scholarly journals Characterization of a Vesivirus Associated with an Outbreak of Acute Hemorrhagic Gastroenteritis in Domestic Dogs

2018 ◽  
Vol 56 (5) ◽  
Author(s):  
Randall W. Renshaw ◽  
Jennifer Griffing ◽  
Jaime Weisman ◽  
Lisa M. Crofton ◽  
Melissa A. Laverack ◽  
...  
Keyword(s):  
Intestinal Tract ◽  
Virus Isolation ◽  
Cho Cell ◽  
Mild Disease ◽  
Reverse Transcription Pcr ◽  
The Brain ◽  
Housing Facility ◽  
Diagnostic Investigations

ABSTRACT Four of eleven affected dogs died despite aggressive treatment during a 2015 focal outbreak of hemorrhagic gastroenteritis following a stay in a pet housing facility. Routine diagnostic investigations failed to identify a specific cause. Virus isolation from fresh necropsy tissues yielded a calicivirus with sequence homology to a vesivirus within the group colloquially known as the vesivirus 2117 strains that were originally identified as contaminants in CHO cell bioreactors. In situ hybridization and reverse transcription-PCR assays of tissues from the four deceased dogs confirmed the presence of canine vesivirus (CaVV) nucleic acids that localized to endothelial cells of arterial and capillary blood vessels. CaVV nucleic acid corresponded to areas of necrosis and hemorrhage primarily in the intestinal tract, but also in the brain of one dog with nonsuppurative meningoencephalitis. This is the first report of an atypical disease association with a putative hypervirulent vesivirus strain in dogs, as all other known strains of CaVV appear to cause nonclinical infections or relatively mild disease. After identification of the CU-296 vesivirus strain from this outbreak, four additional CaVV strains were amplified from unrelated fecal specimens and archived stocks provided by other laboratories. Broader questions include the origins, reservoir(s), and potential for reemergence and spread of these related CaVVs.

scholarly journals Molecular cloning and biochemical characterization of sialidases from zebrafish (Danio rerio)

2007 ◽  
Vol 408 (3) ◽  
pp. 395-406 ◽  
Author(s):  
Marta Manzoni ◽  
Paolo Colombi ◽  
Nadia Papini ◽  
Luana Rubaga ◽  
Natascia Tiso ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Biochemical Characterization ◽  
Chromosome 21 ◽  
Ph Optimum ◽  
Reverse Transcription Pcr ◽  
Genome Wide ◽  
Genome Wide Search ◽  
Putative Orthologues

Sialidases remove sialic acid residues from various sialo-derivatives. To gain further insights into the biological roles of sialidases in vertebrates, we exploited zebrafish (Danio rerio) as an animal model. A zebrafish transcriptome- and genome-wide search using the sequences of the human NEU polypeptides as templates revealed the presence of seven different genes related to human sialidases. neu1 and neu4 are the putative orthologues of the mammalian sialidases NEU1 and NEU4 respectively. Interestingly, the remaining genes are organized in clusters located on chromosome 21 and are all more closely related to mammalian sialidase NEU3. They were thus named neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5. Using RT–PCR (reverse transcription–PCR) we detected transcripts for all genes, apart from neu3.4, and whole-mount in situ hybridization experiments show a localized expression pattern in gut and lens for neu3.1 and neu4 respectively. Transfection experiments in COS7 (monkey kidney) cells demonstrate that Neu3.1, Neu3.2, Neu3.3 and Neu4 zebrafish proteins are sialidase enzymes. Neu3.1, Neu3.3 and Neu4 are membrane-associated and show a very acidic pH optimum below 3.0, whereas Neu3.2 is a soluble sialidase with a pH optimum of 5.6. These results were further confirmed by subcellular localization studies carried out using immunofluorescence. Moreover, expression in COS7 cells of these novel zebrafish sialidases (with the exception of Neu3.2) induces a significant modification of the ganglioside pattern, consistent with the results obtained with membrane-associated mammalian sialidases. Overall, the redundancy of sialidases together with their expression profile and their activity exerted on gangliosides of living cells indicate the biological relevance of this class of enzymes in zebrafish.

scholarly journals Identification and Characterization of Four Autophagy-Related Genes That Are Expressed in Response to Hypoxia in the Brain of the Oriental River Prawn (Macrobrachium nipponense)

