Metabolism of uniformly labeled 13C-eicosapentaenoic acid and 13C-arachidonic acid in young and old men.

PubMed

Léveillé, Pauline; Chouinard-Watkins, Raphaël; Windust, Anthony; Lawrence, Peter; Cunnane, Stephen C; Brenna, J Thomas; Plourde, Mélanie

2017-08-01

Background: Plasma eicosapentaenoic acid (EPA) and arachidonic acid (AA) concentrations increase with age. Objective: The aim of this study was to evaluate EPA and AA metabolism in young and old men by using uniformly labeled carbon-13 ( 13 C) fatty acids. Design: Six young (∼25 y old) and 6 old (∼75 y old) healthy men were recruited. Each participant consumed a single oral dose of 35 mg 13 C-EPA and its metabolism was followed in the course of 14 d in the plasma and 28 d in the breath. After the washout period of ≥28 d, the same participants consumed a single oral dose of 50 mg 13 C-AA and its metabolism was followed for 28 d in plasma and breath. Results: There was a time × age interaction for 13 C-EPA ( P time × age = 0.008), and the shape of the postprandial curves was different between young and old men. The 13 C-EPA plasma half-life was ∼2 d for both young and old men ( P = 0.485). The percentage dose recovered of 13 C-EPA per hour as 13 CO 2 and the cumulative β-oxidation of 13 C-EPA did not differ between young and old men. At 7 d, however, old men had a >2.2-fold higher plasma 13 C-DHA concentration synthesized from 13 C-EPA compared with young men ( P age = 0.03). 13 C-AA metabolism was not different between young and old men. The 13 C-AA plasma half-life was ∼4.4 d in both young and old participants ( P = 0.589). Conclusions: The metabolism of 13 C-AA was not modified by age, whereas 13 C-EPA metabolism was slightly but significantly different in old compared with young men. The higher plasma 13 C-DHA seen in old men may be a result of slower plasma DHA clearance with age. This trial was registered at clinicaltrials.gov as NCT02957188. © 2017 American Society for Nutrition.

Synthesis and characterization of Allium cepa L. as photosensitizer of dye-sensitized solar cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutikno, E-mail: smadnasri@yahoo.com; Afrian, Noverdi; Supriadi,

The synthesis and characterization of Allium cepa L. used as natural pigment for natural dye sensitizer of solar cell has successfully done and anthocyanin is extracted. Anthocynin is color pigment of plant which has characteristic absorption spectrum of photon and excites electrons up to pigment molecules. As the anthocyanin absorbed light increases the excited electrons increase as well. The generated current also increases and it leads to the efficiency increase. The energy conversion efficiency of the cells sensitized with dye of Allium cepa L. was 3,045 x 10{sup −4}%. A simple technique was taken to fabricate dye sensitizer solar cellmore » is spincoating.« less

Effects of Mg{sup 2+}, Co{sup 2+}, and Hg{sup 2+} on the nucleus and nucleolus in root tip cells of allium cepa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, D.; Jiang, W.; Wang, W.

Metal toxicity in plants has been known for a long time. Much importance has increasingly been attached to the problems of metal pollution with the development of modern industry and agriculture. If metals in plants are accumulated to a large extent, it might seriously affect them. The cytological effects of cobalt and mercury have been studied in Allium cepa by documentation of c-mitosis. Also, the quantification of chromosome aberration in Vicia faba root-tip cells treated by magnesium sulphate and in Allium cepa by metyl mercury chloride and mercuric chloride has been reported. Cytological research on the poisoning effects of Mg,more » Co and Hg on the nuclei and nucleoli in root-tip cells of plants has hardly been reported. The aim of this study was to determine the effects of different concentrations of magnesium, cobalt and mercury ions on root growth, and on the nuclei and nucleoli of root tip cells of Allium-cepa. 20 refs., 3 figs.« less

Toxic effects of environmental pollutants: Comparative investigation using Allium cepa L. and Lactuca sativa L.

PubMed

Silveira, Graciele Lurdes; Lima, Maria Gabriela Franco; Reis, Gabriela Barreto Dos; Palmieri, Marcel José; Andrade-Vieria, Larissa Fonseca

2017-07-01

Studies that help understand the mechanisms of action of environmental pollutants are extremely important in environmental toxicology. In this context, assays using plants as models stand out for their simplicity and low performance cost. Among the plants used for this purpose, Allium cepa L. is the model most commonly applied for cytogenotoxic tests, while Lactuca sativa L., already widely used in phytotoxic investigations, has been gaining prominence in cytotoxic analyses. The present study aimed to compare the responses of A. cepa and L. sativa via macroscopic (root growth) and microscopic analyses (cell cycle and DNA fragmentation via TdT-mediated deoxy-uracil nick and labeling (TUNEL) and comet assays) after exposure of their roots to environmental pollutants with known cytogenotoxic mechanisms. Both species presented sensitive and efficient response to the applied tests after exposure to the DNA-alkylating agent Methyl Methanesulfonate (MMS), the heavy metal Cadmium, the aluminum industry waste Spent Potliner (SPL) and the herbicide Atrazine. However, they differed regarding the responses to the evaluated endpoints. Overall, A. cepa was more efficient in detecting clastogenic changes, arising from DNA breakage, while L. sativa rather detected aneugenic alterations, related to chromosome segregation in mitosis. In the tests applied to verify DNA fragmentation (comet and TUNEL assays), A. cepa presented higher sensitivity. In conclusion, both models are efficient to evaluate toxicological risks of environmental pollutants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phytochemical Profiles and Antimicrobial Activities of Allium cepa Red cv. and A. sativum Subjected to Different Drying Methods: A Comparative MS-Based Metabolomics.

PubMed

Farag, Mohamed A; Ali, Sara E; Hodaya, Rashad H; El-Seedi, Hesham R; Sultani, Haider N; Laub, Annegret; Eissa, Tarek F; Abou-Zaid, Fouad O F; Wessjohann, Ludger A

2017-05-08

Plants of the Allium genus produce sulphur compounds that give them a characteristic (alliaceous) flavour and mediate for their medicinal use. In this study, the chemical composition and antimicrobial properties of Allium cepa red cv. and A. sativum in the context of three different drying processes were assessed using metabolomics. Bulbs were dried using either microwave, air drying, or freeze drying and further subjected to chemical analysis of their composition of volatile and non-volatile metabolites. Volatiles were collected using solid phase micro-extraction (SPME) coupled to gas chromatography-mass spectrometry (GC/MS) with 42 identified volatiles including 30 sulphur compounds, four nitriles, three aromatics, and three esters. Profiling of the polar non-volatile metabolites via ultra-performance liquid chromatography coupled to high resolution MS (UPLC/MS) annotated 51 metabolites including dipeptides, flavonoids, phenolic acids, and fatty acids. Major peaks in GC/MS or UPLC/MS contributing to the discrimination between A. sativum and A. cepa red cv. were assigned to sulphur compounds and flavonoids. Whereas sulphur conjugates amounted to the major forms in A. sativum , flavonoids predominated in the chemical composition of A. cepa red cv. With regard to drying impact on Allium metabolites, notable and clear separations among specimens were revealed using principal component analysis (PCA). The PCA scores plot of the UPLC/MS dataset showed closer metabolite composition of microwave dried specimens to freeze dried ones, and distant from air dried bulbs, observed in both A. cepa and A. sativum . Compared to GC/MS, the UPLC/MS derived PCA model was more consistent and better in assessing the impact of drying on Allium metabolism. A phthalate derivative was found exclusively in a commercial garlic preparation via GC/MS, of yet unknown origin. The freeze dried samples of both Allium species exhibited stronger antimicrobial activities compared to dried specimens with A. sativum being in general more active than A. cepa red cv.

Genotypic variation in sulfur assimilation and metabolism of onion (Allium cepa L.) III. Characterization of sulfite reductase

USDA-ARS?s Scientific Manuscript database

Genomic and cDNA sequences corresponding to a ferredoxin-sulfite reductase (SiR) have been cloned from bulb onion (Allium cepa L.) and the expression of the gene and activity of the enzyme characterised with respect to sulfur (S) supply. Cloning, mapping and expression studies revealed that onion ha...

Base-catalyzed hydrolysis and speciation-dependent photolysis of two cephalosporin antibiotics, ceftiofur and cefapirin.

PubMed

Ribeiro, Alyson R; Lutze, Holger V; Schmidt, Torsten C

2018-05-01

Lately, special attention has been given to veterinary cephalosporin antibiotics due to their broad activity spectrum and significant consumption. Indeed, the determination of hydrolytic and photolytic kinetics provides a better comprehension of the undesired persistence of cephalosporins in aqueous matrices. In this work, the two widely used veterinary antibiotics ceftiofur (CEF) and cefapirin (CEPA) showed high instability under alkaline conditions, degrading in few minutes at pH > 11. In buffered solutions at neutral pH and natural temperature (T = 22 ± 1 °C), both drugs presented moderate stability (t½ = 3 d, CEPA and 1.4 d, CEF). Our study also demonstrated that CEPA and CEF speciation did not significantly influence the direct photolysis rates. Using a simulated water disinfection set-up (λ = 254 nm), all ionic species of CEF and CEPA presented fast and similar pseudo-first order degradation rates, k app 0.0095 ± 0.0004 and 0.0092 ± 0.001 cm 2 mJ -1 , respectively. Furthermore, using surface water in hydrolysis experiments, CEF demonstrated significant matrix-dependent stability with a half-life (t½ = 14.7 d) tenfold higher than in buffered solutions. In contrast, CEPA presented a very similar hydrolysis rate in river water (t½ = 4.2 d) and a subtle faster photo-degradation rate in this same matrix (k app 0.0128 ± 0.001 cm 2 mJ -1 ), highlighting the importance of disinfection radiation for cephalosporin depletion in aqueous environments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antioxidant and schistosomicidal effect of Allium sativum and Allium cepa against Schistosoma mansoni different stages.

PubMed

Mantawy, M M; Aly, H F; Zayed, N; Fahmy, Z H

2012-07-01

The schistosomicidal properties of garlic (Allium sativum) and onion (Allium cepa) powder were tested in vitro against Schistosoma mansoni miracidia, schistosomula, cercaria and adult worms. Results indicate their strong biocidal effects against all stages of the parasite and also show scavenging inhibitory effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO). In the present work, the in vivo effects of A. sativum and A. cepa on lipid peroxide and some antioxidant enzymes; thioredoxin reductase (TrxR), sorbitol dehydrogenase (SDH), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) (as they have a crucial role in host protection against invading parasite) were also studied. The data demonstrate that, there was a significant inhibition in SOD, CAT, GR, TrxR and SDH in infected liver while, significant elevation was detected in lipid peroxide as compared to the normal control. The current resultS clearly revealed that, the used both edible plants enhance the host antioxidant system indicated by lowering in lipid peroxide and stimulation of SOD, CAT, GR, TrxR and SDH enzyme levels. Enhancement of such enzymes using A. sativum and A. cepa could in turn render the parasite vulnerable to damage by the host and may play a role in the antischistosomal potency of the used food ingredients.

Evaluation of allium and its seasoning on toxigenic, nutritional, and sensorial profiles of groundnut oil.

PubMed

Murugan, Kasi; Anandaraj, K; Al-Sohaibani, Saleh A

2014-04-01

Mitigation of xerophilic storage fungi-associated aflatoxin threat in culinary oil will be a new technology advantage to food industries. Groundnut oil isolate Aspergillus flavus MTCC 10680 susceptibility to Allium species (A. sativum L., A. cepa L., and A. cepa var. aggregatum) extracts, composition, and in silico confirmation of extract's phytoconstituent aflatoxin synthesis inhibition were determined. The behavior of seasoning carrier medium groundnut oil in the presence of Allium was also determined. All the Allium species extracts exhibited concentration dependent in vitro inhibition on mycelial biomass, radial growth, and toxin elaboration. The gas chromatography-mass spectrometry revealed the presence of 28, 16, and 9 compounds in the extracts of A. sativum, A. cepa, A. cepa var. aggregatum, respectively. The Allium phytocostituents-like hexadecanoic acid, 5-Octanoyl-2,4,6(1H,3H,5H)-pyrimidinetrione, Guanosine, and so on, showed higher binding energy with aflatoxin synthesis key enzyme ver1. Allium seasoning increased the typical nutty odor of the groundnut oil with sweet aroma note as well as intensification of pale yellow color. Allium seasoning exhibited the highest aflatoxin detoxification and aroma development without any nutritional loss. Culinary oil Allium seasoning has anti-aflatoxin and food additive potential for use in food industries. © 2014 Institute of Food Technologists®

Efficacy of Allium cepa test system for screening cytotoxicity and genotoxicity of industrial effluents originated from different industrial activities.

PubMed

Pathiratne, Asoka; Hemachandra, Chamini K; De Silva, Nimal

2015-12-01

Efficacy of Allium cepa test system for screening cytotoxicity and genotoxicity of treated effluents originated from four types of industrial activities (two textile industries, three rubber based industries, two common treatment plants of industrial zones, and two water treatment plants) was assessed. Physico-chemical parameters including the heavy metal/metalloid levels of the effluents varied depending on the industry profile, but most of the measured parameters in the effluents were within the specified tolerance limits of Sri Lankan environmental regulations for discharge of industrial effluents into inland surface waters. In the A. cepa test system, the undiluted effluents induced statistically significant root growth retardation, mitosis depression, and chromosomal aberrations in root meristematic cells in most cases in comparison to the dilution water and upstream water signifying effluent induced cytotoxicity and genotoxicity. Ethyl methane sulphonate (a mutagen, positive control) and all the effluents under 1:8 dilution significantly induced total chromosomal aberrations in root meristematic cells in comparison to the dilution water and upstream water indicating inadequacy of expected 1:8 dilutions in the receiving waters for curtailing genotoxic impacts. The results support the use of a practically feasible A. cepa test system for rapid screening of cytotoxicity and genotoxicity of diverse industrial effluents discharging into inland surface waters.

Mutagenic and antimutagenic effects of aqueous extract of rosemary (Rosmarinus officinalis L.) on meristematic cells of Allium cepa.

PubMed

Felicidade, I; Lima, J D; Pesarini, J R; Monreal, A C D; Mantovani, M S; Ribeiro, L R; Oliveira, R J

2014-11-28

Polyphenolic compounds present in rosemary were found to have antioxidant properties, anticarcinogenic activity, and to increase the detoxification of pro-carcinogens. The aim of the study was to determine the effect the aqueous extract of rosemary (AER) on mutagenicity induced by methylmethane sulfonate in meristematic cells of Allium cepa, as well as to describe its mode of action. Anti-mutagenicity experiments were carried out with 3 different concentrations of AER, which alone showed no mutagenic effects. In antimutagenicity experiments, AER showed chemopreventive activity in cultured meristematic cells of A. cepa against exposure to methylmethane sulfonate. Additionally, post-treatment and simultaneous treatment using pre-incubation protocols were the most effective. Evaluation of different protocols and the percent reduction in DNA indicated bioantimutagenic as well desmutagenic modes of action for AER. AER may be chemopreventive and antimutagenic.

Biodegradability, toxicity and mutagenicity of detergents: Integrated experimental evaluations.

PubMed

Pedrazzani, Roberta; Ceretti, Elisabetta; Zerbini, Ilaria; Casale, Rosario; Gozio, Eleonora; Bertanza, Giorgio; Gelatti, Umberto; Donato, Francesco; Feretti, Donatella

2012-10-01

The widespread use of detergents has raised concern with regard to the environmental pollution caused by their active ingredients, which are biorefractory, toxic and persistent. Since detergents are complex mixtures of different substances, in which synergistic effects may occur, we aimed to assess the mutagenicity of different detergent formulations, taking into account aquatic toxicity and ready biodegradability. We performed a ready biodegradability test (OECD 301 F), Daphnia magna and Vibrio fischeri toxicity tests, and mutagenicity tests (Salmonella/microsome test, Allium cepa test and comet assay). Six detergent formulations were examined, 3 pre-manufacture and 3 commercially available. All detergents presented ready biodegradability. EC50 values varied for all products, according to the marker organism used, but were always higher than the more stringent value considered for aquatic toxicity assessment (V. fischeri 10-60 mg/L; D. magna 25-300 mg/L; A. cepa 250-2000 mg/L). None of the detergents caused mutations in bacteria. However, one commercial ecolabelled product induced an increase in micronucleus frequency in A. cepa root cells. All pre-manufacture detergents and one commercial one, which gave negative results in the Ames and A. cepa tests, induced DNA damage in human leukocytes. A more accurate evaluation of the environmental impact of complex mixtures such as detergents requires a battery of tests to describe degradation, as well as toxicological and mutagenic features. Copyright © 2012 Elsevier Inc. All rights reserved.

Hybrid Correlation Energy (HyCE): An Approach Based on Separate Evaluations of Internal and External Components.

PubMed

Ivanic, Joseph; Schmidt, Michael W

2018-06-04

A novel hybrid correlation energy (HyCE) approach is proposed that determines the total correlation energy via distinct computation of its internal and external components. This approach evolved from two related studies. First, rigorous assessment of the accuracies and size extensivities of a number of electron correlation methods, that include perturbation theory (PT2), coupled-cluster (CC), configuration interaction (CI), and coupled electron pair approximation (CEPA), shows that the CEPA(0) variant of the latter and triples-corrected CC methods consistently perform very similarly. These findings were obtained by comparison to near full CI results for four small molecules and by charting recovered correlation energies for six steadily growing chain systems. Second, by generating valence virtual orbitals (VVOs) and utilizing the CEPA(0) method, we were able to partition total correlation energies into internal (or nondynamic) and external (or dynamic) parts for the aforementioned six chain systems and a benchmark test bed of 36 molecules. When using triple-ζ basis sets it was found that per orbital internal correlation energies were appreciably larger than per orbital external energies and that the former showed far more chemical variation than the latter. Additionally, accumulations of external correlation energies were seen to proceed smoothly, and somewhat linearly, as the virtual space is gradually increased. Combination of these two studies led to development of the HyCE approach, whereby the internal and external correlation energies are determined separately by CEPA(0)/VVO and PT2/external calculations, respectively. When applied to the six chain systems and the 36-molecule benchmark test set it was found that HyCE energies followed closely those of triples-corrected CC and CEPA(0) while easily outperforming MP2 and CCSD. The success of the HyCE approach is more notable when considering that its cost is only slightly more than MP2 and significantly cheaper than the CC approaches.

Production and characterization of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome(s) of Japanese bunching onion (A. fistulosum L.).

PubMed

Hang, Tran Thi Minh; Shigyo, Masayoshi; Yamauchi, Naoki; Tashiro, Yosuke

2004-10-01

First and second backcrosses of amphidiploid hybrids (2n = 4x = 32, genomes AAFF) between shallot (Allium cepa Aggregatum group) and A. fistulosum were conducted to produce A. cepa - A. fistulosum alien addition lines. When shallot (A. cepa Aggregatum group) was used as a pollinator, the amphidiploids and allotriploids set germinable BC(1) and BC(2) seeds, respectively. The 237 BC(1) plants mainly consisted of 170 allotriploids (2n = 3x = 24, AAF) and 42 hypo-allotriploids possessing 23 chromosomes, i.e., single-alien deletions (2n = 3x-1 = 23, AAF-nF). The single-alien deletions in the BC(1) progeny showed dwarfing characteristics and were discriminated from the allotriploids (2n = 24) and hyper-allotriploids (2n = 25) by means of flow cytometric analysis. The chromosome numbers of 46 BC(2) seedlings varied from 16 to 24. Eight monosomic additions (2n = 2x+1 = 17, AA+nF) and 20 single-alien deletions were found in these BC(2) seedlings. Consequently, six kinds of A. cepa - A. fistulosum alien chromosome additions possessing different chromosome numbers (2n = 17, 18, 20, 21, 22, 23) were recognized in the BC(1) and BC(2) populations. A total of 79 aneuploids, including 62 single-alien deletions, were analyzed by a chromosome 6F-specific isozyme marker (Got-2) in order to recognize its existence in their chromosome complements. This analysis revealed that two out of 62 single-alien deletions did not possess 6F. One (AAF-6F) out of the possible eight single-alien deletions could be identified at first. The present study is a first step toward the development of a useful tool, such as a complete set of eight different single-alien deletions, for the rapid chromosomal assignment of genes and genetic markers in A. fistulosum.

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips.

PubMed

Unal, Meral; Palavan-Unsal, Narcin; Tufekci, M A

2008-03-01

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

Evaluation of Toxic, Cytotoxic, Mutagenic, and Antimutagenic Activities of Natural and Technical Cashew Nut Shell Liquids Using the Allium cepa and Artemia salina Bioassays

PubMed Central

Leite, Aracelli de Sousa; Oliveira, George Laylson da Silva; Gomes Júnior, Antonio L.; de Lima, Sidney Gonçalo; Citó, Antônia Maria das Graças Lopes; de Freitas, Rivelilson M.; Melo-Cavalcante, Ana Amélia de C.; Dantas Lopes, José Arimateia

2015-01-01

The cashew nut releases a substance that is known as cashew nut shell liquid (CNSL). There are both natural (iCNSL) and technical (tCNSL) cashew nut shell liquids. This study used an Artemia salina bioassay to evaluate the toxic effects of iCNSL and tCNSL cashew nut shell liquids. It also evaluated the toxicity, cytotoxicity, and mutagenicity of CNSL and its effects on the damage induced by copper sulfate (CuSO4·5H2O) on the meristems' root of Allium cepa. Effects of the damage induced by CuSO4·5H2O were evaluated before (pre-), during (co-), and after (post-) treatments. The iCNSL contained 94.5% anacardic acid, and the tCNSL contained 91.3% cardanol. The liquids were toxic to A. salina. Toxicity, cytotoxicity, and mutagenicity were observed with iCNSL compared with the negative control. Similarly, iCNSL failed to inhibit the toxicity and cytotoxicity of CuSO4·5H2O. The tCNSL was not toxic, cytotoxic, or mutagenic in any of the concentrations. However, the lowest iCNSL concentrations and all of the tCNSL concentrations had preventive, antimutagenic, and reparative effects on micronuclei and on chromosomal aberrations in the A. cepa. Therefore, protective, modulating, and reparative effects may be observed in the A. cepa, depending on the concentration and type of CNSL used. PMID:25861638

Evaluation of toxic, cytotoxic, mutagenic, and antimutagenic activities of natural and technical cashew nut shell liquids using the Allium cepa and Artemia salina bioassays.

PubMed

Leite, Aracelli de Sousa; Dantas, Alisson Ferreira; Oliveira, George Laylson da Silva; Gomes Júnior, Antonio L; de Lima, Sidney Gonçalo; Citó, Antônia Maria das Graças Lopes; de Freitas, Rivelilson M; Melo-Cavalcante, Ana Amélia de C; Dantas Lopes, José Arimateia

2015-01-01

The cashew nut releases a substance that is known as cashew nut shell liquid (CNSL). There are both natural (iCNSL) and technical (tCNSL) cashew nut shell liquids. This study used an Artemia salina bioassay to evaluate the toxic effects of iCNSL and tCNSL cashew nut shell liquids. It also evaluated the toxicity, cytotoxicity, and mutagenicity of CNSL and its effects on the damage induced by copper sulfate (CuSO4·5H2O) on the meristems' root of Allium cepa. Effects of the damage induced by CuSO4·5H2O were evaluated before (pre-), during (co-), and after (post-) treatments. The iCNSL contained 94.5% anacardic acid, and the tCNSL contained 91.3% cardanol. The liquids were toxic to A. salina. Toxicity, cytotoxicity, and mutagenicity were observed with iCNSL compared with the negative control. Similarly, iCNSL failed to inhibit the toxicity and cytotoxicity of CuSO4·5H2O. The tCNSL was not toxic, cytotoxic, or mutagenic in any of the concentrations. However, the lowest iCNSL concentrations and all of the tCNSL concentrations had preventive, antimutagenic, and reparative effects on micronuclei and on chromosomal aberrations in the A. cepa. Therefore, protective, modulating, and reparative effects may be observed in the A. cepa, depending on the concentration and type of CNSL used.

Combination of Cymbopogon citratus and Allium cepa essential oils increased antibacterial activity in leafy vegetables.

PubMed

Ortega-Ramirez, Luis A; Silva-Espinoza, Brenda A; Vargas-Arispuro, Irasema; Gonzalez-Aguilar, Gustavo A; Cruz-Valenzuela, M Reynaldo; Nazzaro, Filomena; Ayala-Zavala, J Fernando

2017-05-01

Cymbopogon citratus and Allium cepa essential oils (EOs) are rich in terpenes and sulfur compounds respectively, both with antibacterial activity and different cell targets, supporting the idea that their combination can increase their efficacy. Major constituents of C. citratus were geranial and neral, while A. cepa presented dipropyl disulfide and dipropyl trisulfide. Cymbopogon citratus and A. cepa EOs inhibited the in vitro growth of Escherichia coli O157:H7 (minimal inhibitory concentrations of 2.21 and 5.13 g L -1 respectively), Salmonella Choleraesuis (3.04 and 1.28 g L -1 ), Listeria monocytogenes (1.33 and 2.56 g L -1 ) and Staphylococcus aureus (0.44 and 5.26 g L -1 ). Application of the EO combination to spinach caused a greater reduction in E. coli (2.34 log colony-forming units (CFU) g -1 ), S. Choleraesuis (2.94 log CFU g -1 ), L. monocytogenes (2.06 log CFU g -1 ) and S. aureus (1.37 log CFU g -1 ) compared with higher doses of individual EOs; a similar effect was observed for romaine lettuce. Individual and combined EOs caused a reduction in flavor acceptability level; however, no significant differences were found among odor acceptability of control vegetables and those treated with the EO combination and C. citratus EO. Leafy vegetables treated with the EO combination showed higher antibacterial protection and odor acceptability compared with individual EO treatments. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Quantification of Quercetin Obtained from Allium cepa Lam. Leaves and its Effects on Streptozotocin-induced Diabetic Neuropathy.

