desipramine, an inhibitor of ASMase, and membrane permeable pCPT-cAMP suppressed the Cr(VI)-induced activation of caspases and DNA fragmentation. These results suggested that accumulation of ceramide play an important role in the Cr(VI)-induced apoptosis of CHO cells through activation of mitochondrial membrane permeability transition.     

Crystal structure of the kainate receptor GluR5 ligand-binding core in complex with (S)-glutamate

The X-ray structure of the ligand-binding core of the kainate receptor GluR5 (GluR5-S1S2) in complex with (S)-glutamate was determined to 1.95 A resolution. The overall GluR5-S1S2 structure comprises two domains and is similar to the related AMPA receptor GluR2-S1S2J. (S)-glutamate binds as in GluR2-S1S2J. Distinct features are observed for Ser741, which stabilizes a highly coordinated network of water molecules and forms an interdomain bridge. The GluR5 complex exhibits a high degree of domain closure (26 degrees) relative to apo GluR2-S1S2J. In addition, GluR5-S1S2 forms a novel dimer interface with a different arrangement of the two protomers compared to GluR2-S1S2J.     

Off-target effect of the Epac agonist 8-pCPT-2′-O-Me-cAMP on P2Y12 receptors in blood platelets

The primary target of the cAMP analogue 8-pCPT-2′-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2′-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2′-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2′O-Me-cAMP binding into the purinergic platelet receptor P2Y₁₂ revealed that the analogue docks similar to the P2Y₁₂ antagonist 2MeSAMP. The 8-pCPT-2′-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2′-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2′-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y₁₂ receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2′-O-Me-cAMP did not affect platelet activation at similar concentrations.