Development of cationic solid lipid nanoparticles incorporating cholesteryl-9-carboxynonanoate (9CCN) for delivery of antagomiRs to macrophages.

Lipid-based nanoparticles are a useful tool for nucleic acids delivery and have been regarded as a promising approach for diverse diseases. However, off-targets effects are a matter of concern and some strategies to improve selectivity of solid lipid nanoparticles (SLNs) were reported. The goal of this study was to test formulations of SLNs incorporating lipid cholesteryl-9-carboxynonanoate (9CCN) as "eat-me" signal to target antagomiR oligonucleotides to macrophages. We formulate four SLNs, and those with a mean diameter of 200 nm and a Z-potential values between 25 and 40 mV, which allowed the antagomiR binding, were selected for in vitro studies. Cell viability, transfection efficiency and cellular uptake assays were performed within in vitro macrophages using flow cytometry and confocal imaging and the SLNs incorporating 25 mg of 9CCN proved to be the best formulation. Subsequently, we used a labeled antagomiR to study tissue distribution in in-vivo ApoE-/- model of atherosclerosis. Using the ApoE-/- model we demonstrated that SLNs with phagocytic signal 9-CCN target macrophages and release the antagomiR cargo in a selective way.

Dysregulated Protein Phosphorylation as Main Contributor of Granulovacuolar Degeneration at the First Stages of Neurofibrillary Tangles Pathology.

The hippocampus of cases with neurofibrillary tangles (NFT) pathology classified as stages I-II, III-IV, and V-VI without comorbidities, and middle-aged (MA) individuals with no NFT pathology, were examined to learn about the composition of granulovacuolar degeneration (GVD). Our results confirm the presence of CK1-δ, p38-P Thr180/Tyr182, SAPK/JNK-P Thr183/Thr185, GSK-3α/ß-P Tyr279/Tyr216, and GSK-3ß Ser9 in the cytoplasmic granules in a subset of neurons of the CA1 and CA2 subfields of the hippocampus. Also, we identify the presence of PKA α/ß-P Thr197, SRC-P Tyr416, PAK1-P Ser199/Ser204, CAMK2A-P Tyr197, and PKCG-P Thr655 in cytoplasmic granules in cases with NFT pathology, but not in MA cases. Our results also confirm the presence of ß-catenin-P Ser45/Thr41, IREα-P Ser274, eIF2α-P Ser51, TDP-43-P Ser403-404 (but absent TDP-43), and ubiquitin in cytoplasmic granules. Other components of the cytoplasmic granules are MAP2-P Thr1620/1623, MAP1B-P Thr1265, ADD1-P Ser726, and ADD1/ADD1-P Ser726/Ser713, in addition to several tau species including 3Rtau, 4Rtau, and tau-P Ser262. The analysis of GVD at progressive stages of NFT pathology reveals the early appearance of phosphorylated kinases and proteins in cytoplasmic granules at stages I-II, before the appearance of pre-tangles and NFTs. Most of these granules are not surrounded by LAMP1-positive membranes. Markers of impaired ubiquitin-protesome system, abnormal reticulum stress response, and altered endocytic and autophagic pathways occur in a subpopulation of neurons containing cytoplasmic granules, and they appear later. These observations suggest early phosphorylation of kinases leading to their activation, and resulting in the abnormal phosphorylation of various substrates, including tau, as a main alteration at the first stages of GVD.

Lung endothelial cells are sensitive to epsilon toxin from Clostridium perfringens.

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.

Involvement of Oligodendrocytes in Tau Seeding and Spreading in Tauopathies.