PubMed

Dureshahwar, Khan; Mubashir, Mohammed; Une, Hemant Devidas

2017-01-01

Antioxidant potential has protective effects in diabetic neuropathy (DN); hence, the present study was designed with an objective to quantify quercetin from shade-dried leaves of Allium cepa Lam. and to study its effects on streptozotocin (STZ)-induced chronic DN. The shade-dried leaves of A. cepa Lam. were extracted with methanol and then fractionated using ethyl acetate (ACEA). The quantification of quercetin in ACEA was evaluated by high-performance thin layer chromatography (HPTLC). The STZ (40 mg/kg) was administered to Sprague-Dawley rats (180-250 g) maintained at normal housing conditions. The STZ was administered once a day for 3 consecutive days. The elevation in blood glucose was monitored for 3 weeks periodically using flavin adenine dinucleotide-glucose dehydrogenase method by Contour TS glucometer. Rats showing blood glucose above 250 mg/dl were selected for the study. Animals were divided into eight groups. ACEA (25, 50, and 100 mg/kg), quercetin (40 mg/kg), metformin (120 mg/kg), and gabapentin (100 mg/kg) were given orally once a day for 2 weeks. The blood glucose level was again measured at the end of treatment to assess DN. Thermal hyperalgesia, cold allodynia, motor incoordination, and neurotoxicity were studied initially and at the end of 2-week treatment. Biochemical parameters were also evaluated after 2-week drug treatment. The quercetin present in ACEA was 4.82% by HPTLC. All the ACEA treatment reduces blood glucose level at the end of the 2-week study and shows a significant neuroprotective effect in STZ-induced DN in the above experimental models. The quercetin present in ACEA proved protective effect in STZ-induced DN. High-performance thin layer chromatography reveals the presence of 4.82% quercetin in Allium cepa ethyl acetate. (ACEA). Its investigation against various diabetic neuropathy biomarkers has proved that ACEA has significant blood glucose reducing action shown neuroprotective action in thermal hyperalgesia, motor incoordination, and biochemical parameters. Abbreviations Used : HPTLC: High-performance thin layer chromatography, TLC: Thin layer chromatography, UV: Ultraviolet, ACEA: Allium cepa ethyl acetate, STZ: Streptozotocin, LDL: Low-density lipids, HDL: High-density lipids.

Mitochondrial and Chromosomal Damage Induced by Oxidative Stress in Zn2+ Ions, ZnO-Bulk and ZnO-NPs treated Allium cepa roots

PubMed Central

Ahmed, Bilal; Dwivedi, Sourabh; Abdin, Malik Zainul; Azam, Ameer; Al-Shaeri, Majed; Khan, Mohammad Saghir; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Musarrat, Javed

2017-01-01

Large-scale synthesis and release of nanomaterials in environment is a growing concern for human health and ecosystem. Therefore, we have investigated the cytotoxic and genotoxic potential of zinc oxide nanoparticles (ZnO-NPs), zinc oxide bulk (ZnO-Bulk), and zinc ions (Zn2+) in treated roots of Allium cepa, under hydroponic conditions. ZnO-NPs were characterized by UV-visible, XRD, FT-IR spectroscopy and TEM analyses. Bulbs of A. cepa exposed to ZnO-NPs (25.5 nm) for 12 h exhibited significant decrease (23 ± 8.7%) in % mitotic index and increase in chromosomal aberrations (18 ± 7.6%), in a dose-dependent manner. Transmission electron microcopy and FT-IR data suggested surface attachment, internalization and biomolecular intervention of ZnO-NPs in root cells, respectively. The levels of TBARS and antioxidant enzymes were found to be significantly greater in treated root cells vis-à-vis untreated control. Furthermore, dose-dependent increase in ROS production and alterations in ΔΨm were observed in treated roots. FT-IR analysis of root tissues demonstrated symmetric and asymmetric P=O stretching of >PO2− at 1240 cm−1 and stretching of C-O ribose at 1060 cm−1, suggestive of nuclear damage. Overall, the results elucidated A. cepa, as a good model for assessment of cytotoxicity and oxidative DNA damage with ZnO-NPs and Zn2+ in plants. PMID:28120857

Water quality of a coastal lagoon (ES, Brazil): abiotic aspects, cytogenetic damage, and phytoplankton dynamics.

PubMed

Duarte, Ian Drumond; Silva, Nayara Heloisa Vieira Fraga; da Costa Souza, Iara; de Oliveira, Larissa Bassani; Rocha, Lívia Dorsch; Morozesk, Mariana; Bonomo, Marina Marques; de Almeida Pereira, Thaís; Dias, Mauro Cesar; de Oliveira Fernandes, Valéria; Matsumoto, Silvia Tamie

2017-04-01

Assessment of water resources requires interdisciplinary studies that include multiple ecosystem aspects. This study evaluated the water quality of Juara Lagoon (ES, Brazil) based on physical and chemical variables, cytogenetic responses in Allium cepa and phytoplankton dynamics. Three sampling sites were defined and water samples were collected during two sampling periods. Analyses such as determination of photic zone, conductivity, and concentrations of nutrients and metals were conducted as well as cytotoxic, mutagenic, and genotoxic potentials using A. cepa test. The main attributes of phytoplankton community, such as total richness, total density, density by class, dominance, and diversity, were also evaluated. Results have revealed that Juara Lagoon has signs of artificial eutrophication at two sampling sites due to high levels of total phosphorus and ammonia nitrogen. Cytotoxic, genotoxic, and mutagenic potentials were detected as well as high concentrations of Fe and Mn. Furthermore, 165 phytoplankton taxa were recorded, with highest richness in Chlorophyceae and Cyanophyceae classes. In addition, Cyanophyceae presented as the highest density class. A. cepa test and phytoplankton community evaluation indicated that the ecological quality of Juara Lagoon is compromised.

Antigenotoxic and antimutagenic effects of Schinus terebinthifolius Raddi in Allium cepa and Swiss mice: a comparative study.

PubMed

Fedel-Miyasato, L E S; Formagio, A S N; Auharek, S A; Kassuya, C A L; Navarro, S D; Cunha-Laura, A L; Monreal, A C D; Vieira, M C; Oliveira, R J

2014-04-30

It is estimated that 60% of anticancer drugs are derived directly or indirectly from medicinal plants. Schinus terebinthifolius Raddi (Anacardiaceae) is traditionally used in Brazilian medicine to treat inflammation, ulcers, and tumors. Because of the need to identify new antimutagenic agents and to determine their mechanism of action, this study evaluated the chemopreventive activity of the methanolic extract from leaves of S. terebinthifolius (MEST) in Allium cepa cells and in Swiss mice analyzing different protocols of MEST in association with DNA-damaging agents. The antigenotoxic and antimutagenic aspects in peripheral blood were evaluated using the comet and micronucleus assays, respectively. The percentage of damage reduction was used to compare the A. cepa and mice results. Our results showed for the first time that MEST can act as a chemopreventive compound that promotes cellular genome integrity by desmutagenic and bioantimutagenic activities in vegetal and animal models. This finding may therefore have therapeutic applications that can indirectly correlate to the prevention and/or treatment of the degenerative diseases such as cancer.

Accumulation of heavy metals in edible parts of vegetables irrigated with waste water and their daily intake to adults and children, District Mardan, Pakistan.

PubMed

Amin, Noor-ul; Hussain, Anwar; Alamzeb, Sidra; Begum, Shumaila

2013-02-15

Green vegetable crops irrigated with wastewater are highly contaminated with heavy metals and are the main source of human exposure to the contaminants. In this study accumulation of eight heavy metals (Cu, Ni, Zn, Cr, Fe, Mn, Co and Pb) in green vegetables like Allium cepa, Allium sativum, Solanum lycopersicum and Solanum melongena, irrigated with wastewater in Mardan are studied using Atomic Absorption spectrophotometer. The studied metals in vegetable grown on wastewater irrigated soil were significantly higher than those of tube well water irrigated soil and WHO/FAO permissible limits (P<0.05). The most heavily contaminated vegetable was wastewater irrigated A. cepa, where the accumulation of Mn (28.05 mg kg(-1)) in the edible parts was 50-fold greater than A. cepa irrigated with tube well water irrigated soil. It may be concluded that both adults and children consuming these vegetables grown in wastewater irrigated soil ingest significant amount of these metals and thus can cause serious health problems. Copyright © 2012 Elsevier Ltd. All rights reserved.

Anti-microbial and anti-oxidant activities of Illicium verum, Crataegus oxyacantha ssp monogyna and Allium cepa red and white varieties.

PubMed

Benmalek, Yamina; Yahia, Ouahiba Ait; Belkebir, Aicha; Fardeau, Marie-Laure

2013-01-01

Illicium verum (badiane or star anise), Crataegus oxyacantha ssp monogyna (hawthorn) and Allium cepa (onion), have traditionnally been used as medicinal plants in Algeria. This study showed that the outer layer of onion is rich in flavonols with contents of 103 ± 7.90 µg/g DW (red variety) and 17.3 ± 0.69 µg/gDW (white variety). We also determined flavonols contents of 14.3 ± 0.21 µg/g 1.65 ± 0.61 µg/g for Crataegus oxyacantha ssp monogyna leaves and berries and 2.37 ± 0.10 µg/g for Illicium verum. Quantitative analysis of anthocyanins showed highest content in Crataegus oxyacantha ssp monogyna berries (5.11 ± 0.266 mg/g), while, inner and outer layers of white onion had the lowest contents with 0.045 ± 0.003mg/g and 0.077 ± 0.001 mg/g respectively.   Flavonols extracts presented high antioxidant activity as compared with anthocyanins and standards antioxidants (ascorbic acid and quercetin). Allium cepa and Crataegus oxyacantha ssp monogyna exhibited the most effective antimicrobial activity.

Hydropower reservoirs: cytotoxic and genotoxic assessment using the Allium cepa root model.

PubMed

Rambo, Cassiano Lazarotto; Zanotelli, Patrícia; Dalegrave, Daniela; De Nez, Dinara; Szczepanik, Jozimar; Carazek, Fábio; Franscescon, Francini; Rosemberg, Denis Broock; Siebel, Anna Maria; Magro, Jacir Dal

2017-03-01

Hydropower offers a reliable source of electricity in several countries, and Brazil supplies its energy needs almost entirely through hydropower plants. Nevertheless, hydropower plants comprise large buildings and water reservoirs and dams, resulting in huge ecological disruptions. Here, we analyzed the impact of four hydropower reservoirs construction in metals and pesticides incidence and the cytotoxic and genotoxic potential of sediment elutriate of rivers from southern Brazil. Our analyses have evidenced the elevated incidence of different metals (lead, iron, cadmium, and chrome) and pesticides (methyl parathion, atrazine, and 2,4-dichlorophenoxyacetic acid). We showed that Allium cepa exposed to sediment elutriates did not change the seed germination rate and mitotic index. However, roots from Allium cepa exposed to reservoirs sediment elutriates showed increased occurrence of chromosomal aberrations and nuclear abnormalities. Therefore, the results obtained in our study indicate that sediment from reservoirs present elevated concentration of metals and pesticides and a significant genotoxic potential. Taken together, our data support that hydropower reservoirs represent an environmental scenario that could impact surrounding wildlife and population.

Sunlight decreased genotoxicity of azadirachtin on root tip cells of Allium cepa and Eucrosia bicolor.

PubMed

Kwankua, W; Sengsai, S; Kuleung, C; Euawong, N

2010-07-01

Utilization of neem plant (Azadirachta indica A. Juss) extract for pest control in agriculture has raised concerns over contamination by the residues to the environment. Such residues, particularly azadirachtin (Aza), may cause deleterious effect to non-target organisms. This investigation was conducted to find out if Aza could be inactivated through exposures to sunlight. Activity of Aza was assessed as its ability to cause cytotoxic and genotoxic effects in the forms of nuclei abnormality and chromosome aberration as measured by mitotic index (MI) and mitotic aberration (MA). Varying concentrations of Aza were tested on Allium cepa and Eucrosia bicolor. It was found that the MI of all root tip meristematic cells of A. cepa and E. bicolor treated with 0.00005%, 0.00010%, 0.00015%, and 0.00020% (w/v) Aza-containing neem extract for 24h, were significantly lower than the controls. Complementary to the lower levels of MI, the Aza-treated groups showed higher MA levels in all cases investigated. Furthermore, the decreasing levels of MI and the increasing levels of MA related well with the increasing concentration of Aza. Microscopic examination of root tip meristematic cells revealed that the anomaly found most often were mitotic disturbances and chromosomal bridges. Exposures of 0.00020% (w/v) Aza to sunlight for 3 days and 7 days decreased Aza ability to induce cytotoxicity and genotoxicity, both in terms of MI and MA, to root tip meristematic cells in A. cepa and E. bicolor. Photodegradation of Aza upon exposure to direct sunlight was confirmed by HPLC. The study implicates that Aza would unlikely cause long term deleterious effects to the environment since it would be inactivated by sunlight. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Analysis of cytotoxicity and genotoxicity on E. coli, human blood cells and Allium cepa suggests a greater toxic potential of hair dye.

PubMed

Maiti, Swati; Sasmal, Kankaayan; Sinha, Sudarson Sekhar; Singh, Mukesh

2016-02-01

Pharmaceuticals and personal care products (PPCPs) are among the most important emerging environmental contaminants in recent time. PPCPs include wide range of cosmetics, among which hair dyes, are immensely popular in modern society. However, impact of hair dye and its residual discharged to the environment in relation to human health and ecological imbalance have not been widely studied. Based on the result of initial survey among the group of populations of eastern India, three most popular and commonly used permanent hair dyes are selected. Working sample of dye is prepared as recommended on the instructions booklet of the hair dye. The effect of three dyes is studied on Escherichia coli, human red blood cells (RBC), white blood cells (WBC) and Allium cepa bulbs by growth inhibition, hemolysis, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and A. cepa micronuclei assays respectively. The Lethal dose (LD) demonstrated significant differences among three dyes and the model systems. In vitro hemolytic assays performed on RBC, and MTT assays on WBC show the cytotoxic effects of hair dye. Significant growth inhibition of E. coli has also been noted. In addition, the root tips of A. cepa treated with the dye have shown major chromosomal abnormalities coupled with cell division retardation. Here low mitotic index confirm cell division retardation. Finally, results of in vitro studies of dye-DNA interactions demonstrate electrostatic interaction. Combing all these results it confirms that hair dyes are cytotoxic and may cause mutagenic effect on living cells irrespective of microbes, plant and animal system. Copyright © 2015 Elsevier Inc. All rights reserved.

[Evaluation of the mutagenicity of detergents by tests on bacteria, plant cells and human leucocytes.].

PubMed

Feretti, Donatella; Pedrazzani, Roberta; Ceretti, Elisabetta; Zerbini, Ilaria; Gozio, Eleonora; Belotti, Caterina; Alias, Carlotta; Donato, Francesco; Gelatti, Umberto

2009-01-01

The aim of this study was to evaluate the mutagenicity of several traditional detergents and that of newer more biodegradable detergents, by using a bacterial test (Ames test), a plant cell test (Allium cepa micronuclei test) and a human leucocyte test (Comet test). All tests were conducted using a wide range of doses (1-2000 mg/l). None of the examined detergents induced mutations in S.typhimurium. One traditional detergent showed a genotoxic effect with the A. cepa test, while all newer detergents and one traditional detergent were shown by the Comet test to be capable of inducing DNA damage.

Systemic control of cell division and endoreduplication by NAA and BAP by modulating CDKs in root tip cells of Allium cepa.

PubMed

Tank, Jigna G; Thaker, Vrinda S

2014-01-01

Molecular mechanism regulated by auxin and cytokinin during endoreduplication, cell division, and elongation process is studied by using Allium cepa roots as a model system. The activity of CDK genes modulated by auxin and cytokinin during cell division, elongation, and endoreduplication process is explained in this research work. To study the significance of auxin and cytokinin in the management of cell division and endoreduplication process in plant meristematic cells at molecular level endoreduplication was developed in root tips of Allium cepa by giving colchicine treatment. There were inhibition of vegetative growth, formation of c-tumor at root tip, and development of endoreduplicated cells after colchicine treatment. This c-tumor was further treated with NAA and BAP to reinitiate vegetative growth in roots. BAP gave positive response in reinitiation of vegetative growth of roots from center of c-tumor. However, NAA gave negative response in reinitiation of vegetative growth of roots from c-tumor. Further, CDKs gene expression analysis from normal, endoreduplicated, and phytohormone (NAA or BAP) treated root tip was done and remarkable changes in transcription level of CDK genes in normal, endoreduplicated, and phytohormones treated cells were observed.

Ecotoxicological and Genotoxic Evaluation of Buenos Aires City (Argentina) Hospital Wastewater

PubMed Central

Juárez, Ángela Beatriz; Dragani, Valeria; Saenz, Magalí Elizabeth; Moretton, Juan

2014-01-01

Hospital wastewater (HWW) constitutes a potential risk to the ecosystems and human health due to the presence of toxic and genotoxic chemical compounds. In the present work we investigated toxicity and genotoxicity of wastewaters from the public hospital of Buenos Aires (Argentina). The effluent from the sewage treatment plant (STP) serving around 10 million inhabitants was also evaluated. The study was carried out between April and September 2012. Toxicity and genotoxicity assessment was performed using the green algae Pseudokirchneriella subcapitata and the Allium cepa test, respectively. Toxicity assay showed that 55% of the samples were toxic to the algae (%I of growth between 23.9 and 54.8). The A. cepa test showed that 40% of the samples were genotoxic. The analysis of chromosome aberrations (CA) and micronucleus (MN) showed no significant differences between days and significant differences between months. The sample from the STP was not genotoxic to A. cepa but toxic to the algae (%I = 41%), showing that sewage treatment was not totally effective. This study highlights the need for environmental control programs and the establishment of advanced and effective effluent treatment plants in the hospitals, which are merely dumping the wastewaters in the municipal sewerage system. PMID:25214834

Anti-microbial and anti-oxidant activities of Illicium verum, Crataegus oxyacantha ssp monogyna and Allium cepa red and white varieties

PubMed Central

Benmalek, Yamina; Yahia, Ouahiba Ait; Belkebir, Aicha; Fardeau, Marie-Laure

2013-01-01

Illicium verum (badiane or star anise), Crataegus oxyacantha ssp monogyna (hawthorn) and Allium cepa (onion), have traditionnally been used as medicinal plants in Algeria. This study showed that the outer layer of onion is rich in flavonols with contents of 103 ± 7.90 µg/g DW (red variety) and 17.3 ± 0.69 µg/gDW (white variety). We also determined flavonols contents of 14.3 ± 0.21 µg/g 1.65 ± 0.61 µg/g for Crataegus oxyacantha ssp monogyna leaves and berries and 2.37 ± 0.10 µg/g for Illicium verum. Quantitative analysis of anthocyanins showed highest content in Crataegus oxyacantha ssp monogyna berries (5.11 ± 0.266 mg/g), while, inner and outer layers of white onion had the lowest contents with 0.045 ± 0.003mg/g and 0.077 ± 0.001 mg/g respectively.   Flavonols extracts presented high antioxidant activity as compared with anthocyanins and standards antioxidants (ascorbic acid and quercetin). Allium cepa and Crataegus oxyacantha ssp monogyna exhibited the most effective antimicrobial activity. PMID:23579100

Potential genotoxic effects of melted snow from an urban area revealed by the Allium cepa test.

PubMed

Blagojević, Jelena; Stamenković, Gorana; Vujosević, Mladen

2009-09-01

The presence of well-known atmospheric pollutants is regularly screened for in large towns but knowledge about the effects of mixtures of different pollutants and especially their genotoxic potential is largely missing. Since falling snow collects pollutants from the air, melted snow samples could be suitable for evaluating potential genotoxicity. For this purpose the Allium cepa anaphase-telophase test was used to analyse melted snow samples from Belgrade, the capital city of Serbia. Samples of snow were taken at two sites, characterized by differences in pollution intensity, in three successive years. At the more polluted site the analyses showed a very high degree of both toxicity and genotoxicity in the first year of the study corresponding to the effects of the known mutagen used as the positive control. At the other site the situation was much better but not without warning signals. The results showed that standard analyses for the presence of certain contaminants in the air do not give an accurate picture of the possible consequences of urban air pollution because the genotoxic potential remains hidden. The A. cepa test has been demonstrated to be very convenient for evaluation of air pollution through analyses of melted snow samples.

Systemic Control of Cell Division and Endoreduplication by NAA and BAP by Modulating CDKs in Root Tip Cells of Allium cepa

PubMed Central

Tank, Jigna G.; Thaker, Vrinda S.

2014-01-01

Molecular mechanism regulated by auxin and cytokinin during endoreduplication, cell division, and elongation process is studied by using Allium cepa roots as a model system. The activity of CDK genes modulated by auxin and cytokinin during cell division, elongation, and endoreduplication process is explained in this research work. To study the significance of auxin and cytokinin in the management of cell division and endoreduplication process in plant meristematic cells at molecular level endoreduplication was developed in root tips of Allium cepa by giving colchicine treatment. There were inhibition of vegetative growth, formation of c-tumor at root tip, and development of endoreduplicated cells after colchicine treatment. This c-tumor was further treated with NAA and BAP to reinitiate vegetative growth in roots. BAP gave positive response in reinitiation of vegetative growth of roots from center of c-tumor. However, NAA gave negative response in reinitiation of vegetative growth of roots from c-tumor. Further, CDKs gene expression analysis from normal, endoreduplicated, and phytohormone (NAA or BAP) treated root tip was done and remarkable changes in transcription level of CDK genes in normal, endoreduplicated, and phytohormones treated cells were observed. PMID:24955358

A comparative profile of methanol extracts of Allium cepa and Allium sativum in diabetic neuropathy in mice

PubMed Central

Bhanot, Abhishek; Shri, Richa

2010-01-01

Introduction: Diabetic Neuropathy (DN) is a major microvascular complication of uncontrolled diabetes. This may result from increased oxidative stress that accompanies diabetes. Hence plants with antioxidant action play an important role in management of diabetes and its complications. Materials and Methods: This study was designed to evaluate preventive as well as curative effect of methanol extracts of outer scales and edible portions of two plants with established antioxidant action - Allium cepa and Allium sativum, in induced DN in albino mice. Mice were divided into control, diabetic and test extracts treated groups. Test extracts were administered daily at a dose of 200 mg/kg p.o. for 21 days, in the preventive group prior to onset of DN, and in the curative group after the onset of DN. Hyperalgesia and oxidative stress markers were assessed. STZ-diabetic mice showed a significant thermal hyperalgesia (as assessed by the tail-flick test), indicating development of DN. Results: Treatment with test extracts prevented loss in body weight, decreased plasma glucose level, and significantly ameliorated the hyperalgesia, TBARS, serum nitrite and GSH levels in diabetic mice. Conclusion: Methanol extract of outer scales of onion has shown most significant improvement; may be due to higher content of phenolic compounds in outer scales of A. cepa. PMID:21713142

Aquatic humic substances inhibit clastogenic events in germinating seeds of herbaceous plants.

PubMed

Ferrara, G; Loffredo, E; Senesi, N

2001-03-01

One humic acid (HA) and two fulvic acids (FAs) of aquatic origin have been tested for their capacity to inhibit clastogenic events caused by maleic hydrazide (MH) in germinating seeds of the herbaceous plant species Allium cepa and Vicia faba. Either HA or FA at concentrations of 50 and 500 mg L(-)(1) was interacted with 10 mg L(-)(1) MH for 24 h before addition to the seeds. The evaluation of genotoxic activity was made by counting micronuclei (MN) and aberrant anatelophases (AT) in root tip cells after treatment with HA or FA alone, MH alone, and interacted HA + MH and FA + MH. Regular AT were also counted as an index of mitotic activity. In all cases HA and FA interacted with MH showed an evident anticlastogenic action indicated by the marked reduction of genetic anomalies. In A. cepa, the anticlastogenic effect of HA and FA was more significant for aberrant AT than for MN, whereas the opposite was true in the case of V. faba. The protective effect exhibited for both anomalies by HA was slightly higher than that of the corresponding FA in A. cepa, whereas no significant differences between these HA and FA treatments were observed in the case of V. faba. The two FAs generally showed similar anticlastogenic behaviors with slight quantitative differences observed as a function of the type of anomaly and the plant species. The effects of HA and FA concentration differed depending on the type of anomaly observed, the plant species, and FA origin. In V. faba, cell division, that is, the number of regular AT, was generally depressed by HA and FA at either concentration with respect to the control. In A. cepa, HA and FA produced either stimulating or inhibiting effects on regular AT depending on their nature, origin, and concentration.

Glu-Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes.