2019 ◽  
Vol 20 (8) ◽  
pp. 1856 ◽  
Author(s):  
Shengming Sun ◽  
Ying Wu ◽  
Hongtuo Fu ◽  
Xianping Ge ◽  
Hongzheng You ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Open Reading Frames ◽  
Dependent Manner ◽  
Macrobrachium Nipponense ◽  
Oriental River Prawn ◽  
Adverse Environmental Conditions ◽  
The Brain ◽  
Identification And Characterization

Autophagy is a cytoprotective mechanism triggered in response to adverse environmental conditions. Herein, we investigated the autophagy process in the oriental river prawn (Macrobrachium nipponense) following hypoxia. Full-length cDNAs encoding autophagy-related genes (ATGs) ATG3, ATG4B, ATG5, and ATG9A were cloned, and transcription following hypoxia was explored in different tissues and developmental stages. The ATG3, ATG4B, ATG5, and ATG9A cDNAs include open reading frames encoding proteins of 319, 264, 268, and 828 amino acids, respectively. The four M. nipponense proteins clustered separately from vertebrate homologs in phylogenetic analysis. All four mRNAs were expressed in various tissues, with highest levels in brain and hepatopancreas. Hypoxia up-regulated all four mRNAs in a time-dependent manner. Thus, these genes may contribute to autophagy-based responses against hypoxia in M. nipponense. Biochemical analysis revealed that hypoxia stimulated anaerobic metabolism in the brain tissue. Furthermore, in situ hybridization experiments revealed that ATG4B was mainly expressed in the secretory and astrocyte cells of the brain. Silencing of ATG4B down-regulated ATG8 and decreased cell viability in juvenile prawn brains following hypoxia. Thus, autophagy is an adaptive response protecting against hypoxia in M. nipponense and possibly other crustaceans. Recombinant MnATG4B could interact with recombinant MnATG8, but the GST protein could not bind to MnATG8. These findings provide us with a better understanding of the fundamental mechanisms of autophagy in prawns.

scholarly journals In situ characterization of the brain–microdevice interface using Device Capture Histology

2011 ◽  
Vol 201 (1) ◽  
pp. 67-77 ◽  
Author(s):  
Andrew J. Woolley ◽  
Himanshi A. Desai ◽  
Mitchell A. Steckbeck ◽  
Neil K. Patel ◽  
Kevin J. Otto
Keyword(s):  
In Situ Characterization ◽  
The Brain

scholarly journals Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake

2017 ◽  
Vol 114 (25) ◽  
pp. E5006-E5015 ◽  
Author(s):  
Andrea Chicca ◽  
Simon Nicolussi ◽  
Ruben Bartholomäus ◽  
Martina Blunder ◽  
Alejandro Aparisi Rey ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Cannabinoid Receptor ◽  
Facilitated Diffusion ◽  
Chemical Probes ◽  
Analgesic Effects ◽  
Complex Process ◽  
Arachidonoyl Glycerol ◽  
The Brain

The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derivedN-substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC50= 10 nM) inhibitorN-(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.

scholarly journals Characterization of a novel alloherpesvirus from Atlantic cod (Gadus morhua)

2011 ◽  
Vol 24 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Mar Marcos-Lopez ◽  
Thomas B. Waltzek ◽  
Ronald P. Hedrick ◽  
Dolores V. Baxa ◽  
Amber F. Garber ◽  
...  
Keyword(s):  
Gadus Morhua ◽  
Virus Isolation ◽  
Atlantic Cod ◽  
International Committee ◽  
Atpase Subunit ◽  
Transmission Electron ◽  
Viral Particles ◽  
Infected Cells

Alloherpesviruses affect freshwater and marine fish species. The aim of the current study was to characterize a novel alloherpesvirus in Atlantic cod ( Gadus morhua). Samples were processed for histopathology, transmission electron microscopy (TEM), virus isolation, molecular characterization, and in situ hybridization (ISH). Histopathology revealed that the infection was restricted to the gills and that it induced cytomegaly in infected cells. By TEM, numerous viral particles with morphology compatible with a herpesvirus were observed inside the cytomegalic cells. To characterize this new agent, polymerase chain reaction amplified regions of the ATPase subunit of the terminase, and DNA polymerase genes were sequenced. Phylogenetic analysis revealed strongest similarity with alloherpesviruses belonging to the genus Ictalurivirus and Salmonivirus. The ISH showed specific labeling of nuclear inclusions in the cytomegalic cells. While virus isolation was unsuccessful, the results obtained through different diagnostic tests in the present study confirm the discovery of a new alloherpesvirus affecting Atlantic cod. The authors propose the formal species designation Gadid herpesvirus 1 (GaHV-1) to be considered for approval by the International Committee on the Taxonomy of Viruses.