Introduction: Human tau seeding and spreading occur following intracerebral inoculation into different gray matter regions of brain homogenates obtained from tauopathies in transgenic mice expressing wild or mutant tau, and in wild-type (WT) mice. However, little is known about tau propagation following inoculation in the white matter. Objectives: The present study is geared to learning about the patterns of tau seeding and cells involved following unilateral inoculation in the corpus callosum of homogenates from sporadic Alzheimer's disease (AD), primary age-related tauopathy (PART: neuronal 4Rtau and 3Rtau), pure aging-related tau astrogliopathy (ARTAG: astroglial 4Rtau with thorn-shaped astrocytes TSAs), globular glial tauopathy (GGT: 4Rtau with neuronal tau and specific tau inclusions in astrocytes and oligodendrocytes, GAIs and GOIs, respectively), progressive supranuclear palsy (PSP: 4Rtau with neuronal inclusions, tufted astrocytes and coiled bodies), Pick's disease (PiD: 3Rtau with characteristic Pick bodies in neurons and tau containing fibrillar astrocytes), and frontotemporal lobar degeneration linked to P301L mutation (FTLD-P301L: 4Rtau familial tauopathy). Methods: Adult WT mice were inoculated unilaterally in the lateral corpus callosum with sarkosyl-insoluble fractions or with sarkosyl-soluble fractions from the mentioned tauopathies; mice were killed from 4 to 7 months after inoculation. Brains were fixed in paraformaldehyde, embedded in paraffin and processed for immunohistochemistry. Results: Tau seeding occurred in the ipsilateral corpus callosum and was also detected in the contralateral corpus callosum. Phospho-tau deposits were found in oligodendrocytes similar to coiled bodies and in threads. Moreover, tau deposits co-localized with active (phosphorylated) tau kinases p38 and ERK 1/2, suggesting active tau phosphorylation of murine tau. TSAs, GAIs, GOIs, tufted astrocytes, and tau-containing fibrillar astrocytes were not seen in any case. Tau deposits were often associated with slight myelin disruption and the presence of small PLP1-immunoreactive globules and dots in the ipsilateral corpus callosum 6 months after inoculation of sarkosyl-insoluble fractions from every tauopathy. Conclusions: Seeding and spreading of human tau in the corpus callosum of WT mice occurs in oligodendrocytes, thereby supporting the idea of a role of oligodendrogliopathy in tau seeding and spreading in the white matter in tauopathies. Slight differences in the predominance of threads or oligodendroglial deposits suggest disease differences in the capacity of tau seeding and spreading among tauopathies.

Glutamate Transporter GLT1 Expression in Alzheimer Disease and Dementia With Lewy Bodies.

Glutamate transporter solute carrier family 1, member 2 (GLT1/EAAT2), a major modulator of glutamate homeostasis in astrocytes, is assessed in post-mortem human brain samples of frontal cortex area 8 in advanced stages of Alzheimer disease (AD) and terminal stages of dementia with Lewy bodies (DLB) in order to gain understanding of astrogliopathy in diseases manifested by dementia. Glial fibrillary acidic protein (GFAP) mRNA expression is significantly increased in AD but not in DLB, whereas GLT1, vesicular glutamate transporter 1 (vGLUT1) and aldehyde dehydrogenase 1 family member 1 (ALDH1L1) are not modified in AD and DLB when compared with controls. GLT1 protein levels are not altered in AD and DLB but GFAP and ALDH1L1 are significantly increased in AD, and GFAP in DLB. As a result, a non-significant decrease in the ratio between GLT1 and GFAP, and between GLT1 and ALDH1L1, is found in both AD and DLB. Double-labeling immunofluorescence and confocal microscopy revealed no visible reduction of GLT1 immunoreactivity in relation to ß-amyloid plaques in AD. These data suggest a subtle imbalance between GLT1, and GFAP and ALDH1L1 expression, with limited consequences in glutamate transport.

Characterization of ecto-nucleotidases in human oviducts with an improved approach simultaneously identifying protein expression and in situ enzyme activity.