PubMed

Lee, Yu Geon; Cho, Jeong-Yong; Hwang, Eom Ji; Jeon, Tae-Il; Moon, Jae-Hak

2017-07-01

A Glu-Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization-mass (ESI-MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography-ESI-MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.

Comparison of cytotoxic and genotoxic effects of plutonium-239 alpha particles and mobile phone GSM 900 radiation in the Allium cepa test.

PubMed

Pesnya, Dmitry S; Romanovsky, Anton V

2013-01-20

The goal of this study was to compare the cytotoxic and genotoxic effects of plutonium-239 alpha particles and GSM 900 modulated mobile phone (model Sony Ericsson K550i) radiation in the Allium cepa test. Three groups of bulbs were exposed to mobile phone radiation during 0 (sham), 3 and 9h. A positive control group was treated during 20min with plutonium-239 alpha-radiation. Mitotic abnormalities, chromosome aberrations, micronuclei and mitotic index were analyzed. Exposure to alpha-radiation from plutonium-239 and exposure to modulated radiation from mobile phone during 3 and 9h significantly increased the mitotic index. GSM 900 mobile phone radiation as well as alpha-radiation from plutonium-239 induced both clastogenic and aneugenic effects. However, the aneugenic activity of mobile phone radiation was more pronounced. After 9h of exposure to mobile phone radiation, polyploid cells, three-groups metaphases, amitoses and some unspecified abnormalities were detected, which were not registered in the other experimental groups. Importantly, GSM 900 mobile phone radiation increased the mitotic index, the frequency of mitotic and chromosome abnormalities, and the micronucleus frequency in a time-dependent manner. Due to its sensitivity, the A. cepa test can be recommended as a useful cytogenetic assay to assess cytotoxic and genotoxic effects of radiofrequency electromagnetic fields. Copyright © 2012 Elsevier B.V. All rights reserved.

Genotoxicity evaluation of two metallic-insecticides using Allium cepa and Tradescantia pallida: A new alternative against leaf-cutting ants.

PubMed

de Souza, Raphael Bastão; de Souza, Cleiton Pereira; Bueno, Odair Correa; Fontanetti, Carmem Silvia

2017-02-01

In order to combat leaf-cutting ants, the pesticide sulfluramid used to be the most widely utilized active ingredient. However, its use was banned in 2009 by the Stockholm Convention, although some countries were allowed to continue using it. As an effective alternative to its replacement, researchers developed a metallic-insecticide system, which is a natural product linked to metal complexes. Thus, the aim of this study was to evaluate the ability of these new metallic-insecticides in change the genetic material of non-target organisms. The tests were performed utilizing chromosomal aberrations and micronucleus tests in the Allium cepa test system and the Trad-MCN test in Tradescantia pallida. To better understand the results, one of the components of the formula, 5-methyl-phenanthroline, was also analyzed according to the same parameters. To A. cepa, the results showed that one of the metallic insecticides induced cytotoxicity and genotoxicity at different concentrations, while the other metallic-insecticide showed chromosomal instability only at the highest concentration. The analysis of 5-methyl-phenanthroline revealed that it can be related with the positive results, since genotoxic effects were induced. In the Trad-MCN test, none of the metallic-insecticides showed genotoxic activity, although one of them induced more micronucleus formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mutagenic potential of water from Pelotas Creek in Rio Grande do Sul, Brazil.

PubMed

Santos, T C O; Maciel, L F; Leal, K S; Bender, A E N; Paiva, T S; Garcias, G L; Martino-Roth, M G

2009-09-01

Water resource degradation is one of mankind's greatest worries, as it causes direct and indirect damage to the associated biota. We initiated a water monitoring study in Pelotas Creek in 2003 in order to assess the mutagenic effect of the creek's waters. Allium cepa cells exposed to water samples and a chronically exposed macrophyte were analyzed, through evaluation of the mitotic index, mitotic anomalies, interphase anomalies, and total anomalies. Five points were chosen along the lower course of Pelotas Creek, from which water samples and floating pennywort (Hydrocotyle ranunculoides, Apiaceae) were collected in 2006 and 2007. The enteric bacterium Escherichia coli was found at all sampling points; in the physical-chemical analysis, a few variables exceeded permitted limits, pH (from 6 to 9), chloride (250 mg/L), hardness (from 10 to 200 mg CaCO(3)/L), and conductivity (100 microOmega/cm). There was an increased number of cytogenetic anomalies in exposed A. cepa cells and in the pennywort in 2006 relative to 2007, which may be explained by the increased rainfall, which was three times greater in 2007 at some stations than in 2006.Omega/cm). There was an increased number of cytogenetic anomalies in exposed A. cepa cells and in the pennywort in 2006 relative to 2007, which may be explained by the increased rainfall, which was three times greater in 2007 at some stations than in 2006.

Controlled green synthesis of silver nanoparticles by Allium cepa and Musa acuminata with strong antimicrobial activity

NASA Astrophysics Data System (ADS)

Sahni, Geetika; Panwar, Amit; Kaur, Balpreet

2015-02-01

A controlled "green synthesis" approach to synthesize silver nanoparticles by Allium cepa and Musa acuminata plant extract has been reported. The effect of different process parameters, such as pH, temperature and time, on synthesis of Ag nanoparticles from plant extracts has been highlighted. The work reports an easy approach to control the kinetics of interaction of metal ions with reducing agents, stabilized by ammonia to achieve sub-10 nm particles with narrow size distribution. The nanoparticles have been characterized by UV-Visible spectra and TEM analysis. Excellent antimicrobial activity at extremely low concentration of the nanoparticles was observed against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Fusarium oxysporum which may allow their exploitation as a new generation nanoproduct in biomedical and agricultural applications.

Effects of aluminum on nucleoli in root tip cells and selected physiological and biochemical characters in Allium cepa var. agrogarum L

PubMed Central

2010-01-01

Background Increased Al concentration causes reduction of mitotic activity, induction of nucleolar alteration, increase of the production of ROS and alteration of several antioxidant enzyme activities in plant cells. Allium cepa is an excellent plant and a useful biomarker for environmental monitoring. Limited information is available about the effects of Al on nucleoli, antioxidant enzyme system, contents of MDA and soluble protein in A. cepa. Therefore, we carried out the investigation in order to better understand the effects of Al on the growth, nucleoli in root tip cells and selected physiological and biochemical characters. Results The results showed that the root growth exposed to 50 μM Al was inhibited significantly. 50 μM Al could induce some particles of argyrophilic proteins scattered in the nuclei and extruded from the nucleoli into the cytoplasm. The nucleolus did not disaggregate normally and still remained its characteristic structure during metaphase. Nucleolar reconstruction was inhibited. 50 μM Al induced high activities of SOD and POD in leaves and roots significantly (P < 0.05) when compared with control, whereas the level of CAT was low significantly (P < 0.05). At 50 μM Al the content of MDA in leaves was high significantly (P < 0.05) at 9th day and in roots increased (P < 0.05) with prolonging the treatment time during 6-12 days. The soluble protein content in leaves treated with 50 μM Al was high significantly (P < 0.05) at 6th day and increased with prolonging the treatment time. Conclusions We suggest that variations in nucleoli and the alterations of antioxidant enzyme activities, MDA and soluble protein contents in Allium cepa can serve as useful biomarkers, which can provide valuable information for monitoring and forecasting effects of exposure to Al in real scenarios conditions. Among the antioxidant enzymes SOD and POD appear to play a key role in the antioxidant defense mechanism under Al toxicity condition. Data from MDA concentration show that Al indirectly produces superoxide radicals, resulting in increased lipid peroxidative products and oxidative stress. PMID:20964828

Effects of aluminum on nucleoli in root tip cells and selected physiological and biochemical characters in Allium cepa var. agrogarum L.

PubMed

Qin, Rong; Jiao, Yunqiu; Zhang, Shanshan; Jiang, Wusheng; Liu, Donghua

2010-10-21

Increased Al concentration causes reduction of mitotic activity, induction of nucleolar alteration, increase of the production of ROS and alteration of several antioxidant enzyme activities in plant cells. Allium cepa is an excellent plant and a useful biomarker for environmental monitoring. Limited information is available about the effects of Al on nucleoli, antioxidant enzyme system, contents of MDA and soluble protein in A. cepa. Therefore, we carried out the investigation in order to better understand the effects of Al on the growth, nucleoli in root tip cells and selected physiological and biochemical characters. The results showed that the root growth exposed to 50 μM Al was inhibited significantly. 50 μM Al could induce some particles of argyrophilic proteins scattered in the nuclei and extruded from the nucleoli into the cytoplasm. The nucleolus did not disaggregate normally and still remained its characteristic structure during metaphase. Nucleolar reconstruction was inhibited. 50 μM Al induced high activities of SOD and POD in leaves and roots significantly (P < 0.05) when compared with control, whereas the level of CAT was low significantly (P < 0.05). At 50 μM Al the content of MDA in leaves was high significantly (P < 0.05) at 9(th) day and in roots increased (P < 0.05) with prolonging the treatment time during 6-12 days. The soluble protein content in leaves treated with 50 μM Al was high significantly (P < 0.05) at 6(th) day and increased with prolonging the treatment time. We suggest that variations in nucleoli and the alterations of antioxidant enzyme activities, MDA and soluble protein contents in Allium cepa can serve as useful biomarkers, which can provide valuable information for monitoring and forecasting effects of exposure to Al in real scenarios conditions. Among the antioxidant enzymes SOD and POD appear to play a key role in the antioxidant defense mechanism under Al toxicity condition. Data from MDA concentration show that Al indirectly produces superoxide radicals, resulting in increased lipid peroxidative products and oxidative stress.

Mutagenic screening of some commonly used medicinal plants in Nigeria.

PubMed

Akintonwa, Alade; Awodele, Olufunsho; Afolayan, Gbenga; Coker, Herbert A B

2009-09-25

The uses of medicinal plants have always been part of human culture. The World Health Organization estimates that up to 80% of the world's population relies on traditional medicinal system for some aspect of primary health care. However, there are few reports on the toxicological properties of most medicinal plants especially, their mutagenicity and carcinogenicity. Therefore, this research is to determine the mutagenic potentials of Morinda lucida [Oruwo (Root)], Azadirachta indica [Dongoyaro (Leaf)], Terapluera tetraptera [Aridan (Fruit)], Plumbago zeylanica [Inabiri (Root)], Xylopia aethiopica [Erunje (Fruit)], Newbouldia laevis [Akoko (Leaf)], Alstonia boonei [Ahun (Bark)], Enantia chlorantha [Awopa (Bark)], and Rauvolfia vomitoria [Asofeyeje (Root)] using the Allium cepa Linn. model and the modified Ames assay. Allium cepa model was used to determine the mean root length, mitotic index and chromosomal aberrations effects of these plants on onion bulbs using 0.1, 1, 5 and 10mg/ml concentration of the plant extracts. The modified Ames test which is a modification of the standard Ames test as described by Ames et al. [Ames, B.N., McCann, J., Yamasaki, E., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Research 31, 347-364] was done using Escherichia coli (0157:H7) that has the phenotypic characteristics of glucose and lactose fermentation, motile, urease negative, indole positive and citrate negative. The results obtained from Allium cepa assay showed increasing root growth inhibition with increased concentration, decreasing mitotic index with increased concentration and chromosomal aberrations. The modified Ames test showed an alteration in the biochemical characteristics of Escherichia coli (0157:H7) for all plants except Rauvolfia vomitoria and Plumbago zeylanica. Three of the medicinal plants altered at least three of the normal biochemical characteristics thus demonstrating mutagenic potentials. The results of internationally accepted Allium cepa were comparable with the modified Ames test. However, a long term in vivo and dose dependent study should be carried out to validate these results and the findings should be communicated to drug and food regulatory body and also to the general public.

Genotoxic evaluation of an industrial effluent from an oil refinery using plant and animal bioassays.

PubMed

Rodrigues, Fernando Postalli; Angeli, José Pedro Friedmann; Mantovani, Mário Sérgio; Guedes, Carmen Luisa Barbosa; Jordão, Berenice Quinzani

2010-01-01

Polycyclic aromatic hydrocarbons (PAHs) are genotoxic chemicals commonly found in effluents from oil refineries. Bioassays using plants and cells cultures can be employed for assessing environmental safety and potential genotoxicity. In this study, the genotoxic potential of an oil refinery effluent was analyzed by means of micronucleus (MN) testing of Alium cepa, which revealed no effect after 24 h of treatment. On the other hand, primary lesions in the DNA of rat (Rattus norvegicus) hepatoma cells (HTC) were observed through comet assaying after only 2 h of exposure. On considering the capacity to detect DNA damage of a different nature and of these cells to metabolize xenobiotics, we suggest the association of the two bioassays with these cell types, plant (Allium cepa) and mammal (HTC) cells, for more accurately assessing genotoxicity in environmental samples.

Economical and environmentally-friendly approaches for usage of onion (Allium cepa L.) waste.

PubMed

Sharma, Kavita; Mahato, Neelima; Nile, Shivraj Hariram; Lee, Eul Tal; Lee, Yong Rok

2016-08-10

Onion (Allium cepa L.) is one of the most commonly cultivated crops across the globe, and its production is increasing every year due to increasing consumer demand. Simultaneously, huge amounts of waste are produced from different parts of the onion, which ultimately affect the environment in various ways. Hence, proper usage as well as disposal of this waste is important from the environmental aspect. This review summarizes various usage methods of onion waste material, and processes involved to achieve maximum benefits. Processing industries produce the largest amount of onion waste. Other sources are storage systems, domestic usage and cultivation fields. Particular emphasis has been given to the methods used for better extraction and usage of onion waste under specific topics: viz. organic synthesis, production of biogas, absorbent for pollutants and value added products.

Sulphur management in onion (Allium cepa) cultivation in hills of Himachal Pradesh.

PubMed

Jaggi, R C; Sharma, R K

2010-05-01

Field experiment were conducted at CSK HPKV Research Farm, Palampur during Rabi seasons of 2000-01 and 2001-02, to study the response of onion (Allium cepa var Patna red) at four sulphur levels (0, 15, 30 and 60 kg ha(-1)) applied through Gypsum and S95. The analysis was done to allocate the limited availability of sulphur for maximizing net profit over fertilizer cost. The results show that the dose of sulphur under its full availability is 43.02 kg ha(-1). But under its scarce availability the maximum benefit would occur when it is applied up to 32.11 kg ha(-1) followed by even distribution of fertilizer i.e. 20 kg ha(-1). The returns following sulphur application at these rates, would be Rs 69340, 73092 and 68700 ha(-1) respectively.

Addition of a combination of onion (Allium cepa) and coconut (Cocos nucifera) to food of sheep stops gastrointestinal helminthic infections.

PubMed

Mehlhorn, Heinz; Al-Quraishy, Saleh; Al-Rasheid, Khaled A S; Jatzlau, Antje; Abdel-Ghaffar, Fathy

2011-04-01

Sheep with gastrointestinal nematodes and cestodes were fed on three farms with a combination of specially prepared extracts of onion (Allium cepa) and coconut (Cocos nucifera) for 8 days containing each 60 g coconut and onion extract, combined with milk powder and/or polyethylene glycol (PEG) propylencarbonate (PC). In all cases, the worm stages disappeared from the feces and were also not found 9 and 20 days after the end of the feeding with this plant combination. Since all treated animals increased their body weight considerably (when compared to untreated animals), worm reduction was apparently as effective as it was shown in previous laboratory trials with rats and mice (Klimpel et al., Parasitol Res, in press, 2010; Abdel-Ghaffar et al., Parasitol Res, in press, 2010; in this volume).

The biphasic interphase-mitotic polarity of cell nuclei induced under DNA replication stress seems to be correlated with Pin2 localization in root meristems of Allium cepa.

PubMed

Żabka, Aneta; Trzaskoma, Paweł; Winnicki, Konrad; Polit, Justyna Teresa; Chmielnicka, Agnieszka; Maszewski, Janusz

2015-02-01

Long-term treatment of Allium cepa seedlings with low concentration of hydroxyurea (HU) results in a disruption of cell cycle checkpoints, leading root apex meristem (RAM) cells to an abnormal organization of nuclear structures forming interphase (I) and mitotic (M) domains of chromatin at opposite poles of the nucleus. Thus far, both critical cell length and an uneven distribution of cyclin B-like proteins along the nuclear axis have been recognized as essential factors needed to facilitate the formation of biphasic interphase-mitotic (IM) cells. Two new aspects with respect to their emergence are investigated in this study. The first concerns a relationship between the polarity of increasing chromatin condensation (IM orientation) and the acropetal (base→apex) alignment of RAM cell files. The second problem involves the effects of auxin (IAA), on the frequency of IM cells. We provide evidence that there is an association between the advanced M-poles of the IM cell nuclei and the polarized accumulation sites of auxin efflux carriers (PIN2 proteins) and IAA. Furthermore, our observations reveal exclusion regions for PIN2 proteins in the microtubule-rich structures, such as preprophase bands (PPBs) and phragmoplast. The current and previous studies have prompted us to formulate a hypothetical mechanism linking PIN2-mediated unilateral localization of IAA and the induction of bipolar IM cells in HU-treated RAMs of A. cepa. Copyright © 2014 Elsevier GmbH. All rights reserved.

Mitogen-activated protein kinase signal transduction and DNA repair network are involved in aluminum-induced DNA damage and adaptive response in root cells of Allium cepa L.

PubMed Central

Panda, Brahma B.; Achary, V. Mohan M.

2014-01-01

In the current study, we studied the role of signal transduction in aluminum (Al3+)-induced DNA damage and adaptive response in root cells of Allium cepa L. The root cells in planta were treated with Al3+ (800 μM) for 3 h without or with 2 h pre-treatment of inhibitors of mitogen-activated protein kinase (MAPK), and protein phosphatase. Also, root cells in planta were conditioned with Al3+ (10 μM) for 2 h and then subjected to genotoxic challenge of ethyl methane sulfonate (EMS; 5 mM) for 3 h without or with the pre-treatment of the aforementioned inhibitors as well as the inhibitors of translation, transcription, DNA replication and repair. At the end of treatments, roots cells were assayed for cell death and/or DNA damage. The results revealed that Al3+ (800 μM)-induced significant DNA damage and cell death. On the other hand, conditioning with low dose of Al3+ induced adaptive response conferring protection of root cells from genotoxic stress caused by EMS-challenge. Pre-treatment of roots cells with the chosen inhibitors prior to Al3+-conditioning prevented or reduced the adaptive response to EMS genotoxicity. The results of this study suggested the involvement of MAPK and DNA repair network underlying Al-induced DNA damage and adaptive response to genotoxic stress in root cells of A. cepa. PMID:24926302

Cytogenetic effects of three commercially formulated pesticides on somatic and germ cells of Allium cepa.

PubMed

Kuchy, Aashiq H; Wani, Aijaz A; Kamili, Azra N

2016-04-01

Cytological effects of Endosri-ES (endosulfan), Nuvan-NU (dichlorvos), and Kvistin-KS (carbendazim) were evaluated on mitotic and meiotic cells of Allium cepa. Test concentrations were chosen by calculating EC50 values of formulated ES, NU, and KS, which turned to be 60, 200, and 500 ppm (parts per million), respectively. Cytological studies were undertaken on root meristem cells of A. cepa using EC50, 1/2 × EC50, and 2 × EC50 of these pesticides for 24 and 48 h. Similarly, a meiotic study was conducted by applying the pesticides at the aforesaid concentrations from seedling to bud stage. A set of onion bulbs exposed to tap water was run parallel for negative control and maleic hydrazide (112.09 ppm) as positive control. During the study period, mitotic index (MI) decreased at all the pesticide concentrations compared to the negative control. Among various chromosomal aberrations, chromatin bridges, breaks, stickiness, laggard, vagrant chromosomes, fragments, C-mitosis, multipolarity, ring chromosome as well as micronuclei were observed in mitotic preparations. In contrast, meiotic aberrations revealed comparatively less frequency of chromosomal aberrations and the most frequent were lagging chromosome, stray bivalents, secondary association, chromatin bridge, disturbed anaphase, and stickiness. Comparative analysis of the pesticides showed that NU was highly toxic to plant cells than KS, while as ES showed intermediate effects between the two. Further, our study revealed that all the three pesticides produce genotoxic effects which can cause health risks to the human populations. Graphical Abstract ᅟ.

Dissection of Trichoderma longibrachiatum-induced defense in onion (Allium cepa L.) against Fusarium oxysporum f. sp. cepa by target metabolite profiling.

PubMed

Abdelrahman, Mostafa; Abdel-Motaal, Fatma; El-Sayed, Magdi; Jogaiah, Sudisha; Shigyo, Masayoshi; Ito, Shin-Ichi; Tran, Lam-Son Phan

2016-05-01

Trichoderma spp. are versatile opportunistic plant symbionts that can cause substantial changes in the metabolism of host plants, thereby increasing plant growth and activating plant defense to various diseases. Target metabolite profiling approach was selected to demonstrate that Trichoderma longibrachiatum isolated from desert soil can confer beneficial agronomic traits to onion and induce defense mechanism against Fusarium oxysporum f. sp. cepa (FOC), through triggering a number of primary and secondary metabolite pathways. Onion seeds primed with Trichoderma T1 strain displayed early seedling emergence and enhanced growth compared with Trichoderma T2-treatment and untreated control. Therefore, T1 was selected for further investigations under greenhouse conditions, which revealed remarkable improvement in the onion bulb growth parameters and resistance against FOC. The metabolite platform of T1-primed onion (T1) and T1-primed onion challenged with FOC (T1+FOC) displayed significant accumulation of 25 abiotic and biotic stress-responsive metabolites, representing carbohydrate, phenylpropanoid and sulfur assimilation metabolic pathways. In addition, T1- and T1+FOC-treated onion plants showed discrete antioxidant capacity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) compared with control. Our findings demonstrated the contribution of T. longibrachiatum to the accumulation of key metabolites, which subsequently leads to the improvement of onion growth, as well as its resistance to oxidative stress and FOC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Marinobufagin, a molecule from poisonous frogs, causes biochemical, morphological and cell cycle changes in human neoplasms and vegetal cells.

PubMed

Machado, Kátia da Conceição; Sousa, Lívia Queiroz de; Lima, Daisy Jereissati Barbosa; Soares, Bruno Marques; Cavalcanti, Bruno Coêlho; Maranhão, Sarah Sant'Anna; Noronha, Janaina da Costa de; Rodrigues, Domingos de Jesus; Militão, Gardenia Carmen Gadelha; Chaves, Mariana Helena; Vieira-Júnior, Gerardo Magela; Pessoa, Cláudia; Moraes, Manoel Odorico de; Sousa, João Marcelo de Castro E; Melo-Cavalcante, Ana Amélia de Carvalho; Ferreira, Paulo Michel Pinheiro

2018-03-15

Skin toad secretion present physiologically active molecules to protect them against microorganisms, predators and infections. This work detailed the antiproliferative action of marinobufagin on tumor and normal lines, investigate its mechanism on HL-60 leukemia cells and its toxic effects on Allium cepa meristematic cells. Initially, cytotoxic action was assessed by colorimetric assays. Next, HL-60 cells were analyzed by morphological and flow cytometry techniques and growing A. cepa roots were examined after 72 h exposure. Marinobufagin presented high antiproliferative action against all human tumor lines [IC 50 values ranging from 0.15 (leukemia) to 7.35 (larynx) μM] and it failed against human erythrocytes and murine lines. Human normal peripheral blood mononuclear cells (PBMC) were up to 72.5-fold less sensitive [IC 50: 10.88 μM] to marinobufagin than HL-60 line, but DNA strand breaks were no detected. Leukemia treaded cells exhibited cell viability reduction, DNA fragmentation, phosphatidylserine externalization, binucleation, nuclear condensation and cytoplasmic vacuoles. Marinobufagin also reduced the growth of A. cepa roots (EC 50 : 7.5 μM) and mitotic index, caused cell cycle arrest and chromosomal alterations (micronuclei, delays and C-metaphases) in meristematic cells. So, to find out partially targeted natural molecules on human leukemia cells, like marinobufagin, is an amazing and stimulating way to continue the battle against cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of the antimutagenic activity and mode of action of carrageenan fiber in cultured meristematic cells of Allium cepa.

PubMed

Nantes, C I; Pesarini, J R; Mauro, M O; Monreal, A C D; Ramires, A D; Oliveira, R J

2014-11-12

In this study, we evaluated the mutagenic and antimutagenic activities of carrageenan, a sulfated polysaccharide, and described its mode of action by using an Allium cepa assay. The results indicate that carrageenan is not mutagenic, rather it has significant chemopreventive potential that is mediated by both demutagenic and bio-antimutagenic activities. This compound can adsorb agents that are toxic to DNA and inactivate them. Additionally, carrageenan can modulate enzymes of the DNA repair system. The percentage of damage reduction ranged from 62.54 to 96.66%, reflecting the compound's high efficiency in preventing the type of mutagenic damage that may be associated with tumor development. Based on these findings and information available in the literature, we conclude that carrageenan is an important fiber that should be considered as a possible base for functional foods and/or diets with potential anticancer activity.

Nuclei of plants as a sink for flavanols.