Cerebral Ovine Herpesvirus 2 Infection of Cattle Is Associated With a Variable Neuropathological Phenotype

2020 ◽  
pp. 030098582097049
Author(s):  
Melanie M. Hierweger ◽  
Céline L. Boujon ◽  
Ronja V. Kauer ◽  
Mireille Meylan ◽  
Torsten Seuberlich ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Viral Encephalitis ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
The Central Nervous System ◽  
Consistent Feature ◽  
Polymerase Chain ◽  
The Brain

Cross-species infection with ovine herpesvirus 2 (OvHV-2) in cattle causes malignant catarrhal fever (MCF). MCF may involve the central nervous system (CNS) with necrotizing arteritis and/or vasculitis described to be unique to MCF and discriminatory compared to other viral CNS infections. However, a systematic histopathological characterization of the neural form of MCF in cattle is lacking. We examined medulla oblongata ( n = 9) or the entire brain ( n = 9) of 18 cattle in which OvHV-2 was identified by quantitative polymerase chain reaction (qPCR), in order to pinpoint potential variations in neuropathology. In 2/18 animals (11%) no lesions were identified, while 16/18 cattle (89%) had brain lesions of varying severity. Presence and quantities of OvHV-2 nucleic acid were determined by in situ hybridization and qPCR, respectively, and were related to the severity of lesions. Fifteen of 18 animals (83%) showed vasculitis, which was mainly of the lymphohistiocytic type, while pathognomonic necrotizing arteritis was only rarely present. Neuroparenchymal lesions included gliosis and/or neuronal changes in 7/16 brains with lesions (44%). The number of CD3+ lymphocytes was highest in animals with simultaneous vascular and neuroparenchymal lesions and high viral genome load. In one animal, OvHV-2 was exclusively observed in CD3+ lymphocytes but not in neurons or microglia. In conclusion, the neuropathological phenotype of bovine MCF in the brain was variable. In some cases, lesions mimicked neurotropic viral encephalitis, while pathognomonic necrotizing arteritis was not a consistent feature of neural MCF. Therefore, molecular detection of OvHV-2 is warranted in the presence of nonsuppurative encephalitis and in the absence of necrotizing arteritis.

scholarly journals Isolation and functional characterization of two dioxygenasese putatively involved in bixin biosynthesis in annatto (Bixa orellana L.)

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7064 ◽  
Author(s):  
Victor Manuel Carballo-Uicab ◽  
Yair Cárdenas-Conejo ◽  
Alba Adriana Vallejo-Cardona ◽  
Margarita Aguilar-Espinosa ◽  
Jacobo Rodríguez-Campos ◽  
...  
Keyword(s):  
Food Industry ◽  
Developmental Stages ◽  
Functional Characterization ◽  
Economic Value ◽  
Bixa Orellana ◽  
Reverse Transcription Pcr ◽  
Bona Fide

Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world’s food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthesis. We used several approaches in order to discriminate the best candidates with CCDs genes. A reverse transcription-PCR (RT-qPCR) expression analysis was carried out in five developmental stages of two accessions of B. orellana seeds with different bixin contents: (P13W, low bixin producer and N4P, high bixin producer). The results showed that three BoCCDs (BoCCD4-1, BoCCD4-3, and BoCCD1-1) had an expression pattern consistent with bixin accumulation during seed development. Additionally, an alignment of the CCD enzyme family and homology models of proteins were generated to verify whether the newly proposed CCD enzymes were bona fide CCDs. The study confirmed that these three enzymes were well-preserved and belonged to the CCD family. In a second selection round, the three CCD genes were analyzed by in situ RT-qPCR in seed tissue. Results indicated that BoCCD4-3 and BoCCD1-1 exhibited tissue-specific expressions in the seed aril. To test whether the two selected CCDs had enzymatic activity, they were expressed in Escherichia coli; activity was determined by identifying their products in the crude extract using UHPLC-ESI-QTOF-MS/MS. The cleavage product (bixin aldehyde) was also analyzed by Fourier transform infrared. The results indicated that both BoCCD4-3 and BoCCD1-1 cleave lycopene in vitro at 5,6-5′,6′.