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those taking place in oviducts, including contraction, beating of cilia, and maintenance of fluid composition that, in turn, influences sperm capacitation and hyperactivation, as well as oocyte and embryo nourishing. Ecto-nucleotidases are the enzymes that regulate extracellular ATP and adenosine levels, thus playing a role in reproduction. We have optimized a convenient method for characterizing ecto-nucleotidases that simultaneously localizes the protein and its associated enzyme activity in the same tissue slice and characterizes ecto-nucleotidases in human oviducts. The technique combines immunofluorescence and in situ histochemistry, allowing precise identification of ecto-nucleotidases at a subcellular level. In oviducts, remarkably, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, with the ability to hydrolyze ATP to AMP, are expressed in ciliated epithelial cells but with different subcellular localization. Ecto-5'nucleotidase/CD73 is also expressed apically in ciliated cells. CD73, together with alkaline phosphatase, also expressed apically in oviductal epithelium, complete the hydrolysis sequence by dephosphorylating AMP to adenosine. The concerted action of these enzymes would contribute to the local increase of adenosine concentration necessary for sperm capacitation. The use of this method would be an asset for testing new potential therapeutic drugs with inhibitory potential, which is of great interest presently in the field of oncology and in other clinical disciplines.

Limited Unfolded Protein Response and Inflammation in Neuroserpinopathy.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare disease characterized by the deposition of multiple intracytoplasmic neuronal inclusions that contain mutated neuroserpin. Tg-Syracuse (Tg-Syr) mice express Ser49Pro mutated neuroserpin and develop clinical and neuropathological features of human FENIB. We used 8-, 34-, 45- and 80-week-old Tg-Syr mice to characterize neuroinflammation and the unfolded protein response (UPR) in a neurodegenerative disease in which abnormal protein aggregates accumulate within the endoplasmic reticulum (ER). There were scattered neuroserpin inclusions in Tg-Syr mice at 8 weeks of age; the numbers of neurons involved and the amount of neuroserpin per neuron increased with age throughout the CNS to 80 weeks of age; no similar inclusions were found in wild type (Tg-WT) mice at any age. Increases in numbers of astrocytes and microglia occurred at advanced disease stages. Among 22 markers in 80-week-old Tg-Syr mice, only II1b and II10rb mRNAs in the somatosensory cortex and CxCl10 and Il10rb mRNAs in the olfactory bulb were upregulated when compared with Tg-WT mice indicating a limited relationship between neuroserpin inclusions and inflammatory responses. The changes were accompanied by a transient increase in expression of Xbp1 spliced at 45 weeks and increased ERdJ4 mRNAs at 80 weeks. The sequestration of UPR activators GRP78 and GRP94 in neuroserpin inclusions might explain the limited UPR responses despite the accumulation of neuroserpin in the ER in this FENIB mouse model.

New cardiac and skeletal protein aggregate myopathy associated with combined MuRF1 and MuRF3 mutations.

Protein aggregate myopathies (PAMs) define muscle disorders characterized by protein accumulation in muscle fibres. We describe a new PAM in a patient with proximal muscle weakness and hypertrophic cardiomyopathy, whose muscle fibres contained inclusions containing myosin and myosin-associated proteins, and aberrant distribution of microtubules. These lesions appear as intact A- and M-bands lacking thin filaments and Z-discs. These features differ from inclusions in myosin storage myopathy (MSM), but are highly similar to those in mice deficient for the muscle-specific RING finger proteins MuRF1 and MuRF3. Sanger sequencing excluded mutations in the MSM-associated gene MYH7 but identified mutations in TRIM63 and TRIM54, encoding MuRF1 and MuRF3, respectively. No mutations in other potentially disease-causing genes were identified by Sanger and whole exome sequencing. Analysis of seven family members revealed that both mutations segregated in the family but only the homozygous TRIM63 null mutation in combination with the heterozygous TRIM54 mutation found in the proband caused the disease phenotype. Both MuRFs are microtubule-associated proteins localizing to sarcomeric M-bands and Z-discs. They are E3 ubiquitin ligases that play a role in degradation of sarcomeric proteins, stabilization of microtubules and myogenesis. Lack of ubiquitin and the 20S proteasome subunit in the inclusions found in the patient suggested impaired turnover of thick filament proteins. Disruption of microtubules in cultured myotubes was rescued by transient expression of wild-type MuRF1. The unique features of this novel myopathy point to defects in homeostasis of A-band proteins in combination with instability of microtubules as cause of the disease.