PubMed

Feucht, W; Polster, J

2001-01-01

Onion cepa (L.) and Tsuga canadensis (L.) Carr. were investigated histochemically on the association of flavanols to nuclei. The young roots of Onion cepa are totally devoid of flavanol structures. Therefore, the excised roots tips were directly incubated into different solutions of flavanols. After 3 h of incubation a flavanol binding on the nuclei was recognizable, as seen by a yellowish-brown tanning reaction. Still to ensure the presence of flavanols on the nuclei, subsequent staining with the p-dimethylaminocinnamaldehyde reagent (DMACA) resulted in an intense blue colouration. Tsuga canadensis has significant amounts of vacuolar flavanol deposits in all parts of the tree as indicated by the DMACA reagent. It is obvious that also the nuclei were associated strongly with flavanols which can be demonstrated particularly elegant in the cells of the seed wings by histochemical methods. However, the mode of flavanol release from the original deposits is not yet clear.

Arbuscular mycorrhiza improves yield and nutritional properties of onion (Allium cepa).

PubMed

Rozpądek, Piotr; Rąpała-Kozik, Maria; Wężowicz, Katarzyna; Grandin, Anna; Karlsson, Stefan; Ważny, Rafał; Anielska, Teresa; Turnau, Katarzyna

2016-10-01

Improving the nutritional value of commonly cultivated crops is one of the most pending problems for modern agriculture. In natural environments plants associate with a multitude of fungal microorganisms that improve plant fitness. The best described group are arbuscular mycorrhizal fungi (AMF). These fungi have been previously shown to improve the quality and yield of several common crops. In this study we tested the potential utilization of Rhizophagus irregularis in accelerating growth and increasing the content of important dietary phytochemicals in onion (Allium cepa). Our results clearly indicate that biomass production, the abundance of vitamin B1 and its analogues and organic acid concentration can be improved by inoculating the plant with AM fungi. We have shown that improved growth is accompanied with up-regulated electron transport in PSII and antioxidant enzyme activity. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Phenol-quinone tautomerism in (arylazo)naphthols and the analogous Schiff bases: benchmark calculations.

PubMed

Ali, S Tahir; Antonov, Liudmil; Fabian, Walter M F

2014-01-30

Tautomerization energies of a series of isomeric [(4-R-phenyl)azo]naphthols and the analogous Schiff bases (R = N(CH3)2, OCH3, H, CN, NO2) are calculated by LPNO-CEPA/1-CBS using the def2-TZVPP and def2-QZVPP basis sets for extrapolation. The performance of various density functionals (B3LYP, M06-2X, PW6B95, B2PLYP, mPW2PLYP, PWPB95) as well as MP2 and SCS-MP2 is evaluated against these results. M06-2X and SCS-MP2 yield results close to the LPNO-CEPA/1-CBS values. Solvent effects (CCl4, CHCl3, CH3CN, and CH3OH) are treated by a variety of bulk solvation models (SM8, IEFPCM, COSMO, PBF, and SMD) as well as explicit solvation (Monte Carlo free energy perturbation using the OPLSAA force field).

Morphotoxicity and cytogenotoxicity of pendimethalin in the test plant Allium cepa L. - A biomarker based study.

PubMed

Verma, Sonam; Srivastava, Alka

2018-04-30

Pesticides have brought tremendous benefits to mankind by increasing food production and controlling various crop diseases. But their prolonged and extensive use has been reported to induce toxicity. Biological markers used for the evaluation of toxic effects of pesticides have increased these days. The aim of this study was to determine the morphotoxic and cytogenotoxic effects of pesticide pendimethalin applied to the soil by using morphological and genotoxic biomarkers in the test plant Allium cepa L. A pot experiment was set up in which pendimethalin was added to soil at the rate of 0, 0.033, 0.044, 0.055 and 0.066 g kg -1 soil. Similar sized onion bulbs were planted in each pot and 3 replicates were maintained for each dose of pendimethalin at 1, 7, 15, 30 and 45 days after treatment. Average root number (ARN) and average length of roots (ALR) of onion bulbs were recorded and on the day 3 of sowing roots were harvested and fixed for cytological analysis. Morphological biomarkers revealed significant concentration and duration dependent inhibition of ARN and ALR as compared to control which shows the morphotoxicity of pendimethalin. The results also showed inhibitory effect on the mitotic index (%) of A. cepa while relative abnormality rate (%) increased. Further, we observed aberrations in both the dividing and non-dividing cells along with spotting of few ring chromosomes. Reduced mitotic index, increased relative abnormality rate; various chromosomal and interphase nuclear aberrations all being mitosis endpoint markers reflect the cytogenotoxicity of pendimethalin, even at lower concentrations. Copyright © 2018 Elsevier Ltd. All rights reserved.

Calcium movement, graviresponsiveness and the structure of columella cells and columella tissues in roots of Allium cepa L

NASA Technical Reports Server (NTRS)

Moore, R.

1985-01-01

Roots of Allium cepa L. cv. Yellow are differentially responsive to gravity. Long (e.g. 40 mm) roots are strongly graviresponsive, while short (c.g. 4 mm) roots are minimally responsive to gravity. Although columella cells of graviresponsive roots are larger than those of nongraviresponsive roots, they partition their volumes to cellular organelles similarly. The movement of amyloplasts and nuclei in columella cells of horizontally-oriented roots correlates positively with the onset of gravicurvature. Furthermore, there is no significant difference in the rates of organellar redistribution when graviresponsive and nongraviresponsive roots are oriented horizontally. The more pronounced graviresponsiveness of longer roots correlates positively with (1) their caps being 9-6 times more voluminous, (2) their columella tissues being 42 times more voluminous, (3) their caps having 15 times more columella cells, and (4) their columella tissues having relative volumes 4.4 times larger than those of shorter, nongraviresponsive roots. Graviresponsive roots that are oriented horizontally are characterized by a strongly polar movement of 45Ca2+ across the root tip from the upper to the lower side, while similarly oriented nongraviresponsive roots exhibit only a minimal polar transport of 45Ca2+. These results indicate that the differential graviresponsiveness of roots of A. cepa is probably not due to either (1) ultrastructural differences in their columella cells, (2) differences in the rates of organellar redistribution when roots are oriented horizontally. Rather, these results indicate the graviresponsiveness may require an extensive columella tissue, which, in turn, may be necessary for polar movement of 45Ca2+ across the root tip.

Investigation of viability of plant tissue in the environmental scanning electron microscopy.

PubMed

Zheng, Tao; Waldron, K W; Donald, Athene M

2009-11-01

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.

ABILITY OF ECOSAR, TOPKAT, NEURAL NETWORKS, AND ASTER TO PREDICT TOXICITY OF CHEMICALS TO AQUATIC BIOTA

EPA Science Inventory

The Canadian Environmental Protection Act (CEPA) which provides the basis for assessing and managing toxic substances in Canada, is being revised. Several new mandates have been introduced in the Act...

Revisiting Bioaccumulation Criteria

EPA Science Inventory

The objective of workgroup 5 was to revisit the B(ioaccumulation) criteria that are currently being used to identify POPs under the Stockholm Convention and PBTs under CEPA, TSCA, REACh and other programs. Despite the lack of a recognized definition for a B substance, we defined ...

Morphological observation of the stria vascularis in midkine and pleiotrophin knockout mice.

PubMed

Sone, Michihiko; Muramatsu, Hisako; Muramatsu, Takashi; Nakashima, Tsutomu

2011-02-01

Midkine and Pleiotrophin are low molecular weight basic proteins with closely related structures and serve as growth/differentiation factors. They have been reported to be expressed in the cochlea during the embryonic and perinatal periods. In the present study, we focused on the roles of midkine and pleiotrophin in the stria vascularis and investigated morphological changes using mice deficient in these genes. Midkine knockout, pleiotrophin knockout, and double knockout mice were used and compared to wild-type mice. Auditory brain stem responses (ABRs) and cochlear blood flows were measured in each type of mice. Pathological changes in the stria vascularis were examined by light microscopy, including immunohistochemical staining with anti-Kir4.1 antibody, and electron microscopy. Hearing thresholds examined by ABRs were significantly higher in midkine knockout and pleiotrophin knockout mice than in wild-type mice. Double knockout mice showed higher thresholds compared to midkine knockout and pleiotrophin knockout mice. Blood flow in the lateral walls did not significantly differ and light microscopy examination showed an almost normal appearance of the stria vascularis in these knockout mice. However, the expression of Kir4.1 was weak in the knockout mice and severe vacuolar degeneration was observed by electron microscopy in the intermediate cells of the double knockout mice. The present study demonstrates that midkine and pleiotrophin play some roles for the morphological maintenance of intermediate cell in the stria vascularis. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Microscopic examination on cytological changes in Allium cepa and shift in phytoplankton population at different doses of Atrazine

NASA Astrophysics Data System (ADS)

Ghosh, Nabarun; Finger, Kristen; Usnick, Samantha; Rogers, William J.; Das, A. B.; Smith, Don W.

2010-06-01

Atrazine is a wide-range herbicide. For over 50 years, atrazine has been used as a selective broadleaf herbicide in many capacities, from pre-plant to pre-emergence to post-emergence, depending on the crop and application. Currently, 96% of all atrazine used is for commercial applications in fields for the control of broadleaf and grassy weeds in crops such as sorghum, corn, sugarcane, pineapple and for the control of undesirable weeds in rangeland. Many panhandle wells have also detected atrazine in samples taken. The concern for the public is the long-term effect of atrazine with its increasing popularity, and the impact on public health. We investigated the effect of different concentrations of atrazine on Allium cepa (onion), a standard plant test system. We established a control with the Allium bulbs grown on hydroponics culture. Varying concentrations of atrazine was used on the standard plant test system, Allium cepa grown hydroponically. The mitotic indices varied and with higher doses, we observed various chromosomal abnormalities including sticky bridges, early and late separations, and lag chromosomes with higher doses of treatments. In the second part of the experiment, 0.1ppb, 1ppb, 10ppb, and 100ppb concentrations of atrazine were applied to established phytoplankton cultures from the Lake Tanglewood, Texas. Study with a Sedgwick-Rafter counter, a BX-40 Olympus microscope with DP-70 camera revealed a gradual shift in the phytoplankton community from obligatory to facultative autotroph and finally to a parasitic planktonic community. This explains the periodic fish kill in the lakes after applications of atrazine in crop fields.

Combination of physico-chemical analysis, Allium cepa test system and Oreochromis niloticus erythrocyte based comet assay/nuclear abnormalities tests for cyto-genotoxicity assessments of treated effluents discharged from textile industries.

PubMed

Hemachandra, Chamini K; Pathiratne, Asoka

2016-09-01

Bioassays for cyto-genotoxicity assessments are generally not required in current textile industry effluent discharge management regulations. The present study applied in vivo plant and fish based toxicity tests viz. Allium cepa test system and Oreochromis niloticus erythrocyte based comet assay and nuclear abnormalities tests in combination with physico-chemical analysis for assessing potential cytotoxic/genotoxic impacts of treated textile industry effluents reaching a major river (Kelani River) in Sri Lanka. Of the treated effluents tested from two textile industries, color in the Textile industry 1 effluents occasionally and color, biochemical oxygen demand and chemical oxygen demand in the Textile industry 2 effluents frequently exceeded the specified Sri Lankan tolerance limits for discharge of industrial effluents into inland surface waters. Exposure of A. cepa bulbs to 100% and 12.5% treated effluents from both industries resulted in statistically significant root growth retardation, mito-depression, and induction of chromosomal abnormalities in root meristematic cells in comparison to the dilution water in all cases demonstrating cyto-genotoxicity associated with the treated effluents. Exposure of O. niloticus to the 100% and 12.5% effluents, resulted in erythrocytic genetic damage as shown by elevated total comet scores and induction of nuclear abnormalities confirming the genotoxicity of the treated effluents even with 1:8 dilution. The results provide strong scientific evidence for the crucial necessity of incorporating cyto-genotoxicity impact assessment tools in textile industry effluent management regulations considering human health and ecological health of the receiving water course under chronic exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

The nature of arms in spiral galaxies. III. Azimuthal profiles

NASA Astrophysics Data System (ADS)

del Rio, M. S.; Cepa, J.

1998-12-01

In this paper we analyse the structure of a small sample of galaxies using a set of CCD images in standard photometric bands presented in a previous paper (del Rio & Cepa 1998a, hereafter \\cite{p2}). The galaxies are NGC 157, 753, 895, 4321, 6764, 6814, 6951, 7479 and 7723, and the selected bands were B and I. Seven galaxies are grand design, i.e. they have two long and symmetric arms, second in the classification of \\cite{ee87} (1987), and are the best laboratories for testing the predictions of the spiral density wave (SDW) theory. Two of the galaxies have intermediate arms, i.e., they are not so well defined. They are selected to compare the results with those found in the grand design spirals. Using the method of analyse the azimuthal flux profiles presented by \\cite{c88} (1988) and Beckman & Cepa (1990) (hereafter \\cite{bc90}) and assuming that star formation is triggered by a spiral density wave, we look for evidence of the existence of a corotation radius, as predicted by the SDW theory. We have determined the corotation radius in all but two grand design galaxies, and, tentatively, in the other four. Galaxies with very weak arms (such as NGC 753 and NGC 6951) or arms which are not well defined (such as NGC 6764 and NGC 7723) present difficulties when employing the azimuthal profile method, but even in these cases, the method is powerful enough to give a good estimate of the value of corotation, which must then be confirmed (or discarded) by other independent methods (del Rio & Cepa 1998b, hereafter \\cite{p4}).

PubMed

Müller Velázquez, Lucía; Bobadilla, Fernando; Novosak, Marina; Cortese, Iliana; Laczeski, Margarita

2018-03-20

Streptococcus agalactiae (SGB) es causa de infecciones severas en menores de tres meses. Meningitis, neumonía y sepsis son los principales cuadros en estos niños. Estas infecciones se encuentran entre las más graves que puede sufrir un individuo en sus primeras doce horas de vida. El niño adquiere la infección por transmisión vertical de la madre colonizada. Para prevenir la enfermedad neonatal se recomienda penicilina como droga de elección en la profilaxis intraparto (PIP) en embarazadas colonizadas. Sin embargo, actualmente se han detectado cepas con sensibilidad disminuida a penicilina por lo que resulta importante realizar la vigilancia de la sensibilidad al mismo para asegurar su utilidad durante la profilaxis. El objetivo de este trabajo fue determinar la sensibilidad a penicilina en cepas de SGB recuperados de mujeres embarazadas de 35-37 semanas de gestación. Se estudiaron 96 aislamientos y se determinó la sensibilidad por método epsilométrico Etest® (LIOFILCHEM, Italia), siguiendo las recomendaciones del Clinical Laboratory Standards Institute (CLSI). Se obtuvo la Concentración Inhibitoria Mínima (CIM) para cada aislamiento bacteriano. El 100% (96) de las cepas estudiadas fue sensible a penicilina con valores de CIM comprendidos entre 0,012 y 0,094 µg mL-1. Estos resultados indican que penicilina sigue siendo el antimicrobiano de elección durante la profilaxis intraparto, para la prevención de la enfermedad neonatal causada por SGB en nuestra región. Se destaca la importancia de la vigilancia epidemiológica de la sensibilidad a penicilina y a otros antimicrobianos para alertar sobre nuevos mecanismos de resistencia y adecuar estrategias de tratamiento.

Alleviation of cyclophosphamide-induced immunosuppression in Wistar rats by onion lectin (Allium cepa agglutinin).

PubMed

Kumar, Vaddi P; Venkatesh, Yeldur P

2016-06-20

In various traditional medicines, onion has been classified as an immune-boosting food. Recent studies have claimed this property due to the presence of bioactive organosulfur compounds, prebiotic fructo-oligosaccharides and an immunomodulatory protein, lectin (Allium cepa agglutinin; ACA) (Prasanna and Venkatesh, 2015. Characterization of onion lectin (Allium cepa agglutinin) as an immunomodulatory protein inducing Th1-type immune response in vitro. Int. Immunopharmacol. vol. 26, pp. 304-313). The aim of this study was to evaluate the immunoprotective properties of ACA in normal and cyclophosphamide (CP; 100μg/kg)-induced immunosuppressed Wistar rats. Wistar rats were administrated different doses of ACA (1, 10, and 100μg) to respective groups in normal as well as immunosuppressed animals. The effect of ACA on the status of immune organs was assessed by examining the splenic and thymic indices, and histopathological changes. The biomarkers for humoral immunity (serum IgG and IgA levels) and serum pro-inflammatory markers (COX-2, TNF-α and IL-10) were measured by ELISA. ACA showed immunoprotective properties by significantly promoting the restoration of lymphoid cell count by ~6 fold vs. model control (immunosuppressed animals) and promotes the immune response significantly (~1.5-fold) in CP-induced immunosuppressed animals compared to model control; production of pro-inflammatory molecules (COX-2 and nitric oxide) and expression levels of immune regulatory molecule (TNF-α) were elevated in a dose-dependent manner. The observed in vivo results suggest that ACA has the potential to be used as a nutritional therapeutic to boost the immune status of immunosuppressed subjects brought about by CP administration. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cytogenetic evaluation of gold nanorods using Allium cepa test.

PubMed

Rajeshwari, A; Roy, Barsha; Chandrasekaran, Natarajan; Mukherjee, Amitava

2016-12-01

The current study reveals the impact of gold nanorods (NRs) capped with CTAB (cetyltrimethylammonium bromide) or PEG (polyethylene glycol) on Allium cepa. The morphology and surface charge of CTAB- and PEG-capped gold NRs were characterized by electron microscopic and zeta potential analyses. The chromosomal aberrations like clumped chromosome, chromosomal break, chromosomal bridge, diagonal anaphase, disturbed metaphase, laggard chromosome, and sticky chromosome were observed in the root tip cells exposed to different concentrations (0.1, 1, and 10 μg/mL) of CTAB- and PEG-capped gold NRs. We found that both CTAB- and PEG-capped gold NRs were able to induce toxicity in the plant system after 4-h interaction. At a maximum concentration of 10 μg/mL, the mitotic index reduction induced by CTAB-capped gold NRs was 40-fold higher than that induced by PEG-capped gold NRs. The toxicity of gold NRs was further confirmed by lipid peroxidation and oxidative stress analyses. The unbound CTAB also contributed to the toxicity in root tip cells, while PEG alone shows less toxicity to the cells. The vehicle control CTAB contributed to the toxic effects in root tip cells, while PEG alone did not show any toxicity to the cells. The results revealed that even though both the particles have adverse effects on A. cepa, there was a significant difference in the mitotic index and oxidative stress generation in root cells exposed to CTAB-capped gold NRs. Thus, this study concludes that the surface polymerization of gold NRs by PEG can reduce the toxicity of CTAB-capped gold NRs. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Ground and Surface Water for Drinking: A Laboratory Study on Genotoxicity Using Plant Tests

PubMed Central

Feretti, Donatella; Ceretti, Elisabetta; Gustavino, Bianca; Zerbini, llaria; Zani, Claudia; Monarca, Silvano; Rizzoni, Marco

2012-01-01

Surface waters are increasingly utilized for drinking water because groundwater sources are often polluted. Several monitoring studies have detected the presence of mutagenicity in drinking water, especially from surface sources due to the reaction of natural organic matter with disinfectant. The study aimed to investigate the genotoxic potential of the products of reaction between humic substances, which are naturally present in surface water, and three disinfectants: chlorine dioxide, sodium hypochlorite and peracetic acid. Commercial humic acids dissolved in distilled water at different total organic carbon (TOC) concentrations were studied in order to simulate natural conditions of both ground water (TOC=2.5 mg/L) and surface water (TOC=7.5 mg/L). These solutions were treated with the biocides at a 1:1 molar ratio of C:disinfectant and tested for genotoxicity using the anaphase chromosomal aberration and micronucleus tests in Allium cepa, and the Vicia faba and Tradescantia micronucleus tests. The tests were carried out after different times and with different modes of exposure, and at 1:1 and 1:10 dilutions of disinfected and undisinfected humic acid solutions. A genotoxic effect was found for sodium hypochlorite in all plant tests, at both TOCs considered, while chlorine dioxide gave positive results only with the A.cepa tests. Some positive effects were also detected for PAA (A.cepa and Tradescantia). No relevant differences were found in samples with different TOC values. The significant increase in all genotoxicity end-points induced by all tested disinfectants indicates that a genotoxic potential is exerted even in the presence of organic substances at similar concentrations to those frequently present in drinking water. PMID:25170443

Herbivory by Thrips tabaci

Treesearch

Deborah M. Kendall

1991-01-01

Herbivory by Thrips tabaci (Lindeman) affects both the bulb yield and phytohormone balance in its major host plant, the onion (Alium cepa L.). Seasonal changes in the susceptibility of onion yield to T. tabaci feeding were examined during the three growth stages of onion; prebulbing, bulbing and sizing (Kendall...

Soil Rhizosphere Microbial Communities and Enzyme Activities under Organic Farming

USDA-ARS?s Scientific Manuscript database

This study investigated the activities of ß-glucosidase (C cycling, ß-glucosaminidase (C and N cycling), acid phosphatase (P cycling) and arylsulfatase (S cycling) under lettuce (Lactuca sativa), potato (Solanum Tuberosum), onion (Allium cepa L), broccoli (Brassica oleracea var. botrytis) and Tall f...

Forskolin: genotoxicity assessment in Allium cepa.

PubMed

Mohammed, Khalid Pasha; Aarey, Archana; Tamkeen, Shayesta; Jahan, Parveen

2015-01-01

Forskolin, a diterpene, 7β-acetoxy-8,13-epoxy-1α,6β,9α-trihydroxy-labd-14-en-11-one (C22H34O7) isolated from Coleus forskohlii, exerts multiple physiological effects by stimulating the enzyme adenylate cyclase and increasing cyclic adenosine monophosphate (cAMP) concentrations. Forskolin is used in the treatment of hypertension, congestive heart failure, eczema, and other diseases. A cytogenetic assay was performed in Allium cepa to assess possible genotoxic effects of forskolin. Forskolin was tested at concentrations 5-100 μM for exposure periods of 24 or 48 h. Treated samples showed significant reductions in mitotic index (p < 0.05) and increases in the frequency of chromosome aberrations (p < 0.01) at both exposure times. The treated meristems showed chromosome aberrations including sticky metaphases, sticky anaphases, laggard, anaphase bridges, micronuclei, polyploidy, fragments, breaks, and C-mitosis. Forskolin may cause genotoxic effects and further toxicological evaluations should be conducted to ensure its safety. Copyright © 2014 Elsevier B.V. All rights reserved.

Pulsed Direct Current Electrospray: Enabling Systematic Analysis of Small Volume Sample by Boosting Sample Economy.

PubMed

Wei, Zhenwei; Xiong, Xingchuang; Guo, Chengan; Si, Xingyu; Zhao, Yaoyao; He, Muyi; Yang, Chengdui; Xu, Wei; Tang, Fei; Fang, Xiang; Zhang, Sichun; Zhang, Xinrong

2015-11-17

We had developed pulsed direct current electrospray ionization mass spectrometry (pulsed-dc-ESI-MS) for systematically profiling and determining components in small volume sample. Pulsed-dc-ESI utilized constant high voltage to induce the generation of single polarity pulsed electrospray remotely. This method had significantly boosted the sample economy, so as to obtain several minutes MS signal duration from merely picoliter volume sample. The elongated MS signal duration enable us to collect abundant MS(2) information on interested components in a small volume sample for systematical analysis. This method had been successfully applied for single cell metabolomics analysis. We had obtained 2-D profile of metabolites (including exact mass and MS(2) data) from single plant and mammalian cell, concerning 1034 components and 656 components for Allium cepa and HeLa cells, respectively. Further identification had found 162 compounds and 28 different modification groups of 141 saccharides in a single Allium cepa cell, indicating pulsed-dc-ESI a powerful tool for small volume sample systematical analysis.

Flavonol Glucoside and Antioxidant Enzyme Biosynthesis Affected by Mycorrhizal Fungi in Various Cultivars of Onion (Allium cepa L.).

PubMed

Mollavali, Mohanna; Bolandnazar, Saheb Ali; Schwarz, Dietmar; Rohn, Sascha; Riehle, Peer; Zaare Nahandi, Fariborz

2016-01-13

The objective of this study was to investigate the impact of mycorrhizal symbiosis on qualitative characteristics of onion (Allium cepa L.). For this reason, five onion cultivars with different scale color and three different strains of arbuscular mycorrhizal fungi (Diversispora versiformis, Rhizophagus intraradices, Funneliformis mosseae) were used. Red cultivars, mainly 'Red Azar-shahr', showed the highest content in vitamin C, flavonols, and antioxidant enzymes. Mycorrhizal inoculation increased total phenolic, pyruvic acid, and vitamin C of onion plants. Considerable increase was observed in quercetin-4'-O-monoglucoside and isorhamnetin-4'-O-monoglucoside content in plants inoculated with Diversispora versiformis, but quercetin-3,4'-O-diglucoside was not significantly influenced. Analyses for phenylalanine ammonia-lyase (PAL) and antioxiodant enzyme activities such as polyphenol oxidase (PPO), catalase (CAT), and peroxidase (POD) revealed that all except PPO were enhanced by mycorrhizal inoculation. Overall, these findings suggested that mycorrhizal inoculation influenced biosynthesis of flavonol glucosides and antioxidant enzymes by increasing nutrient uptake or by induction of the plant defense system.

Effect of Cultivar and Cultivation Year on the Metabolite Profile of Onion Bulbs ( Allium cepa L.).