scholarly journals Neurochemical Characterization of Neurons Expressing Estrogen Receptor β in the Hypothalamic Nuclei of Rats Using in Situ Hybridization and Immunofluorescence

2019 ◽  
Vol 21 (1) ◽  
pp. 115 ◽  
Author(s):  
Moeko Kanaya ◽  
Shimpei Higo ◽  
Hitoshi Ozawa
Keyword(s):  
In Situ Hybridization ◽  
High Ratio ◽  
Estrogen Signaling ◽  
Stria Terminalis ◽  
Bed Nucleus ◽  
Hypothalamic Nuclei ◽  
Vasopressin Neurons ◽  
The Brain

Estrogens play an essential role in multiple physiological functions in the brain, including reproductive neuroendocrine, learning and memory, and anxiety-related behaviors. To determine these estrogen functions, many studies have tried to characterize neurons expressing estrogen receptors known as ERα and ERβ. However, the characteristics of ERβ-expressing neurons in the rat brain still remain poorly understood compared to that of ERα-expressing neurons. The main aim of this study is to determine the neurochemical characteristics of ERβ-expressing neurons in the rat hypothalamus using RNAscope in situ hybridization (ISH) combined with immunofluorescence. Strong Esr2 signals were observed especially in the anteroventral periventricular nucleus (AVPV), bed nucleus of stria terminalis, hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, and medial amygdala, as previously reported. RNAscope ISH with immunofluorescence revealed that more than half of kisspeptin neurons in female AVPV expressed Esr2, whereas few kisspeptin neurons were found to co-express Esr2 in the arcuate nucleus. In the PVN, we observed a high ratio of Esr2 co-expression in arginine-vasopressin neurons and a low ratio in oxytocin and corticotropin-releasing factor neurons. The detailed neurochemical characteristics of ERβ-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERβ.

scholarly journals Molecular cloning and characterization of a novel phospholipase C, PLC-η

2005 ◽  
Vol 389 (1) ◽  
pp. 181-186 ◽  
Author(s):  
Jong-Ik HWANG ◽  
Yong-Seok OH ◽  
Kum-Joo SHIN ◽  
Hyun KIM ◽  
Sung Ho RYU ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Phospholipase C ◽  
Neuronal Cell ◽  
Zona Incerta ◽  
Pleckstrin Homology Domain ◽  
Molecular Biological Analysis ◽  
Splicing Variants ◽  
The Brain

PLC (phospholipase C) plays an important role in intracellular signal transduction by hydrolysing phosphatidylinositol 4,5-bisphosphate, a membrane phospholipid. To date, 12 members of the mammalian PLC isoforms have been identified and classified into five isotypes β, γ, δ, ε and ζ, which are regulated by distinct mechanisms. In the present study, we describe the identification of a novel PLC isoform in the brains of human and mouse, named PLC-η, which contains the conserved pleckstrin homology domain, X and Y domains for catalytic activity and the C2 domain. The first identified gene encoded 1002 (human) or 1003 (mouse) amino acids with an estimated molecular mass of 115 kDa. The purified recombinant PLC-η exhibited Ca2+-dependent catalytic activity on phosphatidylinositol 4,5-bisphosphate. Furthermore, molecular biological analysis revealed that the PLC-η gene was transcribed to several splicing variants. Although some transcripts were detected in most of the tissues we examined, the transcript encoding 115 kDa was restricted to the brain and lung. In addition, the expression of the 115 kDa protein was defined in only nerve tissues such as the brain and spinal cord. In situ hybridization analysis with brain revealed that PLC-η was abundantly expressed in various regions including cerebral cortex, hippocampus, zona incerta and cerebellar Purkinje cell layer, which are neuronal cell-enriched regions. These results suggest that PLC-η may perform fundamental roles in the brain.

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