Glial and neuronal tau pathology in tauopathies: characterization of disease-specific phenotypes and tau pathology progression.

Tauopathies are degenerative diseases characterized by the accumulation of phosphorylated tau in neurons and glial cells. With some exceptions, tau deposits in neurons are mainly manifested as pretangles and tangles unrelated to the tauopathy. It is thought that abnormal tau deposition in neurons occurs following specific steps, but little is known about the progression of tau pathology in glial cells in tauopathies. We compared tau pathology in different astrocyte phenotypes and oligodendroglial inclusions with that in neurons in a large series of tauopathies, including progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick disease, frontotemporal lobar degenerations (FTLD) associated with mutations in the tau gene, globular glial tauopathy (GGT), and tauopathy in the elderly. Our findings indicate that disease-specific astroglial phenotypes depend on i) the primary amino acid sequence of tau (mutated tau, 3Rtau, and 4Rtau); ii) phospho-specific sites of tau phosphorylation, tau conformation, tau truncation, and ubiquitination in that order (which parallel tau modifications related to pretangle and tangle stages in neurons); and iii) modifications of the astroglial cytoskeleton. In contrast to astrocytes, coiled bodies in oligodendrocytes have similar characteristics whatever the tauopathy, except glial globular inclusions in GGT, and coiled bodies and globular oligodendroglial inclusions in FTLD-tau/K317M. These observations indicate that tau pathology in glial cells largely parallels, but is not identical to, that in neurons in many tauopathies.

Functional genomics reveals dysregulation of cortical olfactory receptors in Parkinson disease: novel putative chemoreceptors in the human brain.

Parkinson disease (PD) is no longer considered a complex motor disorder but rather a systemic disease with variable nonmotor deficits that may include impaired olfaction, depression, mood and sleep disorders, and altered cortical function. Increasing evidence indicates that multiple metabolic defects occur in regions outside the substantia nigra, including the cerebral cortex, even at premotor stages of the disease. We investigated changes in gene expression in the frontal cortex in PD patient brains using a transcriptomics approach. Functional genomics analysis indicated that cortical olfactory receptors (ORs) and taste receptors (TASRs) are altered in PD patients. Olfactory receptors OR2L13, OR1E1, OR2J3, OR52L1, and OR11H1 and taste receptors TAS2R5 and TAS2R50 were downregulated, but TAS2R10 and TAS2R13 were upregulated at premotor and parkinsonian stages in the frontal cortex area 8 in PD patient brains. Furthermore, we present novel evidence that, in addition to the ORs, obligate downstream components of OR function adenylyl cyclase 3 and olfactory G protein (Gαolf), OR transporters, receptor transporter proteins 1 and 2 and receptor expression enhancing protein 1, and OR xenobiotic removing UDP-glucuronosyltransferase 1 family polypeptide A6 are widely expressed in neurons of the cerebral cortex and other regions of the adult human brain. Together, these findings support the concept that ORs and TASRs in the cerebral cortex may have novel physiologic functions that are affected in PD patients.

Ecto-nucleotidases distribution in human cyclic and postmenopausic endometrium.