PubMed

Böttcher, Christoph; Krähmer, Andrea; Stürtz, Melanie; Widder, Sabine; Schulz, Hartwig

2018-03-28

This study investigated the variation of metabolite profiles of onion bulbs ( Allium cepa L.) depending on genetic and environmental factors. Nine onion cultivars ("Corrado", "Cupido", "Forum", "Hytech", "Picador", "Redlight", "Snowpack", "Stardust", "Sturon") with different scale color and dry matter content were grown in a two-year field trial. Using a recently established metabolite profiling approach based on liquid chromatography-coupled electrospray ionization quadrupole time-of-flight mass spectrometry, 106 polar and semipolar metabolites which belong to compound classes determining nutritional, sensory, and technological quality of onion bulbs such as saccharides, flavonoids, S-substitued cysteine conjugates, amino acids, and derived γ-glutamyl peptides were relatively quantitated in parallel. Statistical analyses of the obtained data indicated that depending on the compound class genetic and environmental factors differently contributed to variation of metabolite levels. For saccharides and flavonoids the genetic factor was the major source of variation, whereas for cysteine sulfoxides, amino acids, and peptides both genetic and environmental factors had a significant impact on corresponding metabolite levels.

Ecotoxicological and genotoxic assessment of hospital laundry wastewaters.

PubMed

Kern, Deivid Ismael; Schwaickhardt, Rômulo de Oliveira; Lutterbeck, Carlos Alexandre; Kist, Lourdes Teresinha; Alcayaga, Eduardo Alexis Lobo; Machado, Ênio Leandro

2015-01-01

The aim of the present study was to assess the ecotoxicity and genotoxicity of hospital laundry wastewaters generated from a regional hospital located in Rio Pardo Valley in the state of Rio Grande do Sul, Brazil. Physicochemical, microbiological, ecotoxicological, and genotoxic analyses were performed, and the results indicate that some parameters were not in accordance with the limit concentrations established by Brazilian and international guidelines for urban wastewaters. Daphnia magna (EC50 2.01%) and Danio rerio (LC50 29.25%) acute toxicity was detected, and sublethal effects were identified in Lactuca sativa (IC25 12.50%) and Allium cepa (IC25 51.25%). Cytotoxicity was observed at the five wastewater concentrations used yielding statistically significant differences (p < 0.05) in the meristematic cells of A. cepa compared with the negative control. The results obtained here warn about the necessity to develop treatment methods that can mitigate the environmental impacts caused by the ecotoxicity and genotoxicity of hospital laundry wastewaters.

Effect of different exposed lights on quercetin and quercetin glucoside content in onion (Allium cepa L.)

PubMed Central

Ko, Eun Young; Nile, Shivraj Hariram; Sharma, Kavita; Li, Guan Hao; Park, Se Won

2014-01-01

Quercetin and quercetin glucosides are the major flavonols present in onion (Allium cepa L.) and are predominantly present as quercetin, quercetin-3,4′-diglucoside and quercetin-4′-glucoside. Effect of different light wavelengths on onion after harvest and storage, with fluorescent, blue, red and ultra violet light influenced the quercetin and quercetin glucosides profile. In a peeled onion, all the light treatments elevated quercetin content in bulb. Among them, particularly fluorescent light effect was more eminent which stimulates the maximum synthesis of quercetin in onion. In case of whole onion bulb, skin and pulp showed different responses to light treatment, respectively. The pulp had the highest quercetin glucosides under blue light, whereas the lowest under fluorescent light. Onion skin showed nearly opposite pattern as compared to the pulp. In particular, light treatment proved to be a better way to increase the level of quercetin content in onions which might be utilized for industrial production of bioactive compounds from onion and onion waste products. PMID:26150744

Effect of different exposed lights on quercetin and quercetin glucoside content in onion (Allium cepa L.).

PubMed

Ko, Eun Young; Nile, Shivraj Hariram; Sharma, Kavita; Li, Guan Hao; Park, Se Won

2015-07-01

Quercetin and quercetin glucosides are the major flavonols present in onion (Allium cepa L.) and are predominantly present as quercetin, quercetin-3,4'-diglucoside and quercetin-4'-glucoside. Effect of different light wavelengths on onion after harvest and storage, with fluorescent, blue, red and ultra violet light influenced the quercetin and quercetin glucosides profile. In a peeled onion, all the light treatments elevated quercetin content in bulb. Among them, particularly fluorescent light effect was more eminent which stimulates the maximum synthesis of quercetin in onion. In case of whole onion bulb, skin and pulp showed different responses to light treatment, respectively. The pulp had the highest quercetin glucosides under blue light, whereas the lowest under fluorescent light. Onion skin showed nearly opposite pattern as compared to the pulp. In particular, light treatment proved to be a better way to increase the level of quercetin content in onions which might be utilized for industrial production of bioactive compounds from onion and onion waste products.

Pituitary-adrenal responses to oxotremorine and acute stress in male and female M1 muscarinic receptor knockout mice: comparisons to M2 muscarinic receptor knockout mice.

PubMed

Rhodes, M E; Rubin, R T; McKlveen, J M; Karwoski, T E; Fulton, B A; Czambel, R K

2008-05-01

Both within the brain and in the periphery, M(1) muscarinic receptors function primarily as postsynaptic receptors and M(2) muscarinic receptors function primarily as presynaptic autoreceptors. In addition to classical parasympathetic effectors, cholinergic stimulation of central muscarinic receptors influences the release of adrenocorticotrophic hormone (ACTH) and corticosterone. We previously reported that oxotremorine administration to male and female M(2) receptor knockout and wild-type mice increased ACTH to a significantly greater degree in knockout males compared to all other groups, and that M(2) knockout mice of both sexes were significantly more responsive to the mild stress of saline injection than were wild-type mice. These results accord with the primary function of M(2) receptors as presynaptic autoreceptors. In the present study, we explored the role of the M(1) receptor in pituitary-adrenal responses to oxotremorine and saline in male and female M(1) knockout and wild-type mice. Because these mice responded differently to the mild stress of saline injection than did the M(2) knockout and wild-type mice, we also determined hormone responses to restraint stress in both M(1) and M(2) knockout and wild-type mice. Male and female M(1) knockout and wild-type mice were equally unresponsive to the stress of saline injection. Oxotremorine increased both ACTH and corticosterone in M(1) wild-type mice to a significantly greater degree than in knockout mice. In both M(1) knockout and wild-type animals, ACTH responses were greater in males compared to females, and corticosterone responses were greater in females compared to males. Hormone responses to restraint stress were increased in M(2) knockout mice and decreased in M(1) knockout mice compared to their wild-type counterparts. These findings suggest that M(1) and M(2) muscarinic receptor subtypes differentially influence male and female pituitary-adrenal responses to cholinergic stimulation and stress. The decreased pituitary-adrenal sensitivity to oxotremorine and restraint stress noted in M(1) knockout mice is consistent with M(1) being primarily a postsynaptic receptor. Conversely, the increased pituitary-adrenal sensitivity to these challenges noted in M(2) knockout mice is consistent with M(2) being primarily a presynaptic autoreceptor.

Educators as "Equity Warriors." CEPA Working Paper No. 17-11

ERIC Educational Resources Information Center

Rochmes, Jane; Penner, Emily; Loeb, Susanna

2017-01-01

Despite the multifaceted causes of educational disparities, schools' responsibility for reducing inequality undergirds American ideals. Educators operate as street-level bureaucrats to accomplish this equalizing work. Surprisingly, we know little about how teachers think about inequality or enact strategies to combat it, even though their…

7 CFR 959.5 - Onions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 8 2012-01-01 2012-01-01 false Onions. 959.5 Section 959.5 Agriculture Regulations of... Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE ONIONS GROWN IN SOUTH TEXAS Order Regulating Handling Definitions § 959.5 Onions. Onions means all varieties of Allium cepa commonly known as onions...

7 CFR 959.5 - Onions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 8 2014-01-01 2014-01-01 false Onions. 959.5 Section 959.5 Agriculture Regulations of... ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE ONIONS GROWN IN SOUTH TEXAS Order Regulating Handling Definitions § 959.5 Onions. Onions means all varieties of Allium cepa commonly known as onions...

7 CFR 959.5 - Onions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 8 2011-01-01 2011-01-01 false Onions. 959.5 Section 959.5 Agriculture Regulations of... Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE ONIONS GROWN IN SOUTH TEXAS Order Regulating Handling Definitions § 959.5 Onions. Onions means all varieties of Allium cepa commonly known as onions...

7 CFR 955.5 - Vidalia onion.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 8 2012-01-01 2012-01-01 false Vidalia onion. 955.5 Section 955.5 Agriculture... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA Definitions § 955.5 Vidalia onion. Vidalia onion means all varieties of Allium cepa of the hybrid yellow...

7 CFR 955.5 - Vidalia onion.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 8 2011-01-01 2011-01-01 false Vidalia onion. 955.5 Section 955.5 Agriculture... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA Definitions § 955.5 Vidalia onion. Vidalia onion means all varieties of Allium cepa of the hybrid yellow...

7 CFR 959.5 - Onions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 8 2013-01-01 2013-01-01 false Onions. 959.5 Section 959.5 Agriculture Regulations of... ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE ONIONS GROWN IN SOUTH TEXAS Order Regulating Handling Definitions § 959.5 Onions. Onions means all varieties of Allium cepa commonly known as onions...

7 CFR 955.5 - Vidalia onion.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Vidalia onion. 955.5 Section 955.5 Agriculture... and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA Definitions § 955.5 Vidalia onion. Vidalia onion means all varieties of Allium cepa of the hybrid yellow...

7 CFR 955.5 - Vidalia onion.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 8 2014-01-01 2014-01-01 false Vidalia onion. 955.5 Section 955.5 Agriculture... AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA Definitions § 955.5 Vidalia onion. Vidalia onion means all varieties of Allium cepa of the hybrid yellow...

7 CFR 959.5 - Onions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Onions. 959.5 Section 959.5 Agriculture Regulations of... Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE ONIONS GROWN IN SOUTH TEXAS Order Regulating Handling Definitions § 959.5 Onions. Onions means all varieties of Allium cepa commonly known as onions...

7 CFR 955.5 - Vidalia onion.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 8 2013-01-01 2013-01-01 false Vidalia onion. 955.5 Section 955.5 Agriculture... AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE VIDALIA ONIONS GROWN IN GEORGIA Definitions § 955.5 Vidalia onion. Vidalia onion means all varieties of Allium cepa of the hybrid yellow...

40 CFR 180.41 - Crop group tables.

Code of Federal Regulations, 2010 CFR

2010-07-01

.... var. bulbiferum L.H. Bailey; A. cepa L. var. viviparum (Metz.) Alef.) 3-07B Onion, Welsh, tops (Allium... grains group. (19) Crop Group 17. Grass Forage, Fodder, and Hay Group. (i) Representative commodities. Bermuda grass; bluegrass; and bromegrass or fescue. (ii) Commodities. The commodities included in Crop...

ESD and the Rio Conventions

ERIC Educational Resources Information Center

Sarabhai, Kartikeya V.; Ravindranath, Shailaja; Schwarz, Rixa; Vyas, Purvi

2012-01-01

Chapter 36 of Agenda 21, a key document of the 1992 Earth Summit, emphasised reorienting education towards sustainable development. While two of the Rio conventions, the Convention on Biological Diversity (CBD) and the United Nations Framework Convention on Climate Change (UNFCCC), developed communication, education and public awareness (CEPA)…

Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity

DTIC Science & Technology

2000-01-01

various organs and to sites of inflammation. They may have additional functions. For example analysis of CXCR4 knockout mice show that CXCR4, which...SDF-1 knockout mice had similar phenotypes (195). Homozygous knockout of CXCR4 or SDF-1 results in embyonic lethality. Though CCR5 appears to be...dispensable, other chemokine receptors have vital functions. CXCR5 knockout mice have B-cell homing defects (118), and CXCR2 knockout mice

San Francisco Unified School District: 2013 Survey Brief

ERIC Educational Resources Information Center

Center for Education Policy Analysis at Stanford University, 2013

2013-01-01

Since the 2009-10 school-year, the Center for Education Policy Analysis (CEPA) at Stanford University administered Teacher, Assistant Principal (AP), and Principal surveys as part of San Francisco Unified School District's (SFUSD's) evaluation of the Quality Teacher and Education Act (passed as Proposition A 2008). The findings from these surveys…

Interactions among cultivation, weeds, and a bio-fungicide in organic Vidalia sweet onion

USDA-ARS?s Scientific Manuscript database

Weed management in organic Vidalia® sweet onion (Allium cepa) is largely dependent on multiple cultivations with a tine weeder. Earlier research suggested cultivation with a tine weeder did not predispose onion bulbs to infection during storage. Trials were conducted from 2012 through 2014 near Ly...

Quantitative trait loci controlling amounts and types of epicuticular waxes in onion

USDA-ARS?s Scientific Manuscript database

Natural variation exists in onion (Allium cepa L.) for amounts and types of epicuticular waxes on leaves. Wild-type waxy onion possesses copious amounts of these waxes, while the foliage of semi-glossy and glossy phenotypes accumulate significantly less wax. Reduced amounts of epicuticular waxes hav...

Host ranges of North American isolates of Penicillium causing blue mold of bulb crops

USDA-ARS?s Scientific Manuscript database

Single isolates of four Penicillium species belonging to series Corymbifera (Penicillium allii, P. hirsutum, P. tulipae, P. venetum) plus an isolate of P. polonicum, all from North American sources, were inoculated individually into Crocus sativus, Allium sativum (garlic), A. cepa (onion), Iris holl...

Recent transmission of drug-resistant Mycobacterium tuberculosis in a prison population in southern Brazil.

PubMed

Reis, Ana Julia; David, Simone Maria Martini de; Nunes, Luciana de Souza; Valim, Andreia Rosane de Moura; Possuelo, Lia Gonçalves

2016-01-01

We conducted a cross-sectional, retrospective study, characterized by classical and molecular epidemiology, involving M. tuberculosis isolates from a regional prison in southern Brazil. Between January of 2011 and August of 2014, 379 prisoners underwent sputum smear microscopy and culture; 53 (13.9%) were diagnosed with active tuberculosis. Of those, 8 (22.9%) presented with isoniazid-resistant tuberculosis. Strain genotyping was carried out by 15-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat analysis; 68.6% of the patients were distributed into five clusters, and 87.5% of the resistant cases were in the same cluster. The frequency of drug-resistant tuberculosis cases and the rate of recent transmission were high. Our data suggest the need to implement an effective tuberculosis control program within the prison system. RESUMO Estudo transversal, retrospectivo, com isolados de M. tuberculosis de pacientes de um presídio regional no sul do Brasil, caracterizado através de epidemiologia clássica e molecular. Entre janeiro de 2011 e agosto de 2014, 379 detentos foram submetidos a baciloscopia e cultura, sendo 53 (13,9%) diagnosticados com tuberculose ativa. Desses, 8 (22,9%) apresentavam tuberculose resistente a isoniazida. A genotipagem das cepas foi realizada por 15-locus mycobacterial interspersed repetitive units-variable number of tandem repeat analysis; 68,6% dos pacientes estavam distribuídos em cinco clusters, e 87,5% dos casos resistentes estavam em um mesmo cluster. Verificou-se uma frequência elevada de casos de resistência e alta taxa de transmissão recente. Estes dados sugerem a necessidade da implantação de um programa efetivo de controle da tuberculose no sistema prisional.

Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1α-hydroxylase

PubMed Central

Ohnishi, Mutsuko; Nakatani, Teruyo; Lanske, Beate; Razzaque, M. Shawkat

2011-01-01

Changes in the expression of klotho, a β-glucuronidase, contribute to the development of features that resemble those of premature aging, as well as chronic renal failure. Klotho knockout mice have increased expression of the sodium/phosphate cotransporter (NaPi2a) and 1α-hydroxylase in their kidneys, along with increased serum levels of phosphate and 1,25-dihydroxyvitamin D. These changes are associated with widespread soft-tissue calcifications, generalized tissue atrophy, and a shorter lifespan in the knockout mice. To determine the role of the increased vitamin D activities in klotho knockout animals, we generated klotho and 1α-hydroxylase double-knockout mice. These double mutants regained body weight and developed hypophosphatemia with a complete elimination of the soft-tissue and vascular calcifications that were routinely found in klotho knockout mice. The markedly increased serum fibroblast growth factor 23 and the abnormally low serum parathyroid hormone levels, typical of klotho knockout mice, were significantly reversed in the double-knockout animals. These in vivo studies suggest that vitamin D has a pathologic role in regulating abnormal mineral ion metabolism and soft-tissue anomalies of klotho-deficient mice. PMID:19225558

San Francisco Unified School District Survey Report II: Teacher Hiring

ERIC Educational Resources Information Center

Gao, Niu

2013-01-01

This survey report contains the results of the 2009-10, 2010-11, and 2011-12 Principal Surveys conducted by Stanford's Center for Education Policy Analysis (CEPA). The surveys were administered to principals in the San Francisco Unified School District (SFUSD). This report appears in two parts, each highlighting some of the aggregated responses…

The Causal Effects of Cultural Relevance: Evidence from an Ethnic Studies Curriculum. CEPA Working Paper No. 16-01

ERIC Educational Resources Information Center

Dee, Thomas; Penner, Emily

2016-01-01

An extensive theoretical and qualitative literature stresses the promise of instructional practices and content aligned with the cultural experiences of minority students. Ethnic studies courses provide a growing but controversial example of such "culturally relevant pedagogy." However, the empirical evidence on the effectiveness of…

Nitrogen requirements at bulb initiation for production of intermediate-day onions

USDA-ARS?s Scientific Manuscript database

The effect of nitrogen application on growth, nitrogen (N) uptake, yield, and quality of intermediate-day onion (Allium cepa L. ‘Guimar’) was evaluated in the field in southern Portugal. Plants were fertilized with 30 kg/ha N at transplanting, 10 kg/ha N at 29 days after transplanting (DAT) during ...

Identification of Narcissus yellow stripe virus and a closely-related potyvirus isolate in plants of Allium carinatum

USDA-ARS?s Scientific Manuscript database

A survey of varieties and species of ornamental Allium revealed the presence of multiple viruses, including potyviruses, carlaviruses, and allexiviruses. Most of these viruses have been previously identified in A. sativum (garlic), A. cepa (onion), A. porrum (synonym A. ampeloprasum var. porrum; lee...

Income Segregation between Schools and School Districts. CEPA Working Paper No. 16-04

ERIC Educational Resources Information Center

Owens, Ann; Reardon, Sean F.; Jencks, Christopher

2016-01-01

Although trends in the racial segregation of schools are well documented, less is known about trends in "income" segregation. We use multiple data sources to document trends in income segregation between schools and school districts. Between-district income segregation of families with children enrolled in public school increased by over…

40 CFR 180.41 - Crop group tables.

Code of Federal Regulations, 2011 CFR

2011-07-01

.... var. bulbiferum L.H. Bailey; A. cepa L. var. viviparum (Metz.) Alef.) 3-07B Onion, Welsh, tops (Allium... Tahiti lime, Citrus latifolia (Yu. Tanaka) Tanaka 10-10B Tangelo, Citrus x tangelo J.W. Ingram & H.E... straw of all commodities included in the group cereal grains group. (22) Crop Group 17. Grass Forage...

Structural analyses and immunomodulatory properties of fructo-oligosaccharides from onion (Allium cepa).

PubMed

Kumar, V Prasanna; Prashanth, K V Harish; Venkatesh, Y P

2015-03-06

Onion (Allium cepa) is an immune-boosting food rich in fructans. The major aim of this study is to characterize and investigate the immunomodulatory properties of onion fructo-oligosaccharides (FOS). FOS was isolated from onion bulbs by hot 80% ethanol extraction (yield: ∼4.5 g/100 g fw) followed by gel permeation chromatography. NMR of onion FOS revealed unusual β-D-Glc terminal residue at the non-reducing end. TLC and ESI-MS analyses showed that onion FOS ranged from trisaccharides to hexasaccharides. Onion FOS (50 μg/mL) significantly increased (∼3-fold) the proliferation of mouse splenocytes/thymocytes vs. control. Further, onion FOS enhanced (∼2.5-fold) the production of nitric oxide by peritoneal exudates cells (PECs) from Wistar rats; intracellular free radicals production and phagocytic activity of isolated murine PECs were also augmented. Our structural and in vitro results indicate that onion FOS comprising of tri- to hexasaccharide units belongs to inulin-type fructans, and possess immunostimulatory activities towards murine lymphocytes and macrophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

Investigation of Volatiles Emitted from Freshly Cut Onions (Allium cepa L.) by Real Time Proton-Transfer Reaction-Mass Spectrometry (PTR-MS)

PubMed Central

Løkke, Mette Marie; Edelenbos, Merete; Larsen, Erik; Feilberg, Anders

2012-01-01

Volatile organic compounds (VOCs) in cut onions (Allium cepa L.) were continuously measured by PTR-MS during the first 120 min after cutting. The headspace composition changed rapidly due to the very reactive volatile sulfurous compounds emitted from onion tissue after cell disruption. Mass spectral signals corresponding to propanethial S-oxide (the lachrymatory factor) and breakdown products of this compound dominated 0–10 min after cutting. Subsequently, propanethiol and dipropyl disulfide predominantly appeared, together with traces of thiosulfinates. The concentrations of these compounds reached a maximum at 60 min after cutting. Propanethiol was present in highest concentrations and had an odor activity value 20 times higher than dipropyl disulfide. Thus, propanethiol is suggested to be the main source of the characteristic onion odor. Monitoring the rapid changes of VOCs in the headspace of cut onion necessitates a high time resolution, and PTR-MS is demonstrated to be a very suitable method for monitoring the headspace of freshly cut onions directly after cutting without extraction or pre-concentration. PMID:23443367

Cytotoxicity and mutagenicity of fluoxetine hydrochloride (Prozac), with or without vitamins A and C, in plant and animal model systems.

PubMed

Düsman, E; Almeida, I V; Mariucci, R G; Mantovani, M S; Vicentini, V E P

2014-01-28

Fluoxetine, commonly known as Prozac, is the first representative of the so-called new generation of antidepressants that promise efficacy, with few side effects, against deep depression, nervous bulimia, and anxiety. As there is a growing number of people suffering from anxiety and depression; consequently, the use of fluoxetine is also increasing. Verifying absence of drug effects such as cytotoxicity or mutagenicity is of great importance. Certain vitamins, such as vitamin A (retinol, retinoids) and vitamin C (ascorbic acid) protect and are extremely active against mutagens. We evaluated the cytotoxic and mutagenic activity of fluoxetine, with and without concomitant administration of vitamin A or C, in Allium cepa meristem cells and Wistar rat bone marrow cells. The A. cepa meristem cells showed fluoxetine cytotoxicity; concomitant treatment with vitamin A or C proved non-protective. Treatment of Wistar rats with fluoxetine intraperitoneally or via gavage did not affect cell division or cause clastogenic effects. Vitamin A and C did not affect the cytotoxicity or mutagenicity of fluoxetine in the rat cells.

Cytotoxicity of aluminum oxide nanoparticles on Allium cepa root tip--effects of oxidative stress generation and biouptake.

PubMed

Rajeshwari, A; Kavitha, S; Alex, Sruthi Ann; Kumar, Deepak; Mukherjee, Anita; Chandrasekaran, Natarajan; Mukherjee, Amitava

2015-07-01

The commercial usage of Al2O3 nanoparticles (Al2O3 NPs) has gone up significantly in the recent times, enhancing the risk of environmental contamination with these agents and their consequent adverse effects on living systems. The current study has been designed to evaluate the cytogenetic potential of Al2O3 NPs in Allium cepa (root tip cells) at a range of exposure concentrations (0.01, 0.1, 1, 10, and 100 μg/mL), their uptake/internalization profile, and the oxidative stress generated. We noted a dose-dependent decrease in the mitotic index (42 to 28 %) and an increase in the number of chromosomal aberrations. Various chromosomal aberrations, e.g. sticky, multipolar and laggard chromosomes, chromosomal breaks, and the formation of binucleate cells, were observed by optical, fluorescence, and confocal laser scanning microscopy. FT-IR analysis demonstrated the surface chemical interaction between the nanoparticles and root tip cells. The biouptake of Al2O3 in particulate form led to reactive oxygen species generation, which in turn probably contributed to the induction of chromosomal aberrations.

Correlation of toxicity with lead content in root tip cells (Allium cepa L.).

PubMed

Carruyo, Ingrid; Fernández, Yusmary; Marcano, Letty; Montiel, Xiomara; Torrealba, Zaida

2008-12-01

The present study determines lead content in onion root tip cells (Allium cepa L.), correlating it with its toxicity. The treatment was carried at 25 +/- 0.5 degrees C using aqueous solutions of lead chloride at 0.1, 0.25, 0.50, 0.75, and 1 ppm for 12, 24, 48, and 72 h. For each treatment, a control where the lead solution was substituted by distilled water was included. After treatment, the meristems were fixed with a mixture of alcohol-acetic acid (3:1) and colored according to the technique of Feulgen. Lead content was quantified by graphite furnace absorption atomic spectrometry. The lead content in the roots ranged from 3.25 to 244.72 microg/g dry weight, with a direct relation with the concentration and time of exposure. A significant negative correlation was presented (r = -0.3629; p < 0.01) among lead content and root growth increment, and a positive correlation (r = 0.7750; p < 0.01) with the induction of chromosomic aberrations. In conclusion, lead is able to induce a toxic effect in the exposed roots, correlated with its content.