Extracellular ATP and its hydrolysis product, adenosine, acting through specific receptors collectively named purinergic receptors, regulate female fertility by influencing the endometrial fluid microenvironment. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phospho-diesterases (E-NPPs), ecto-5'-nucleotidase (5'NT), and alkaline phosphatases (APs). The aim of the present work is to characterize the expression and distribution of ecto-nucleotidases in human endometrium along the menstrual cycle and after menopause, to evaluate their potential utility as fertility markers. We examined proliferative, secretory and atrophic endometria from women without endometrial pathology undergoing hysterectomy. We show that the ecto-nucleotidases are mainly present at endometrial epithelia, both luminal and glandular, and that their expression fluctuates along the cycle and also changes after menopause. An important result was identifying NPP3 as a new biological marker of tubal metaplasia. Our results emphasize the relevance of the study of purinergic signaling in human fertility.

Cerebellar amyloid-ß plaques: disturbed cortical circuitry in AßPP/PS1 transgenic mice as a model of familial Alzheimer's disease.

Cerebellar amyloid-ß (Aß) deposition in the form of neuritic plaques and Purkinje cell loss are common in certain pedigrees of familial Alzheimer's disease (FAD) mainly linked to PS1 mutations. AßPP/PS1 transgenic mice, here used as a model of FAD, show a few Aß plaques in the molecular layer of the cerebellum at 6 months, and which increase in number with age. Motor impairment is apparent in transgenic mice aged 12 months. Combined methods have shown degenerated parallel fibers as the main component of dystrophic neurites of Aß plaques, loss of synaptic contacts between parallel fibers and dendritic spines of Purkinje cells, and degeneration of granule cells starting at 12 months and increasing in mice 18/20 months old. In addition, abnormal mitochondria and focal loss of Purkinje and basket cells, together with occasional axonal torpedoes and increased collaterals of Purkinje cells in mice aged 18/20 months, is suggested to be a concomitant defect presumably related to soluble extracellular or intracellular Aß. These observations demonstrate serious deterioration of the neuronal circuitry in the cerebellum of AßPP/PS1 transgenic mice, and they provide support for the interpretation of similar alterations occurring in certain pedigrees with FAD.

Glucose-dependent changes in SNARE protein levels in pancreatic ß-cells.

Prolonged exposure to high glucose concentration alters the expression of a set of proteins in pancreatic ß-cells and impairs their capacity to secrete insulin. The cellular and molecular mechanisms that lie behind this effect are poorly understood. In this study, three either in vitro or in vivo models (cultured rat pancreatic islets incubated in high glucose media, partially pancreatectomized rats, and islets transplanted to streptozotozin-induced diabetic mice) were used to evaluate the dependence of the biological model and the treatment, together with the cell location (insulin granule or plasma membrane) of the affected proteins and the possible effect of sustained insulin secretion, on the glucose-induced changes in protein expression. In all three models, islets exposed to high glucose concentrations showed a reduced expression of secretory granule-associated vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptobrevin/vesicle-associated membrane protein 2 and cellubrevin but minor or no significant changes in the expression of the membrane-associated target-SNARE proteins syntaxin1 and synaptosomal-associated protein-25 and a marked increase in the expression of synaptosomal-associated protein-23 protein. The inhibition of insulin secretion by the L-type voltage-dependent calcium channel nifedipine or the potassium channel activator diazoxide prevented the glucose-induced reduction in islet insulin content but not in vesicle-SNARE proteins, indicating that the granule depletion due to sustained exocytosis was not involved in the changes of protein expression induced by high glucose concentration. Altogether, the results suggest that high glucose has a direct toxic effect on the secretory pathway by decreasing the expression of insulin granule SNARE-associated proteins.

TAR DNA-Binding protein 43 accumulation in protein aggregate myopathies.

Protein aggregate myopathies, including myofibrillar myopathies and sporadic inclusion body myositis (sIBM), are characterized by abnormal protein aggregates composed of various muscular and ectopic proteins. Previous studies have shown the crucial role ofdysregulated transcription factors such as neuron-restrictive silencerfactor in the expression of aberrant proteins in myotilinopathies. Here, we assessed possible aberrant expression of TAR DNA-bindingprotein 43 (TDP-43), another factor involved in transcription regulation. TDP-43-immunoreactive intracytoplasmic inclusions were seen in all cases examined of myotilinopathy, desminopathy, and sIBM, and in 1 case of inclusion body myositis with Paget disease of bone and frontotemporal degeneration (IBMPFD). TAR DNA-binding protein 43 colocalized with myotilin and valosin in myotilinopathies and IBMPFD, respectively, but only occasionally colocalized with ubiquitin in myotilinopathies, desminopathies, sIBM, and IBMPFD; this indicates that accumulated TDP-43 is largely not ubiquitinated. Moreover, phosphorylated TDP-43 at Ser403/404 and Ser409/410 accumulated in the cytoplasm of vulnerable fibers but did not always colocalize with nonphosphorylated TDP-43. Cytoplasmic deposition was accompanied by decreased TDP-43 localization in the nuclei of affected fibers. These findings indicate that TDP-43 not only is another protein accumulated in myofibrillar myopathies, sIBM, and IBMPFD but also likely has a role through altered microRNA processing in the abnormal protein production, modification, and accumulation in protein aggregate myopathies.

Dystrophic neurites of senile plaques in Alzheimer's disease are deficient in cytochrome c oxidase.

Double-labeling immunofluorescence and confocal microscopy have been used to learn about the local relationship between amyloid, mitochondria, and cytochrome c oxidase (COX) in dystrophic neurites of senile plaques in the frontal cortex in Alzheimer's disease (AD). Dystrophic neurites surrounding amyloid plaques are filled with mitochondrial porin-immunoreactive structures. In contrast with tangle-bearing and non-tangle-bearing neurons, which express mitochondrial porin and COX subunit 4, porin-immunoreactive neurites of senile plaques lack COX subunit 4. Parallel western blot studies in mitochondria-enriched fractions of the frontal cortex in the same cases disclosed reduced expression levels of COX, but not of prohibitin, in AD stages VB/C of Braak. Co-localization of porin and lysosomal associated protein 1, as revealed by double-labeling immunofluorescence and confocal microscopy, suggests that mitochondria may be engulfed by lysosomes in dystrophic neurites. These findings support a local link between amyloid deposition, abnormal mitochondria and impaired respiratory chain function (resulting from decrease of COX expression) in dystrophic neurites of senile plaques in AD.

Mutant ubiquitin and p62 immunoreactivity in cases of combined multiple system atrophy and Alzheimer's disease.

Recent studies have shown the co-existence of alpha-synuclein and phosphorylated tau (pTau) in several neurodegenerative diseases. Here, we report two autopsy cases of combined multiple system atrophy (MSA) and Alzheimer's disease (AD). In both cases, abundant alpha-synuclein-positive glial and neuronal cytoplasmic inclusions were found in the brainstem, amygdala and hippocampal formation. pTau-positive neurofibrillary tangles (NFTs) were widely distributed in case 1 (Braak stage VI) and moderate in case 2 (Braak stage III). Although alpha-synuclein and pTau pathology co-occurred in the hippocampus and entorhinal cortex, only a few neurons showed co-existence of these two proteins. Immunoreactivity for p62, a ubiquitin proteasome system related protein, was found in the majority of NFTs, but in only a small proportion of neuronal alpha-synuclein inclusions. In addition, UBB+1, a mutant form of ubiquitin and a marker for proteasomal dysfunction, was present in the majority of NFTs, whereas co-existence of alpha-synuclein and UBB+1 was found in only a few neurons. These findings indicate that alpha-synuclein and phosphorylated tau co-occur in certain brain regions in cases of combined MSA and AD and that the proteasomal pathways differ between alpha-synuclein- and pTau-bearing neurons.

Cloning, molecular characterization and expression of ecto-nucleoside triphosphate diphosphohydrolase-1 from Torpedo electric organ.

During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme. In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.