Evidence of reactive astrocytes but not peripheral immune system activation in a mouse model of Fragile X Syndrome

PubMed Central

Yuskaitis, Christopher J.; Beurel, Eleonore; Jope, Richard S.

2010-01-01

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is one of the few known genetic causes of autism. FXS results from the loss of Fmr1 gene function, thus Fmr1 knockout mice provide a model to study impairments associated with FXS and autism and to test potential therapeutic interventions. The inhibitory serine-phosphorylation of glycogen synthase kinase-3 (GSK3) is lower in brain regions of Fmr1 knockout mice than wild-type mice and the GSK3 inhibitor lithium rescues several behavioral impairments in Fmr1 knockout mice. Therefore, we examined if the serine-phosphorylation of GSK3 in Fmr1 knockout mice also was altered outside the brain and if administration of lithium ameliorated the macroorchidism phenotype. Additionally, since GSK3 regulates numerous functions of the immune system and immune alterations have been associated with autism, we tested if immune function is altered in Fmr1 knockout mice. The inhibitory serine-phosphorylation of GSK3 was significantly lower in the testis and liver of Fmr1 knockout mice than wild-type mice, and chronic lithium treatment reduced macroorchidism in Fmr1 knockout mice. No alterations in peripheral immune function were identified in Fmr1 knockout mice. However, examination of glia, the immune cells of the brain, revealed reactive astrocytes in several brain regions of Fmr1 knockout mice and treatment with lithium reduced this in the striatum and cerebellum. These results provide further evidence of the involvement of dysregulated GSK3 in FXS, and demonstrate that lithium administration reduces macroorchidism and reactive astrocytes in Fmr1 knockout mice. PMID:20600866

De Novo Assembly and Transcriptome Analysis of Bulb Onion (Allium cepa L.) during Cold Acclimation Using Contrasting Genotypes

PubMed Central

Natarajan, Sathishkumar; Park, Jong-In; Chung, Mi-Young; Nou, Ill-Sup

2016-01-01

Bulb onion (Allium cepa) is the second most widely cultivated and consumed vegetable crop in the world. During winter, cold injury can limit the production of bulb onion. Genomic resources available for bulb onion are still very limited. To date, no studies on heritably durable cold and freezing tolerance have been carried out in bulb onion genotypes. We applied high-throughput sequencing technology to cold (2°C), freezing (-5 and -15°C), and control (25°C)-treated samples of cold tolerant (CT) and cold susceptible (CS) genotypes of A. cepa lines. A total of 452 million paired-end reads were de novo assembled into 54,047 genes with an average length of 1,331 bp. Based on similarity searches, these genes were aligned with entries in the public non-redundant (nr) database, as well as KEGG and COG database. Differentially expressed genes (DEGs) were identified using log10 values with the FPKM method. Among 5,167DEGs, 491 genes were differentially expressed at freezing temperature compared to the control temperature in both CT and CS libraries. The DEG results were validated with qRT-PCR. We performed GO and KEGG pathway enrichment analyses of all DEGs and iPath interactive analysis found 31 pathways including those related to metabolism of carbohydrate, nucleotide, energy, cofactors and vitamins, other amino acids and xenobiotics biodegradation. Furthermore, a large number of molecular markers were identified from the assembled genes, including simple sequence repeats (SSRs) 4,437 and SNP substitutions of transition and transversion types of CT and CS. Our study is the first to provide a transcriptome sequence resource for Allium spp. with regard to cold and freezing stress. We identified a large set of genes and determined their DEG profiles under cold and freezing conditions using two different genotypes. These data represent a valuable resource for genetic and genomic studies of Allium spp. PMID:27627679

Genotoxicity assessment of raw and treated water samples using Allium cepa assay: evidence from Perak River, Malaysia.

PubMed

Malakahmad, Amirhossein; Manan, Teh Sabariah Binti Abd; Sivapalan, Subarna; Khan, Taimur

2018-02-01

Allium cepa assay was carried out in this study to evaluate genotoxic effects of raw and treated water samples from Perak River in Perak state, Malaysia. Samples were collected from three surface water treatment plants along the river, namely WTPP, WTPS, and WTPK. Initially, triplicates of equal size Allium cepa (onions) bulbs, 25-30 mm in diameter and average weight of 20 g, were set up in distilled water for 24 h at 20 ± 2 °C and protected from direct sunlight, to let the roots to grow. After germination of roots (0.5-1.0 cm in length), bulbs were transferred to collected water samples each for a 96-h period of exposure. The root physical deformations were observed. Genotoxicity quantification was based on mitotic index and genotoxicity level. Statistical analysis using cross-correlation function for replicates from treated water showed that root length has inverse correlation with mitotic indices (r = - 0.969) and frequencies of cell aberrations (r = - 0.976) at lag 1. Mitotic indices and cell aberrations of replicates from raw water have shown positive correlation at lag 1 (r = 0.946). Genotoxicity levels obtained were 23.4 ± 1.98 (WTPP), 26.68 ± 0.34 (WTPS), and 30.4 ± 1.13 (WTPK) for treated water and 17.8 ± 0.18 (WTPP), 37.15 ± 0.17 (WTPS), and 47.2 ± 0.48 (WTPK) for raw water. The observed cell aberrations were adherence, chromosome delay, C-metaphase, chromosome loss, chromosome bridge, chromosome breaks, binucleated cell, mini cell, and lobulated nuclei. The morphogenetic deformations obtained were likely due to genotoxic substances presence in collected water samples. Thus, water treatment in Malaysia does not remove genotoxic compounds.

Accurate, precise, and efficient theoretical methods to calculate anion-π interaction energies in model structures.

PubMed

Mezei, Pál D; Csonka, Gábor I; Ruzsinszky, Adrienn; Sun, Jianwei

2015-01-13

A correct description of the anion-π interaction is essential for the design of selective anion receptors and channels and important for advances in the field of supramolecular chemistry. However, it is challenging to do accurate, precise, and efficient calculations of this interaction, which are lacking in the literature. In this article, by testing sets of 20 binary anion-π complexes of fluoride, chloride, bromide, nitrate, or carbonate ions with hexafluorobenzene, 1,3,5-trifluorobenzene, 2,4,6-trifluoro-1,3,5-triazine, or 1,3,5-triazine and 30 ternary π-anion-π' sandwich complexes composed from the same monomers, we suggest domain-based local-pair natural orbital coupled cluster energies extrapolated to the complete basis-set limit as reference values. We give a detailed explanation of the origin of anion-π interactions, using the permanent quadrupole moments, static dipole polarizabilities, and electrostatic potential maps. We use symmetry-adapted perturbation theory (SAPT) to calculate the components of the anion-π interaction energies. We examine the performance of the direct random phase approximation (dRPA), the second-order screened exchange (SOSEX), local-pair natural-orbital (LPNO) coupled electron pair approximation (CEPA), and several dispersion-corrected density functionals (including generalized gradient approximation (GGA), meta-GGA, and double hybrid density functional). The LPNO-CEPA/1 results show the best agreement with the reference results. The dRPA method is only slightly less accurate and precise than the LPNO-CEPA/1, but it is considerably more efficient (6-17 times faster) for the binary complexes studied in this paper. For 30 ternary π-anion-π' sandwich complexes, we give dRPA interaction energies as reference values. The double hybrid functionals are much more efficient but less accurate and precise than dRPA. The dispersion-corrected double hybrid PWPB95-D3(BJ) and B2PLYP-D3(BJ) functionals perform better than the GGA and meta-GGA functionals for the present test set.

BTEX biodegradation by bacteria from effluents of petroleum refinery.

PubMed

Mazzeo, Dânia Elisa Christofoletti; Levy, Carlos Emílio; de Angelis, Dejanira de Franceschi; Marin-Morales, Maria Aparecida

2010-09-15

Groundwater contamination with benzene, toluene, ethylbenzene and xylene (BTEX) has been increasing, thus requiring an urgent development of methodologies that are able to remove or minimize the damages these compounds can cause to the environment. The biodegradation process using microorganisms has been regarded as an efficient technology to treat places contaminated with hydrocarbons, since they are able to biotransform and/or biodegrade target pollutants. To prove the efficiency of this process, besides chemical analysis, the use of biological assessments has been indicated. This work identified and selected BTEX-biodegrading microorganisms present in effluents from petroleum refinery, and evaluated the efficiency of microorganism biodegradation process for reducing genotoxic and mutagenic BTEX damage through two test-systems: Allium cepa and hepatoma tissue culture (HTC) cells. Five different non-biodegraded BTEX concentrations were evaluated in relation to biodegraded concentrations. The biodegradation process was performed in a BOD Trak Apparatus (HACH) for 20 days, using microorganisms pre-selected through enrichment. Although the biodegradation usually occurs by a consortium of different microorganisms, the consortium in this study was composed exclusively of five bacteria species and the bacteria Pseudomonas putida was held responsible for the BTEX biodegradation. The chemical analyses showed that BTEX was reduced in the biodegraded concentrations. The results obtained with genotoxicity assays, carried out with both A. cepa and HTC cells, showed that the biodegradation process was able to decrease the genotoxic damages of BTEX. By mutagenic tests, we observed a decrease in damage only to the A. cepa organism. Although no decrease in mutagenicity was observed for HTC cells, no increase of this effect after the biodegradation process was observed either. The application of pre-selected bacteria in biodegradation processes can represent a reliable and effective tool in the treatment of water contaminated with BTEX mixture. Therefore, the raw petroleum refinery effluent might be a source of hydrocarbon-biodegrading microorganisms. Copyright 2010 Elsevier B.V. All rights reserved.

Cytogenotoxicity screening of source water, wastewater and treated water of drinking water treatment plants using two in vivo test systems: Allium cepa root based and Nile tilapia erythrocyte based tests.

PubMed

Hemachandra, Chamini K; Pathiratne, Asoka

2017-01-01

Biological effect directed in vivo tests with model organisms are useful in assessing potential health risks associated with chemical contaminations in surface waters. This study examined the applicability of two in vivo test systems viz. plant, Allium cepa root based tests and fish, Oreochromis niloticus erythrocyte based tests for screening cytogenotoxic potential of raw source water, water treatment waste (effluents) and treated water of drinking water treatment plants (DWTPs) using two DWTPs associated with a major river in Sri Lanka. Measured physico-chemical parameters of the raw water, effluents and treated water samples complied with the respective Sri Lankan standards. In the in vivo tests, raw water induced statistically significant root growth retardation, mitodepression and chromosomal abnormalities in the root meristem of the plant and micronuclei/nuclear buds evolution and genetic damage (as reflected by comet scores) in the erythrocytes of the fish compared to the aged tap water controls signifying greater genotoxicity of the source water especially in the dry period. The effluents provoked relatively high cytogenotoxic effects on both test systems but the toxicity in most cases was considerably reduced to the raw water level with the effluent dilution (1:8). In vivo tests indicated reduction of cytogenotoxic potential in the tested drinking water samples. The results support the potential applications of practically feasible in vivo biological test systems such as A. cepa root based tests and the fish erythrocyte based tests as complementary tools for screening cytogenotoxicity potential of the source water and water treatment waste reaching downstream of aquatic ecosystems and for evaluating cytogenotoxicity eliminating efficacy of the DWTPs in different seasons in view of human and ecological safety. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fumigant activity of plant essential oils and components from garlic (Allium sativum) and clove bud (Eugenia caryophyllata) oils against the Japanese termite (Reticulitermes speratus Kolbe).

PubMed

Park, Il-Kwon; Shin, Sang-Chul

2005-06-01

Plant essential oils from 29 plant species were tested for their insecticidal activities against the Japanese termite, Reticulitermes speratus Kolbe, using a fumigation bioassay. Responses varied with plant material, exposure time, and concentration. Good insecticidal activity against the Japanese termite was achived with essential oils of Melaleuca dissitiflora, Melaleuca uncinata, Eucalyptus citriodora, Eucalyptus polybractea, Eucalyptus radiata, Eucalyptus dives, Eucalyptus globulus, Orixa japonica, Cinnamomum cassia, Allium cepa, Illicium verum, Evodia officinalis, Schizonepeta tenuifolia, Cacalia roborowskii, Juniperus chinensis var. horizontalis, Juniperus chinensis var. kaizuka, clove bud, and garlic applied at 7.6 microL/L of air. Over 90% mortality after 3 days was achieved with O. japonica essential oil at 3.5 microL/L of air. E. citriodora, C. cassia, A. cepa, I. verum, S. tenuifolia, C. roborowskii, clove bud, and garlic oils at 3.5 microL/L of air were highly toxic 1 day after treatment. At 2.0 microL/L of air concentration, essential oils of I. verum, C. roborowskik, S. tenuifolia, A. cepa, clove bud, and garlic gave 100% mortality within 2 days of treatment. Clove bud and garlic oils showed the most potent antitermitic activity among the plant essential oils. Garlic and clove bud oils produced 100% mortality at 0.5 microL/L of air, but this decreased to 42 and 67% after 3 days of treatment at 0.25 microL/L of air, respectively. Analysis by gas chromatography-mass spectrometry led to the identification of three major compounds from garlic oil and two from clove bud oils. These five compounds from two essential oils were tested individually for their insecticidal activities against Japanese termites. Responses varied with compound and dose. Diallyl trisulfide was the most toxic, followed by diallyl disulfide, eugenol, diallyl sulfide, and beta-caryophyllene. The essential oils described herein merit further study as potential fumigants for termite control.

De Novo Assembly and Transcriptome Analysis of Bulb Onion (Allium cepa L.) during Cold Acclimation Using Contrasting Genotypes.

PubMed

Han, Jeongsukhyeon; Thamilarasan, Senthil Kumar; Natarajan, Sathishkumar; Park, Jong-In; Chung, Mi-Young; Nou, Ill-Sup

2016-01-01

Bulb onion (Allium cepa) is the second most widely cultivated and consumed vegetable crop in the world. During winter, cold injury can limit the production of bulb onion. Genomic resources available for bulb onion are still very limited. To date, no studies on heritably durable cold and freezing tolerance have been carried out in bulb onion genotypes. We applied high-throughput sequencing technology to cold (2°C), freezing (-5 and -15°C), and control (25°C)-treated samples of cold tolerant (CT) and cold susceptible (CS) genotypes of A. cepa lines. A total of 452 million paired-end reads were de novo assembled into 54,047 genes with an average length of 1,331 bp. Based on similarity searches, these genes were aligned with entries in the public non-redundant (nr) database, as well as KEGG and COG database. Differentially expressed genes (DEGs) were identified using log10 values with the FPKM method. Among 5,167DEGs, 491 genes were differentially expressed at freezing temperature compared to the control temperature in both CT and CS libraries. The DEG results were validated with qRT-PCR. We performed GO and KEGG pathway enrichment analyses of all DEGs and iPath interactive analysis found 31 pathways including those related to metabolism of carbohydrate, nucleotide, energy, cofactors and vitamins, other amino acids and xenobiotics biodegradation. Furthermore, a large number of molecular markers were identified from the assembled genes, including simple sequence repeats (SSRs) 4,437 and SNP substitutions of transition and transversion types of CT and CS. Our study is the first to provide a transcriptome sequence resource for Allium spp. with regard to cold and freezing stress. We identified a large set of genes and determined their DEG profiles under cold and freezing conditions using two different genotypes. These data represent a valuable resource for genetic and genomic studies of Allium spp.

Characterization of onion lectin (Allium cepa agglutinin) as an immunomodulatory protein inducing Th1-type immune response in vitro.

PubMed

Prasanna, Vaddi K; Venkatesh, Yeldur P

2015-06-01

Onion (Allium cepa), a bulb crop of economic importance, is known to have many health benefits. The major objective of the present study is to address the immunomodulatory properties of onion lectin (A. cepa agglutinin; ACA). ACA was purified from onion extract by D-mannose-agarose chromatography (yield: ~1 mg/kg). ACA is non-glycosylated and showed a molecular mass of ~12 kDa under reducing/non-reducing SDS-PAGE; glutaraldehyde cross-linking indicated that ACA is a non-covalent tetramer of ~12 kDa subunits. Its N-terminal sequence (RNVLLNNEGL; UniProt KB Accn. C0HJM8) showed 70-90% homology to mannose-specific Allium agglutinins. ACA showed specific hemagglutination activity of 8200 units/mg and is stable in the pH range 6-10 and up to 45° C. The immunomodulatory activity of ACA was assessed using the macrophage cell line, RAW264.7 and rat peritoneal macrophages; at 0.1 μg/well, it showed a significant increase (6-8-fold vs. control) in the production of nitric oxide at 24h, and significantly stimulated (2-4-fold vs. control) the production of pro-inflammatory cytokines (TNF-α and IL-12) at 24h. ACA (0.1 μg/well) enhanced the proliferation of murine thymocytes by ~4 fold (vs. control) at 24h; however, ACA does not proliferate B cell-enriched rat splenocytes. Further, it significantly elevated the expression levels of cytokines (IFN-γ and IL-2) over the control in murine thymocytes. Taken together, purified ACA induces a Th1-type immune response in vitro. Though present in low amounts, ACA may contribute to the immune-boosting potential of the popular spice onion since considerable amounts are consumed on a daily basis universally. Copyright © 2015 Elsevier B.V. All rights reserved.

Mouse model of fragile X syndrome: behavioral and hormonal response to stressors.

PubMed

Nielsen, Darci M; Evans, Jeffrey J; Derber, William J; Johnston, Kenzie A; Laudenslager, Mark L; Crnic, Linda S; Maclean, Kenneth N

2009-06-01

Fragile X syndrome, a form of mental retardation caused by inadequate levels of fragile X mental retardation protein (FMRP), is characterized by extreme sensitivity to sensory stimuli and increased behavioral and hormonal reactivity to stressors. Fmr1 knockout mice lack FMRP and exhibit abnormal responses to auditory stimuli. This study sought to determine whether Fmr1 knockout mice on an F1 hybrid background are normal in their response to footshock. Knockout mice were also examined for signs of hyperexcitation across an extended trial range, and serum corticosterone levels were evaluated in response to various stressors. The ability to acquire conditioned taste aversion was also assessed. Knockout mice exhibited no impairment in associative aversive learning or memory, since they successfully expressed conditioned taste aversion. Footshock-sensitivity, freezing behavior, and corticosterone response to various stressors did not differ between knockout and wild-type mice. However, knockout mice exhibited significantly increased responses during the extended test. The knockout mice's increased responsiveness to footshock in the extended test may be an indication of increased vulnerability to stress or enhanced emotional reactivity. Copyright (c) 2009 APA, all rights reserved.

San Francisco Unified School District Survey Report I: Teacher Evaluation

ERIC Educational Resources Information Center

Gao, Niu

2013-01-01

This survey report contains the results of the 2009-10, 2010-11, and 2011-12 Teacher and Principal Surveys conducted by Stanford's Center for Education Policy Analysis (CEPA). The surveys were administered to teachers and principals in the San Francisco Unified School District (SFUSD). This report appears in two parts, each highlighting some of…

School Performance, Accountability and Waiver Reforms: Evidence from Louisiana. CEPA Working Paper No. 17-06

ERIC Educational Resources Information Center

Dee, Thomas; Dizon-Ross, Elise

2017-01-01

States that received federal waivers to the No Child Left Behind (NCLB) Act were required to implement reforms in designated "Focus Schools" that contribute to achievement gaps. In this study, we examine the performance effects of such "differentiated accountability" reforms in the state of Louisiana. The Focus School reforms…

Know the Child: The Importance of Teacher Knowledge of Individual Students' Skills (KISS). CEPA Working Paper

ERIC Educational Resources Information Center

York, Benjamin N.

2014-01-01

Teachers require knowledge of the unique skills that each child brings to the classroom in order to effectively target instruction towards students' learning needs. Despite substantial investments in programs aimed at enhancing teacher knowledge of individual students' skills (KISS), we know surprisingly little about how KISS is distributed or how…

The Effects of Accountability Incentives in Early Childhood Education. CEPA Working Paper No. 17-10

ERIC Educational Resources Information Center

Bassok, Daphna; Dee, Thomas S.; Latham, Scott

2017-01-01

In an effort to enhance the quality of early childhood education (ECE) at scale, nearly all U.S. states have recently adopted Quality Rating and Improvement Systems (QRIS). These accountability systems give providers and parents information on program quality and create both reputational and financial incentives for program improvement. However,…

Teacher Turnover, Teacher Quality, and Student Achievement in DCPS. CEPA Working Paper No. 16-03

ERIC Educational Resources Information Center

Adnot, Melinda; Dee, Thomas; Katz, Veronica; Wyckoff, James

2016-01-01

In practice, teacher turnover appears to have negative effects on school quality as measured by student performance. However, some simulations suggest that turnover can instead have large, positive effects under a policy regime in which low-performing teachers can be accurately identified and replaced with more effective teachers. This study…

The Gift of Time? School Starting Age and Mental Health. CEPA Working Paper No. 15-08

ERIC Educational Resources Information Center

Dee, Thomas; Sievertsen, Hans Henrik

2015-01-01

In many developed countries, children now begin their formal schooling at an older age. However, a growing body of empirical studies provides little evidence that such schooling delays improve educational and economic outcomes. This study presents new evidence on whether school starting age influences student outcomes by relying on linked Danish…

The Global Increase in the Socioeconomic Achievement Gap, 1964-2015. CEPA Working Paper No. 17-04

ERIC Educational Resources Information Center

Chmielewski, Anna K.

2017-01-01

The existence of a "socioeconomic achievement gap"--a disparity in academic achievement between students from high- and low-socioeconomic status (SES) backgrounds--is well-known in educational research. The SES achievement gap has been documented across a wide range of countries. What is unknown in most countries is whether the SES…

School Segregation and Racial Academic Achievement Gaps. CEPA Working Paper No. 15-12

ERIC Educational Resources Information Center

Reardon, Sean F.

2015-01-01

Although it is clear that racial segregation is linked to academic achievement gaps, the mechanisms underlying this link have been debated since Coleman published his eponymous 1966 report. In this paper, I examine 16 distinct measures of segregation to determine which is most strongly associated with academic achievement gaps. I find very clear…

The Geography of Racial/Ethnic Test Score Gaps. CEPA Working Paper No. 16-10

ERIC Educational Resources Information Center

Reardon, Sean F.; Kalogrides, Demetra; Shores, Ken

2017-01-01

We estimate racial/ethnic achievement gaps in several hundred metropolitan areas and several thousand school districts in the United States using the results of roughly 200 million standardized math and reading tests administered to public school students from 2009-2013. We show that achievement gaps vary substantially, ranging from nearly 0 in…

Effectiveness of Interactive Satellite-Transmitted Instruction: Experimental Evidence from Ghanaian Primary Schools. CEPA Working Paper No. 17-08

ERIC Educational Resources Information Center

Johnston, Jamie; Ksoll, Christopher

2017-01-01

In lower- and middle-income countries, including Ghana, students in rural areas dramatically underperform their urban peers. Rural schools struggle to attract and retain professionally trained teachers (GES 2012; World Bank 2012). We explore one potential solution to the problem of teacher recruitment: distance instruction. Through a cluster…

Persistence Patterns in Massive Open Online Courses (MOOCs). CEPA Working Paper No. 15-09

ERIC Educational Resources Information Center

Evans, Brent J.; Baker, Rachel B.; Dee, Thomas

2015-01-01

Using a unique dataset of 44 Massive Open Online Courses (MOOCs), this paper examines critical patterns of enrollment, engagement, persistence, and completion among students in online higher education. By leveraging fixed-effects specifications based on over 2.1 million student observations across more than 2,900 lectures, we analyze engagement,…

Mobile Phones, Civic Engagement, and School Performance in Pakistan. CEPA Working Paper No. 16-17

ERIC Educational Resources Information Center

Asim, Minahil; Dee, Thomas

2016-01-01

The effective governance of local public services depends critically on the civic engagement of local citizens. However, recent efforts to promote effective citizen oversight of the public-sector services in developing countries have had mixed results. This study discusses and evaluates a uniquely designed, low-cost, scalable program designed to…

Host ranges of Penicillium species causing blue mold of bulb crops in Washington State and Idaho

USDA-ARS?s Scientific Manuscript database

First reported from the Pacific Northwest (PNW) of U.S.A. as causal agents of blue mold on edible and/or ornamental bulbs are Penicillium albocoremium (from Tulipa sp.; pathogenic on Allium sativum, A. cepa, A. stipitatum, Iris hollandica and Tulipa sp.), P. crustosum (from Narcissus; pathogenic on ...

Teachers' Beliefs about Students' Social Disadvantage and Student Achievement. CEPA Working Paper No. 15-03

ERIC Educational Resources Information Center

Rochmes, Jane

2015-01-01

While progress to close racial achievement gaps has stagnated and income achievement gaps have grown, recent case studies enthusiastically describe "transformational" schools, which claim to establish conditions that enable students--primarily poor students of color--to achieve at levels far higher than their social background predicts.…

Cytoprotective role of autophagy against BH3 mimetic gossypol in ATG5 knockout cells generated by CRISPR-Cas9 endonuclease.

PubMed

Kim, Na-Yeon; Han, Byeal-I; Lee, Michael

2016-01-01

Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

New insight into the role of the β3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout

PubMed Central

Ferguson, Carolyn; Hardy, Steven L; Werner, David F; Hileman, Stanley M; DeLorey, Timothy M; Homanics, Gregg E

2007-01-01

Background The β3 subunit of the γ-aminobutyric acid type A receptor (GABAA-R) has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of β3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated β3 gene was engineered. Results Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the β3 subunit was flanked by loxP sites (i.e., floxed). Crossing the floxed β3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of β3 was achieved by crossing floxed β3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal β3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of β3 was achieved using α CamKII-cre transgenic mice. Palate development was normal in forebrain selective β3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15–25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese. Conclusion Conditional inactivation of the β3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed β3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the β3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes. PMID:17927825

Structural and Functional Analysis of HIV-1 Coreceptors: Roles of Charged Residues and Posttranslational Modifications on Coreceptor Activity

DTIC Science & Technology

2000-01-01

to sites of inflammation. They may have additional functions. For example analysis of CXCR4 knockout mice show that CXCR4, which is chemotactic for... mice had similar phenotypes (195). Homozygous knockout of CXCR4 or SDF-1 results in embyonic lethality. Though CCR5 appears to be dispensable, other...chemokine receptors have vital functions. CXCR5 knockout mice have B-cell homing defects (118), and CXCR2 knockout mice overproduce B-cells and

[Preliminary exploration on knockout drops (Meng Han Agents)].

PubMed

Zhang, Z

1996-05-01

This author points out, based on relevant materials, that knockout drops were vertigo powder. Due to homophonic reasons in Chinese language, the term "mingxuan" was transliterated into the former Chinese term (menghan). Knockout drops for medicinal use were merely made up of compound recipes containing stramonium flowers. The knockout drops in old fictions and opera books were powder of stramonium flower. The ingredients and application of such recipes are discussed here, the anti-remedies for such recipes are also mentioned.

RNaseT2 knockout rats exhibit hippocampal neuropathology and deficits in memory.

PubMed

Sinkevicius, Kerstin W; Morrison, Thomas R; Kulkarni, Praveen; Caffrey Cagliostro, Martha K; Iriah, Sade; Malmberg, Samantha; Sabrick, Julia; Honeycutt, Jennifer A; Askew, Kim L; Trivedi, Malav; Ferris, Craig F

2018-06-27

RNASET2 deficiency in humans is associated with infant cystic leukoencephalopathy, which causes psychomotor impairment, spasticity and epilepsy. A zebrafish mutant model suggests that loss of RNASET2 function leads to neurodegeneration due to the accumulation of non-degraded RNA in the lysosomes. The goal of this study was to characterize the first rodent model of RNASET2 deficiency. The brains of 3- and 12-month-old RNaseT2 knockout rats were studied using multiple magnetic resonance imaging modalities and behavioral tests. While T1- and T2-weighted images of RNaseT2 knockout rats exhibited no evidence of cystic lesions, the prefrontal cortex and hippocampal complex were enlarged in knockout animals. Diffusion-weighted imaging showed altered anisotropy and putative gray matter changes in the hippocampal complex of the RNaseT2 knockout rats. Immunohistochemistry for glial fibrillary acidic protein (GFAP) showed the presence of hippocampal neuroinflammation. Decreased levels of lysosome-associated membrane protein 2 (LAMP2) and elevated acid phosphatase and β-N-acetylglucosaminidase (NAG) activities indicated that the RNASET2 knockout rats likely had altered lysosomal function and potential defects in autophagy. Object recognition tests confirmed that RNaseT2 knockout rats exhibited memory deficits. However, the Barnes maze, and balance beam and rotarod tests indicated there were no differences in spatial memory or motor impairments, respectively. Overall, patients with RNASET2 deficiency exhibited a more severe neurodegeneration phenotype than was observed in the RNaseT2 knockout rats. However, the vulnerability of the knockout rat hippocampus as evidenced by neuroinflammation, altered lysosomal function and cognitive defects indicates that this is still a useful in vivo model to study RNASET2 function. © 2018. Published by The Company of Biologists Ltd.

Deficiency in the manganese efflux transporter SLC30A10 induces severe hypothyroidism in mice.

PubMed

Hutchens, Steven; Liu, Chunyi; Jursa, Thomas; Shawlot, William; Chaffee, Beth K; Yin, Weiling; Gore, Andrea C; Aschner, Michael; Smith, Donald R; Mukhopadhyay, Somshuvra

2017-06-09

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6-8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20-60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50-80%) and profoundly increased thyroid-stimulating hormone levels (∼800-1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism

PubMed Central

Lei, Tianluo; Zhou, Lei; Layton, Anita T.; Zhou, Hong; Zhao, Xuejian; Bankir, Lise

2011-01-01

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts. PMID:21849488

Role of thin descending limb urea transport in renal urea handling and the urine concentrating mechanism.

PubMed

Lei, Tianluo; Zhou, Lei; Layton, Anita T; Zhou, Hong; Zhao, Xuejian; Bankir, Lise; Yang, Baoxue

2011-12-01

Urea transporters UT-A2 and UT-B are expressed in epithelia of thin descending limb of Henle's loop and in descending vasa recta, respectively. To study their role and possible interaction in the context of the urine concentration mechanism, a UT-A2 and UT-B double knockout (UT-A2/B knockout) mouse model was generated by targeted deletion of the UT-A2 promoter in embryonic stem cells with UT-B gene knockout. The UT-A2/B knockout mice lacked detectable UT-A2 and UT-B transcripts and proteins and showed normal survival and growth. Daily urine output was significantly higher in UT-A2/B knockout mice than that in wild-type mice and lower than that in UT-B knockout mice. Urine osmolality in UT-A2/B knockout mice was intermediate between that in UT-B knockout and wild-type mice. The changes in urine osmolality and flow rate, plasma and urine urea concentration, as well as non-urea solute concentration after an acute urea load or chronic changes in protein intake suggested that UT-A2 plays a role in the progressive accumulation of urea in the inner medulla. These results suggest that in wild-type mice UT-A2 facilitates urea absorption by urea efflux from the thin descending limb of short loops of Henle. Moreover, UT-A2 deletion in UT-B knockout mice partially remedies the urine concentrating defect caused by UT-B deletion, by reducing urea loss from the descending limbs to the peripheral circulation; instead, urea is returned to the inner medulla through the loops of Henle and the collecting ducts.

Renal Dysfunction Induced by Kidney-Specific Gene Deletion of Hsd11b2 as a Primary Cause of Salt-Dependent Hypertension.

PubMed

Ueda, Kohei; Nishimoto, Mitsuhiro; Hirohama, Daigoro; Ayuzawa, Nobuhiro; Kawarazaki, Wakako; Watanabe, Atsushi; Shimosawa, Tatsuo; Loffing, Johannes; Zhang, Ming-Zhi; Marumo, Takeshi; Fujita, Toshiro

2017-07-01

Genome-wide analysis of renal sodium-transporting system has identified specific variations of Mendelian hypertensive disorders, including HSD11B2 gene variants in apparent mineralocorticoid excess. However, these genetic variations in extrarenal tissue can be involved in developing hypertension, as demonstrated in former studies using global and brain-specific Hsd11b2 knockout rodents. To re-examine the importance of renal dysfunction on developing hypertension, we generated kidney-specific Hsd11b2 knockout mice. The knockout mice exhibited systemic hypertension, which was abolished by reducing salt intake, suggesting its salt-dependency. In addition, we detected an increase in renal membrane expressions of cleaved epithelial sodium channel-α and T53-phosphorylated Na + -Cl - cotransporter in the knockout mice. Acute intraperitoneal administration of amiloride-induced natriuresis and increased urinary sodium/potassium ratio more in the knockout mice compared with those in the wild-type control mice. Chronic administration of amiloride and high-KCl diet significantly decreased mean blood pressure in the knockout mice, which was accompanied with the correction of hypokalemia and the resultant decrease in Na + -Cl - cotransporter phosphorylation. Accordingly, a Na + -Cl - cotransporter blocker hydrochlorothiazide significantly decreased mean blood pressure in the knockout mice. Chronic administration of mineralocorticoid receptor antagonist spironolactone significantly decreased mean blood pressure of the knockout mice along with downregulation of cleaved epithelial sodium channel-α and phosphorylated Na + -Cl - cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11β-HSD2 leads to salt-dependent hypertension, which is attributed to mineralocorticoid receptor-epithelial sodium channel-Na + -Cl - cotransporter activation in the kidney, and provides evidence that renal dysfunction is essential for developing the phenotype of apparent mineralocorticoid excess. © 2017 American Heart Association, Inc.

Behavioral and neurochemical characterization of mice deficient in the phosphodiesterase-1B (PDE1B) enzyme.

PubMed

Siuciak, J A; McCarthy, S A; Chapin, D S; Reed, T M; Vorhees, C V; Repaske, D R

2007-07-01

PDE1B is a calcium-dependent cyclic nucleotide phosphodiesterase that is highly expressed in the striatum. In order to investigate the physiological role of PDE1B in the central nervous system, PDE1B knockout mice (C57BL/6N background) were assessed in behavioral tests and their brains were assayed for monoamine content. In a variety of well-characterized behavioral tasks, including the elevated plus maze (anxiety-like behavior), forced swim test (depression-like behavior), hot plate (nociception) and two cognition models (passive avoidance and acquisition of conditioned avoidance responding), PDE1B knockout mice performed similarly to wild-type mice. PDE1B knockout mice showed increased baseline exploratory activity when compared to wild-type mice. When challenged with amphetamine (AMPH) and methamphetamine (METH), male and female PDE1B knockout mice showed an exaggerated locomotor response. Male PDE1B knockout mice also showed increased locomotor responses to higher doses of phencyclidine (PCP) and MK-801; however, this effect was not consistently observed in female knockout mice. In the striatum, increased dopamine turnover (DOPAC/DA and HVA/DA ratios) was found in both male and female PDE1B knockout mice. Striatal serotonin (5-HT) levels were also decreased in PDE1B knockout mice, although levels of the metabolite, 5HIAA, were unchanged. The present studies demonstrate increased striatal dopamine turnover in PDE1B knockout mice associated with increased baseline motor activity and an exaggerated locomotor response to dopaminergic stimulants such as methamphetamine and amphetamine. These data further support a role for PDE1B in striatal function.

Development of a Markerless Knockout Method for Actinobacillus succinogenes

PubMed Central

Joshi, Rajasi V.; Schindler, Bryan D.; McPherson, Nikolas R.; Tiwari, Kanupriya

2014-01-01

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain. PMID:24610845

Development of a markerless knockout method for Actinobacillus succinogenes.

PubMed

Joshi, Rajasi V; Schindler, Bryan D; McPherson, Nikolas R; Tiwari, Kanupriya; Vieille, Claire

2014-05-01

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

Hepatic changes in metabolic gene expression in old ghrelin and ghrelin receptor knockout mice

USDA-ARS?s Scientific Manuscript database

Ghrelin knockout (GKO) and ghrelin receptor (growth hormone secretagogue receptor) knockout (GHSRKO) mice exhibit enhanced insulin sensitivity, but the mechanism is unclear. Insulin sensitivity declines with age and is inversely associated with accumulation of lipid in liver, a key glucoregulatory ...

Strategic Staffing? How Performance Pressures Affect the Distribution of Teachers within Schools and Resulting Student Achievement. CEPA Working Paper No. 15-15

ERIC Educational Resources Information Center

Grissom, Jason; Kalogrides, Demetra; Loeb, Susanna

2015-01-01

School performance pressures apply disproportionately to tested grades and subjects. Using longitudinal administrative data and teacher survey data from a large urban school district, we examine schools' responses to those pressures in assigning teachers to high-stakes and low-stakes classrooms. We find that teachers who produce greater student…

Long-Term Trends in Private School Enrollments by Family Income. CEPA Working Paper No. 17-07

ERIC Educational Resources Information Center

Murnane, Richard J.; Reardon, Sean F.

2017-01-01

We use data from multiple national surveys to describe trends in private elementary school enrollment by family income from 1968-2013. We note several important trends. First, the private school enrollment rate of middle-income families declined substantially over the last five decades, while that of high-income families remained quite stable.…

The Effect of Physical Activity on Student Performance in College: An Experimental Evaluation. CEPA Working Paper No. 17-03

ERIC Educational Resources Information Center

Fricke, Hans; Lechner, Michael; Steinmayr, Andreas

2017-01-01

What is the role of physical activity in the process of human capital accumulation? Brain research provides growing evidence of the importance of physical activity for various aspects of cognitive functions. An increasingly sedentary lifestyle could thus be not only harmful to population health, but also disrupt human capital accumulation. This…

Building Teacher Teams: Evidence of Positive Spillovers from More Effective Colleagues. CEPA Working Paper No. 15-20

ERIC Educational Resources Information Center

Sun, Min; Loeb, Susanna; Grissom, Jason

2015-01-01

Student peer effects are well documented. We know far less, however, about peer effects among teachers. We hypothesize that a relatively effective teacher may positively affect the performance of their peers, while a relatively ineffective teacher may negatively impact the performance of other teachers with whom they work closely. Utilizing a…

Biochemistry and Chemotherapy of Malaria and Leishmaniasis

DTIC Science & Technology

1993-12-06

sativum), elephant garlic (Allium scorodoprasum), onion (Allium cepa), and licorice ( Glycyrrhiza glabra). Cells of Leish- mania mexicana 227 and... Glycyrrhiza glabra) inhibited the growth of the leishmanial parasites, but were not toxic to HeLa cells. All of the extracts showed varying inhibitory...root) and Glycyrrhiza glabra (licorice). 10 * Materials & Methods Cultures of Parasitic Protozoa: Promastigotes of Leishmania mexicana Walter Reed

Understanding Loan Aversion in Education: Evidence from High School Seniors, Community College Students, and Adults. CEPA Working Paper No. 16-15

ERIC Educational Resources Information Center

Boatman, Angela; Evans, Brent; Soliz, Adela

2016-01-01

Student loans are a crucial aspect of financing a college education for millions of Americans, yet we have surprisingly little empirical evidence concerning individuals' unwillingness to borrow money for educational purposes. This study provides the first large-scale quantitative evidence of levels of loan aversion in the United States. Using…

Linking U.S. School District Test Score Distributions to a Common Scale. CEPA Working Paper No. 16-09

ERIC Educational Resources Information Center

Reardon, Sean F.; Kalogrides, Demetra; Ho, Andrew D.

2017-01-01

There is no comprehensive database of U.S. district-level test scores that is comparable across states. We describe and evaluate a method for constructing such a database. First, we estimate linear, reliability-adjusted linking transformations from state test score scales to the scale of the National Assessment of Educational Progress (NAEP). We…

Supporting Parenting through Differentiated and Personalized Text-Messaging: Testing Effects on Learning during Kindergarten. CEPA Working Paper No. 16-18

ERIC Educational Resources Information Center

Doss, Christopher; Fahle, Erin M.; Loeb, Susanna; York, Benjamin N.

2017-01-01

Recent studies show that texting-based interventions can produce educational benefits in children across a range of ages. We study the effects of a text-based program for kindergarten parents, distinguishing a general program from one that adds differentiation and personalization based on the child's developmental level. Children in the…

Changing Distributions: How Online College Classes Alter Student and Professor Performance. CEPA Working Paper No. 15-10

ERIC Educational Resources Information Center

Bettinger, Eric; Fox, Lindsay; Loeb, Susanna; Taylor, Eric

2015-01-01

Online college courses are a rapidly expanding feature of higher education, yet little research identifies their effects. Using an instrumental variables approach and data from DeVry University, this study finds that, on average, online course-taking reduces student learning by one-third to one-quarter of a standard deviation compared to…

Tyramide-FISH mapping of single genes for development of an integrated recombination and cytogenetic map of chromosome 5 of Allium cepa

USDA-ARS?s Scientific Manuscript database

Chromosome 5 of onion carries major quantitative trait loci (QTL) of interest to breeders that control dry-matter content, pungency and storability of bulbs, amounts and types of epicuticular waxes, and resistances to abiotic factors. SNPs, SSRs and RFLPs in expressed regions of the onion genome hav...

The Promise of Bilingual and Dual Immersion Education. CEPA Working Paper No. 15-11

ERIC Educational Resources Information Center

Umansky, Ilana M.; Valentino, Rachel A.; Reardon, Sean F.

2015-01-01

One in five school-age children in the U.S. speaks a language other than English at home (Zeigler & Camarota, 2014). Roughly half of these emerging bilingual students (Garcia, 2009) are classified as English learners (ELs) when they enter school, meaning they do not meet state or district criteria for English proficiency (NCES, 2015). As the…

School-Based Healthcare and Academic Performance: Implications of Physical Health Services for Educational Outcomes and Inequality. CEPA Working Paper No. 15-07

ERIC Educational Resources Information Center

Rochmes, Jane E.

2016-01-01

Health and education are reciprocally related, and research indicates that unhealthy students are poorly positioned to learn. Providing services that prevent health problems or help students cope with existing health concerns is one way that schools intervene in the relationship between student background and educational outcomes. Providing health…

Race and Stratification in College Enrollment over Time. CEPA Working Paper No. 16-14

ERIC Educational Resources Information Center

Baker, Rachel; Klasik, Daniel; Reardon, Sean F.

2016-01-01

In this study we examine trends in segregation by race and ethnicity in higher education from 1985 to 2013. We have three key findings. Over the past 30 years, students from different groups have attended college at increasingly similar rates; gaps are decreasing. But these decreases have been driven largely by large increases in minority student…

Lamin-like analogues in plants: the characterization of NMCP1 in Allium cepa

PubMed Central

Moreno Díaz de la Espina, Susana

2013-01-01

The nucleoskeleton of plants contains a peripheral lamina (also called plamina) and, even though lamins are absent in plants, their roles are still fulfilled in plant nuclei. One of the most intriguing topics in plant biology concerns the identity of lamin protein analogues in plants. Good candidates to play lamin functions in plants are the members of the NMCP (nuclear matrix constituent protein) family, which exhibit the typical tripartite structure of lamins. This paper describes a bioinformatics analysis and classification of the NMCP family based on phylogenetic relationships, sequence similarity and the distribution of conserved regions in 76 homologues. In addition, NMCP1 in the monocot Allium cepa characterized by its sequence and structure, biochemical properties, and subnuclear distribution and alterations in its expression throughout the root were identified. The results demonstrate that these proteins exhibit many similarities to lamins (structural organization, conserved regions, subnuclear distribution, and solubility) and that they may fulfil the functions of lamins in plants. These findings significantly advance understanding of the structural proteins of the plant lamina and nucleoskeleton and provide a basis for further investigation of the protein networks forming these structures. PMID:23378381

Genotoxicity of the disinfection by-products resulting from peracetic acid- or hypochlorite-disinfected sewage wastewater.

PubMed

Crebelli, R; Conti, L; Monarca, S; Feretti, D; Zerbini, I; Zani, C; Veschetti, E; Cutilli, D; Ottaviani, M

2005-03-01

Wastewater disinfection is routinely carried out to prevent the spread of human pathogens present in wastewater effluents. To this aim, chemical and physical treatments are applied to the effluents before their emission in water bodies. In this study, the influence of two widely used disinfectants, peracetic acid (PAA) and sodium hypochlorite (NaClO), on the formation of mutagenic by-products was investigated. Wastewater samples were collected before and after disinfection, in winter and in summer, at a pilot plant installed in a municipal wastewater-treatment plant. Samples were adsorbed using silica C18 cartridges and the concentrates were tested for mutagenicity in the Salmonella typhimurium reversion test with strains TA98 and TA100. Non-concentrated water samples were tested with two plant genotoxicity assays (the Allium cepa root anaphase aberration test and the Tradescantia/micronucleus test). Mutagenicity assays in bacteria and in Tradescantia showed borderline mutagenicity in some of the wastewater samples, independent of the disinfection procedure applied. Negative results were obtained in the A. cepa anaphase aberration test. These results indicate that, in the conditions applied, wastewater disinfection with PAA and NaClO does not lead to the formation of significant amounts of genotoxic by-products.

Fluoride toxicity effects in onion (Allium cepa L.) grown in contaminated soils.

PubMed

Jha, S K; Nayak, A K; Sharma, Y K

2009-07-01

A pot experiment was carried out under controlled condition to investigate the accumulation, uptake and toxicity effects of fluoride (F) in onion (Allium cepa L.) grown on the soil contaminated by inorganic fluoride (NaF). Six different levels of soil contamination were used by adding 0, 100, 200, 400, 600 and 800 mg NaFkg(-1) to the soil. The F concentration in shoot, bulb and root varied between 16.3 and 109.1mg Fkg(-1), 15.8 and 54.3mg Fkg(-1) and 18.6 and 151.6 mg Fkg(-1), respectively. The visible symptoms of F toxicity in terms of tip burning and death of the plant was noticed in highly contaminated soils (>400mg NaFkg(-1) soil). The phyto-toxic threshold limit (LC(50)) in onion shoot was found to be 55 mg Fkg(-1), beyond which the biomass yield decreased by 50%. It was also inferred from the study that there is a partitioning of F in onion, with more accumulation in roots and shoots than in bulbs. The order of retention of fluoride in onion found to be roots>shoot>bulb.

The possible involvement of root-cap mucilage in gravitropism and calcium movement across root tips of Allium cepa L

NASA Technical Reports Server (NTRS)

Moore, R.; Fondren, W. M.

1986-01-01

Roots of Allium cepa L. grown in aerated water elongate rapidly, but are not graviresponsive. These roots (1) possess extensive columella tissues comprised of cells containing numerous sedimented amyloplasts, (2) lack mucilage on their tips, and (3) are characterized by a weakly polar movement of calcium (Ca) across their tips. Placing roots in humid air correlates positively with the (1) onset of gravicurvature, (2) appearance of mucilage on tips of the roots, and (3) onset of the ability to transport Ca polarly to the lower side of the root tip. Gravicurvature of roots previously submerged in aerated water is more rapid when roots are oriented vertically for 1-2 h in humid air prior to being oriented horizontally. The more rapid gravicurvature of these roots correlates positively with the accumulation of mucilage at the tips of roots during the time the roots are oriented vertically. Therefore, the onset of gravicurvature and the ability of roots to transport Ca to the lower sides of their tips correlate positively with the presence of mucilage at their tips. These results suggest that mucilage may be important for the transport of Ca across root caps.

Energy-dependent RBE of neutrons to induce micronuclei in root-tip cells of Allium cepa onion irradiated as dry dormant seeds and seedlings.

PubMed

Zhang, Wenyi; Fujikawa, Kazuo; Endo, Satoru; Ishikawa, Masayori; Ohtaki, Megu; Ikeda, Hideo; Hoshi, Masaharu

2003-06-01

The relative biological effectiveness (RBE) of various energy neutrons produced from a Schenkel-type accelerator at the Research Institute for Radiation Biology and Medicine, Hiroshima University (HIRRAC), compared with 60Co gamma-ray radiation was determined. The neutron radiations and gamma-ray radiation produced good linear changes in the frequency of micronuclei induced in the root-tip cells of Allium cepa onion irradiated as dry dormant seeds (seed assay) and seedlings (seedling assay) with varying radiation doses. Therefore the RBE for radiation-induced micronuclei can be calculated as the ratio of the slopes of the fitted linear dose response for the neutron radiations and the 60Co gamma-ray radiation. The RBE values by seed assay and seedling assay decreased to 174 +/- 7, from 216 +/- 9, and to 31.4 +/- 1.0, from 45.3 +/- 1.3 (one standard error), respectively, when neutron energies increased to 1.0 MeV, from 0.2 MeV, in the present study. Furthermore, the ratio of the micronucleus induction rates of seed assay to seedling assay by gamma-ray radiation was much lower than that by neutron radiations.

Lamin-like analogues in plants: the characterization of NMCP1 in Allium cepa.

PubMed

Ciska, Malgorzata; Masuda, Kiyoshi; Moreno Díaz de la Espina, Susana

2013-04-01

The nucleoskeleton of plants contains a peripheral lamina (also called plamina) and, even though lamins are absent in plants, their roles are still fulfilled in plant nuclei. One of the most intriguing topics in plant biology concerns the identity of lamin protein analogues in plants. Good candidates to play lamin functions in plants are the members of the NMCP (nuclear matrix constituent protein) family, which exhibit the typical tripartite structure of lamins. This paper describes a bioinformatics analysis and classification of the NMCP family based on phylogenetic relationships, sequence similarity and the distribution of conserved regions in 76 homologues. In addition, NMCP1 in the monocot Allium cepa characterized by its sequence and structure, biochemical properties, and subnuclear distribution and alterations in its expression throughout the root were identified. The results demonstrate that these proteins exhibit many similarities to lamins (structural organization, conserved regions, subnuclear distribution, and solubility) and that they may fulfil the functions of lamins in plants. These findings significantly advance understanding of the structural proteins of the plant lamina and nucleoskeleton and provide a basis for further investigation of the protein networks forming these structures.

Therapeutic effects of Allium sativum and Allium cepa in Schistosoma mansoni experimental infection.

PubMed

Mantawy, Mona Mohamed; Ali, Hanan Farouk; Rizk, Maha Zaki

2011-01-01

The effects of both garlic (Allium sativum) and onion (Allium cepa) on some biochemical parameters in Schistosoma mansoni infected mice individually and mixed either with or without the currently used drug, praziquantel (PZQ) were investigated. These involved some immunological parameters, namely IgM, IgG, interleukins 2 and 6 (IL-2 and 6) and tumor necrosis factor (TNF-α), some antioxidant enzymes [catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX)]. In addition, parasitological and histopathological investigations were performed. No changes were observed in the normal control mice treated with dry extract of onion or garlic, individually or mixed, with or without PZQ, compared to the normal healthy control group. Infection with S. mansoni showed an increase in IgG, IgM, IL-2, IL-6, TNF-α and catalase enzyme, accompanied with a decrease in GPX and SOD antioxidant enzyme activities. Remarkable amelioration was noticed in the levels of all the measured parameters in S. mansoni infected mice after administration of the studied extracts. Moreover a significant reduction in worm burden, hepatic and intestinal eggs and oogram count was noticed which was reflected in normalization of liver architecture.

Organically fertilized onions (Allium cepa L.): effects of the fertilizer placement method on quercetin content and soil nitrogen dynamics.

PubMed

Mogren, Lars M; Caspersen, Siri; Olsson, Marie E; Gertsson, Ulla E

2008-01-23

Field-cured onions cv. Hyskin ( Allium cepa L.) supplied with organic nitrogen fertilizer were studied. The fertilizer was applied by broadcasting and harrowing, broadcasting and rotary cultivation, or placement between rows. Nitrogen dynamics were monitored throughout the growing season by soil sampling. Variation in quercetin content in the onion scales was analyzed by HPLC. The organically fertilized onions were compared with inorganically fertilized onions grown in the same field. Inoculation with arbuscular mycorrhizal fungi (AMF) in the row at sowing or during commercial transplant production was tested but did not significantly affect mycorrhizal root colonization levels in the field. Onions that received no fertilizer at all or that had fertilizer placed between rows had better establishment, probably due to more favorable soil nitrogen concentrations for seedling emergence. Broadcast application led to higher nitrogen concentration in the root zone, resulting in fewer but larger individual onions. Quercetin levels were not significantly altered as a result of nitrogen fertilizer source (inorganic or organic), application method, or mycorrhizal inoculation. However, variation between years was significant, with quercetin levels in 2004 almost twice as high as those in 2005.

ANTI-QUORUM SENSING ACTIVITY OF SOME MEDICINAL PLANTS.

PubMed

Al-Haidari, Rwaida A; Shaaban, Mona I; Ibrahim, Sabrin R M; Mohamed, Gamal A

2016-01-01

Quorum sensing is the key regulator of virulence factors of Pseudomonas aeruginosa such as biofilm formation, motility, productions of proteases, hemolysin, pyocyanin, and toxins. The aim of this study was to explore the effect of the extracts from some medicinal plants on quorum sensing and related virulence factors of P. aeruginosa . Quorum sensing inhibitory (OSI) effect of the alcohol extracts of 20 medicinal plants was evaluated by Chromobacterium violaceum reporter using agar cup diffusion method. The efficient QSI extracts were tested for their activity against biofilm synthesis, motility, and synthesis of pyocyanin from P. aeruginosa PA14. The extracts of Citrus sinensis, Laurus nobilis, Elettaria cardamomum, Allium cepa , and Coriandrum sativum exhibited potent quorum quenching effect. On the other hand, Psidium guajava and Mentha longifolia extracts showed lower QSI activity. These extracts exhibited significant elimination of pyocyanin formation and biofilm development of Pseudomonas aeruginosa PA14. In addition, they significantly inhibited twitching and swimming motilities of P. aeruginosa PA14. This study illustrated, for the first time, the importance of C. sinensis, L. nobilis, E. cardamomum, A. cepa , and C. sativum as quorum sensing inhibitors and virulence suppressors of P. aeruginosa . Thus, these plants could provide a natural source for the elimination of Pseudomonas pathogenesis.

Global Gene Expression Profiling in PAI-1 Knockout Murine Heart and Kidney: Molecular Basis of Cardiac-Selective Fibrosis

PubMed Central

Ghosh, Asish K.; Murphy, Sheila B.; Kishore, Raj; Vaughan, Douglas E.

2013-01-01

Fibrosis is defined as an abnormal matrix remodeling due to excessive synthesis and accumulation of extracellular matrix proteins in tissues during wound healing or in response to chemical, mechanical and immunological stresses. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1(PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways including Ankrd1, Pi16, Egr1, Scx, Timp1, Timp2, Klf6, Loxl1 and Klotho, were deregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. While the levels of Ankrd1, Pi16 and Timp1 proteins were elevated during EndMT, the level of Timp4 protein was decreased. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in fibrogenesis and several other biological processes. The significance of these observations in the light of heart-specific profibrotic signaling and fibrogenesis are discussed. PMID:23724005

Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts.

PubMed

Denecke, Shane; Fusetto, Roberto; Batterham, Philip

2017-12-01

ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC 50 values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chemical composition, in vitro antioxidant and anti-inflammatory properties of essential oils of four dietary and medicinal plants from Cameroon.

PubMed

Ndoye Foe, Florentine Marie-Chantal; Tchinang, Tatiana Flore Kemegni; Nyegue, Ascencion Maximilienne; Abdou, Jean-Pierre; Yaya, Abel Joel Gbaweng; Tchinda, Alembert Tiabou; Essame, Jean-Louis Oyono; Etoa, François-Xavier

2016-04-07

In the Cameroonian traditional medicine, plants of the Capparidaceae, Euphorbiaceae and Liliaceae families are used to treat several metabolic diseases. These plants are rich in various compounds belonging to the glucosinolates and thiosulfinates family. Till date, very little studies have been done aiming at assessing the antioxidant and inflammatory properties of the essential oils (EOs) of these plants. Essential oils are volatile extracts produced by secondary metabolism. They are usually constituted of terpens and may also contain specific non terpenic components such as glucosinolates and thiosulfinates for the species that are being considered in the present study. This study highlights and compares the chemical composition, antioxidant and anti-inflammatory properties of the essential oils of the stem barks of Drypetes gossweileri (Euphorbiaceae), roots of Pentadiplandra brazzeana (Capparidaceae), red bulbs of Allium cepa and Alium sativum (Liliaceae) collected in Cameroon (Central Africa). The essential oils were extracted by hydrodistillation and analyzed by gas chromatography (GC) and gas chromatography coupled to mass spectrometry (GC-MS). In vitro antioxidant activities were determined using the radical scavenging assay, total phenolic content, ferric reducing antioxidant power (FRAP) assay and determination of antioxidant activity index (AAI) according to the method described by Scherer and Godoy. The anti-inflammatory activities were evaluated using albumin denaturation method. Differences (p < 0.05) between the experimental and the control groups were evaluated using one way analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons. The main components of Allium sativum essential oil were diallyl trisulfide (41.62 %), diallyl disulfide (19.74 %), allyl methyl trisulfide (12.95 %), diallyl sulfide (7.1 %) and diallyl tetrasulfide (4.22 %). Those of Allium cepa essential oil were diallyl trisulfide (22.17 %), dipropyl trisulfide (11.11 %), 2-methyl-3,4-dithiaheptane (9.88 %), methyl propyl trisulfide (8.14 %), dipropyl tetrasulfide (8.07 %) and 2-propenyl propyl disulfide (5.15 %). Drypetes gossweileri and Pentadiplandra brazzeana essential oils presented similar chemical compositions as compared with benzylisothiocyanate content (63.19 and 97.63 % respectively), but differed in benzylcyanide content (35.72 and 0.86 % respectively). The essential oils were rich in phenolic compounds in the following order Allium sativum < Allium cepa < D. gossweileiri < P. brazzeana. The essential oils exhibited high antioxidant and DPPH radical scavenging effect but low ferric reducing power activity. Moreover, the four essential oils showed anti-inflammatory activities (by heat denaturation of Bovine Serum Albumin). The anti-inflammatory activities of P. brazzeana and A. cepa essential oils were comparable but higher than those of D. gossweileri and sodium diclofenac used as a reference non-steroidal anti-inflammatory drug. The essential oils of the plants were rich in organosulfur compounds. These compounds were probably responsible for their appreciable antioxidant and anti-inflammatory activities. Due to their antioxidant and anti-inflammatory properties, the essential oils of some of these plants might be used as natural additives in the pharmaceutical, cosmetic and agro-industries.

Reduced Extinction of Hippocampal-Dependent Memories in CPEB Knockout Mice

ERIC Educational Resources Information Center

Zearfoss, N. Ruth; Richter, Joel D.; Berger-Sweeney, Joanne

2006-01-01

CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of…

Adaptive bi-level programming for optimal gene knockouts for targeted overproduction under phenotypic constraints

PubMed Central

2013-01-01

Background Optimization procedures to identify gene knockouts for targeted biochemical overproduction have been widely in use in modern metabolic engineering. Flux balance analysis (FBA) framework has provided conceptual simplifications for genome-scale dynamic analysis at steady states. Based on FBA, many current optimization methods for targeted bio-productions have been developed under the maximum cell growth assumption. The optimization problem to derive gene knockout strategies recently has been formulated as a bi-level programming problem in OptKnock for maximum targeted bio-productions with maximum growth rates. However, it has been shown that knockout mutants in fact reach the steady states with the minimization of metabolic adjustment (MOMA) from the corresponding wild-type strains instead of having maximal growth rates after genetic or metabolic intervention. In this work, we propose a new bi-level computational framework--MOMAKnock--which can derive robust knockout strategies under the MOMA flux distribution approximation. Methods In this new bi-level optimization framework, we aim to maximize the production of targeted chemicals by identifying candidate knockout genes or reactions under phenotypic constraints approximated by the MOMA assumption. Hence, the targeted chemical production is the primary objective of MOMAKnock while the MOMA assumption is formulated as the inner problem of constraining the knockout metabolic flux to be as close as possible to the steady-state phenotypes of wide-type strains. As this new inner problem becomes a quadratic programming problem, a novel adaptive piecewise linearization algorithm is developed in this paper to obtain the exact optimal solution to this new bi-level integer quadratic programming problem for MOMAKnock. Results Our new MOMAKnock model and the adaptive piecewise linearization solution algorithm are tested with a small E. coli core metabolic network and a large-scale iAF1260 E. coli metabolic network. The derived knockout strategies are compared with those from OptKnock. Our preliminary experimental results show that MOMAKnock can provide improved targeted productions with more robust knockout strategies. PMID:23368729

Adaptive bi-level programming for optimal gene knockouts for targeted overproduction under phenotypic constraints.

PubMed

Ren, Shaogang; Zeng, Bo; Qian, Xiaoning

2013-01-01

Optimization procedures to identify gene knockouts for targeted biochemical overproduction have been widely in use in modern metabolic engineering. Flux balance analysis (FBA) framework has provided conceptual simplifications for genome-scale dynamic analysis at steady states. Based on FBA, many current optimization methods for targeted bio-productions have been developed under the maximum cell growth assumption. The optimization problem to derive gene knockout strategies recently has been formulated as a bi-level programming problem in OptKnock for maximum targeted bio-productions with maximum growth rates. However, it has been shown that knockout mutants in fact reach the steady states with the minimization of metabolic adjustment (MOMA) from the corresponding wild-type strains instead of having maximal growth rates after genetic or metabolic intervention. In this work, we propose a new bi-level computational framework--MOMAKnock--which can derive robust knockout strategies under the MOMA flux distribution approximation. In this new bi-level optimization framework, we aim to maximize the production of targeted chemicals by identifying candidate knockout genes or reactions under phenotypic constraints approximated by the MOMA assumption. Hence, the targeted chemical production is the primary objective of MOMAKnock while the MOMA assumption is formulated as the inner problem of constraining the knockout metabolic flux to be as close as possible to the steady-state phenotypes of wide-type strains. As this new inner problem becomes a quadratic programming problem, a novel adaptive piecewise linearization algorithm is developed in this paper to obtain the exact optimal solution to this new bi-level integer quadratic programming problem for MOMAKnock. Our new MOMAKnock model and the adaptive piecewise linearization solution algorithm are tested with a small E. coli core metabolic network and a large-scale iAF1260 E. coli metabolic network. The derived knockout strategies are compared with those from OptKnock. Our preliminary experimental results show that MOMAKnock can provide improved targeted productions with more robust knockout strategies.

Continuum analyzing power for 4He(p-->,p') at 100 MeV

NASA Astrophysics Data System (ADS)

Lawrie, J. J.; Whittal, D. M.; Cowley, A. A.

1990-08-01

Distorted-wave impulse approximation calculations of the continuum analyzing power for the inclusive reaction 4He(p-->,p') at an incident energy of 100 MeV are presented. In addition to the quasifree knockout of nucleons, contributions from the knockout of deuteron, triton, and helion clusters are taken into account, together with a breakup component. Whereas nucleon knockout by itself does not account for the experimentally observed analyzing power, the inclusion of clusters has a large effect. Thus a simple knockout model is able to provide a reasonable description of the experimental continuum analyzing power.

Genetic deletion of CB1 receptors improves non-associative learning.

PubMed

Degroot, Aldemar; Salhoff, Craig; Davis, Richard J; Nomikos, George G

2005-07-01

Habituation (a form of non-associative learning) was measured by assessing locomotion in novel activity monitors in CB1 receptor knockout mice and juxtaposed to habituation measured in muscarinic M2, M4, and double M2/M4 receptor knockout mice. M2 and M2/M4, but not M4, receptor knockout mice appeared to have an impaired ability to habituate, whereas CB1 receptor knockout mice showed enhanced habituation compared to wild-type animals. We conclude that CB1 receptor gene invalidation improves habituation tentatively through an increase in cholinergic neurotransmission.

Reduced extinction of hippocampal-dependent memories in CPEB knockout mice.

PubMed

Berger-Sweeney, Joanne; Zearfoss, N Ruth; Richter, Joel D

2006-01-01

CPEB is a sequence-specific RNA binding protein that regulates translation at synapses. In neurons of CPEB knockout mice, synaptic efficacy is reduced. Here, we have performed a battery of behavioral tests and find that relative to wild-type animals, CPEB knockout mice, although similar on many baseline behaviors, have reduced extinction of memories on two hippocampal-dependent tasks. A corresponding microarray analysis reveals that about 0.14% of hippocampal genes have an altered expression in the CPEB knockout mouse. These data suggest that CPEB-dependent local protein synthesis may be an important cellular mechanism underlying extinction of hippocampal-dependent memories.

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level

PubMed Central

Yuen, Garmen; Khan, Fehad J.; Gao, Shaojian; Stommel, Jayne M.; Batchelor, Eric; Wu, Xiaolin

2017-01-01

Abstract CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. PMID:29036671

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

PubMed

Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

2017-11-16

CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Pyviko: an automated Python tool to design gene knockouts in complex viruses with overlapping genes.

PubMed

Taylor, Louis J; Strebel, Klaus

2017-01-07

Gene knockouts are a common tool used to study gene function in various organisms. However, designing gene knockouts is complicated in viruses, which frequently contain sequences that code for multiple overlapping genes. Designing mutants that can be traced by the creation of new or elimination of existing restriction sites further compounds the difficulty in experimental design of knockouts of overlapping genes. While software is available to rapidly identify restriction sites in a given nucleotide sequence, no existing software addresses experimental design of mutations involving multiple overlapping amino acid sequences in generating gene knockouts. Pyviko performed well on a test set of over 240,000 gene pairs collected from viral genomes deposited in the National Center for Biotechnology Information Nucleotide database, identifying a point mutation which added a premature stop codon within the first 20 codons of the target gene in 93.2% of all tested gene-overprinted gene pairs. This shows that Pyviko can be used successfully in a wide variety of contexts to facilitate the molecular cloning and study of viral overprinted genes. Pyviko is an extensible and intuitive Python tool for designing knockouts of overlapping genes. Freely available as both a Python package and a web-based interface ( http://louiejtaylor.github.io/pyViKO/ ), Pyviko simplifies the experimental design of gene knockouts in complex viruses with overlapping genes.

Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways.

PubMed

Kato, Hideki; Ishida, Junji; Matsusaka, Taiji; Ishimaru, Tomohiro; Tanimoto, Keiji; Sugiyama, Fumihiro; Yagami, Ken-Ichi; Nangaku, Masaomi; Fukamizu, Akiyoshi

2015-01-01

The renin-angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control.

Thyroid Hormone Receptor α- and β-Knockout Xenopus tropicalis Tadpoles Reveal Subtype-Specific Roles During Development.

PubMed

Nakajima, Keisuke; Tazawa, Ichiro; Yaoita, Yoshio

2018-02-01

Thyroid hormone (TH) binds TH receptor α (TRα) and β (TRβ) to induce amphibian metamorphosis. Whereas TH signaling has been well studied, functional differences between TRα and TRβ during this process have not been characterized. To understand how each TR contributes to metamorphosis, we generated TRα- and TRβ-knockout tadpoles of Xenopus tropicalis and examined developmental abnormalities, histology of the tail and intestine, and messenger RNA expression of genes encoding extracellular matrix-degrading enzymes. In TRβ-knockout tadpoles, tail regression was delayed significantly and a healthy notochord was observed even 5 days after the initiation of tail shortening (stage 62), whereas in the tails of wild-type and TRα-knockout tadpoles, the notochord disappeared after ∼1 day. The messenger RNA expression levels of genes encoding extracellular matrix-degrading enzymes (MMP2, MMP9TH, MMP13, MMP14, and FAPα) were obviously reduced in the tail tip of TRβ-knockout tadpoles, with the shortening tail. The reduction in olfactory nerve length and head narrowing by gill absorption were also affected. Hind limb growth and intestinal shortening were not compromised in TRβ-knockout tadpoles, whereas tail regression and olfactory nerve shortening appeared to proceed normally in TRα-knockout tadpoles, except for the precocious development of hind limbs. Our results demonstrated the distinct roles of TRα and TRβ in hind limb growth and tail regression, respectively. Copyright © 2018 Endocrine Society.

Engaging Teachers: Measuring the Impact of Teachers on Student Attendance in Secondary School. CEPA Working Paper No. 17-01

ERIC Educational Resources Information Center

Liu, Jing; Loeb, Susanna

2017-01-01

Both anecdotal and systematic evidence points to the importance of teachers for students' long-run success. Previous research on effective teachers has focused almost exclusively on student test score gains in math and reading. For this study we are able to link middle and high school teachers to the class-attendance of students in their…

The Causes and Consequences of Test Score Manipulation: Evidence from the New York Regents Examinations. CEPA Working Paper No. 16-08

ERIC Educational Resources Information Center

Dee, Thomas S.; Dobbie, Will; Jacob, Brian A.; Rockoff, Jonah

2016-01-01

In this paper, we show that the design and decentralized, school-based scoring of New York's high school exit exams--the Regents Examinations--led to the systematic manipulation of test sores just below important proficiency cutoffs. Our estimates suggest that teachers inflate approximately 40 percent of test scores near the proficiency cutoffs.…

The Impact of the Great Recession on Student Achievement: Evidence from Population Data. CEPA Working Paper No. 17-09

ERIC Educational Resources Information Center

Shores, Kenneth; Steinberg, Matthew P.

2017-01-01

The Great Recession was the most severe economic downturn in the United States since the Great Depression. Using newly available population-level achievement data from the Stanford Education Data Archive (SEDA), we estimate the impact of the Great Recession on the math and English language arts (ELA) achievement of all grade 3-8 students in the…

The Effects of Class Size in Online College Courses: Experimental Evidence. CEPA Working Paper No. 15-14

ERIC Educational Resources Information Center

Bettinger, Eric; Doss, Christopher; Loeb, Susanna; Taylor, Eric

2015-01-01

Class size is a first-order consideration in the study of education production and education costs. How larger or smaller classes affect student outcomes is especially relevant to the growth and design of online classes. We study a field experiment in which college students were quasi-randomly assigned to either a large or a small class. All…

Can Socioeconomic Status Substitute for Race in Affirmative Action College Admissions Policies? Evidence from a Simulation Model. CEPA Working Paper No. 15-04

ERIC Educational Resources Information Center

Reardon, Sean F.; Baker, Rachel; Kasman, Matt; Klasik, Daniel; Townsend, Joseph

2017-01-01

This paper simulates a system of socioeconomic status (SES)-based affirmative action in college admissions and examines the extent to which it can produce racial diversity in selective colleges. Using simulation models, we investigate the potential relative effects of race- and/or SES-based affirmative action policies, alongside targeted,…

New report of Lolium multiflorum and Rumex crispus as weed hosts of epiphytic populations of Psuedomonas sp., causal agent of yellow bud in onion in Geogia, USA

USDA-ARS?s Scientific Manuscript database

Yellow bud, an emerging bacterial disease of onion (Allium cepa L.), has been spreading throughout the Vidalia onion-growing region in Georgia since 2007. Symptoms of yellow bud include intense chlorosis in emerging leaves and severe blight in the older leaves leading to stand loss and reduced bulb ...

Knockout of the Na,K-ATPase α2-isoform in cardiac myocytes delays pressure overload-induced cardiac dysfunction

PubMed Central

Rindler, Tara N.; Lasko, Valerie M.; Nieman, Michelle L.; Okada, Motoi; Lorenz, John N.

2013-01-01

The α2-isoform of the Na,K-ATPase (α2) is the minor isoform of the Na,K-ATPase expressed in the cardiovascular system and is thought to play a critical role in the regulation of cardiovascular hemodynamics. However, the organ system/cell type expressing α2 that is required for this regulation has not been fully defined. The present study uses a heart-specific knockout of α2 to further define the tissue-specific role of α2 in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model using the Cre/loxP system to generate a tissue-specific knockout of α2 in the heart using β-myosin heavy chain Cre. We have achieved a 90% knockout of α2 expression in the heart of the knockout mice. Interestingly, the heart-specific knockout mice exhibit normal basal cardiac function and systolic blood pressure, and in addition, these mice develop ACTH-induced hypertension in response to ACTH treatment similar to control mice. Surprisingly, the heart-specific knockout mice display delayed onset of cardiac dysfunction compared with control mice in response to pressure overload induced by transverse aortic constriction; however, the heart-specific knockout mice deteriorated to control levels by 9 wk post-transverse aortic constriction. These results suggest that heart expression of α2 does not play a role in the regulation of basal cardiovascular function or blood pressure; however, heart expression of α2 plays a role in the hypertrophic response to pressure overload. This study further emphasizes that the tissue localization of α2 determines its unique roles in the regulation of cardiovascular function. PMID:23436327

Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension

PubMed Central

Li, Wencheng; Peng, Hua; Mehaffey, Eamonn P.; Kimball, Christie D.; Grobe, Justin L.; van Gool, Jeanette M.G.; Sullivan, Michelle N.; Earley, Scott; Danser, A.H. Jan; Ichihara, Atsuhiro; Feng, Yumei

2013-01-01

The (pro)renin receptor, which binds both renin and prorenin, is a newly discovered component of the renin angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, non-proteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate salt induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. (Pro)renin receptor expression, detected by immunostaining and RT-PCR, was significantly decreased in the brains of knockout compared with wide-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild type mice. This hypertensive response was abolished in (pro)renin receptor knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate salt increased (pro)renin receptor expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in (pro)renin receptor knockout mice. (Pro)renin receptor knockout in neurons prevented the development of Deoxycorticosterone acetate salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, non-proteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate salt-induced hypertension, possibly through diminished angiotensin II formation. PMID:24246383

Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress.

PubMed

Spiekerkoetter, U; Tokunaga, C; Wendel, U; Mayatepek, E; Exil, V; Duran, M; Wijburg, F A; Wanders, R J A; Strauss, A W

2004-03-01

In humans with deficiency of the very long-chain acyl-CoA dehydrogenase (VLCAD), C14-C18 acylcarnitines accumulate. In this paper we have used the VLCAD knockout mouse as a model to study changes in blood carnitine and acylcarnitine profiles under stress. VLCAD knockout mice exhibit stress-induced hypoglycaemia and skeletal myopathy; symptoms resembling human VLCADD. To study the extent of biochemical derangement in response to different stressors, we determined blood carnitine and acylcarnitine profiles after exercise on a treadmill, fasting, or exposure to cold. Even in a nonstressed, well-fed state, knockout mice presented twofold higher C14-C18 acylcarnitines and a lower free carnitine of 72% as compared to wild-type littermates. After 1 h of intense exercise, the C14-C18 acylcarnitines in blood significantly increased, but free carnitine remained unchanged. After 8 h of fasting at 4 degrees C, the long-chain acylcarnitines were elevated 5-fold in knockout mice in comparison with concentrations in unstressed wild-type mice (P < 0.05), and four out of 12 knockout mice died. Free carnitine decreased to 44% as compared with unstressed wild-type mice. An increase in C14-C18 acylcarnitines and a decrease of free carnitine were also observed in fasted heterozygous and wild-type mice. Long-chain acylcarnitines in blood increase in knockout mice in response to different stressors and concentrations correlate with the clinical condition. A decrease in blood free carnitine in response to severe stress is observed in knockout mice but also in wild-type littermates. Monitoring blood acylcarnitine profiles in response to different stressors may allow systematic analysis of therapeutic interventions in VLCAD knockout mice.

Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex.

PubMed

Ventura, R; Pascucci, T; Catania, M V; Musumeci, S A; Puglisi-Allegra, S

2004-09-01

Fragile X syndrome is an X-linked form of mental retardation including, among others, symptoms such as stereotypic behaviour, hyperactivity, hyperarousal, and cognitive deficits. We hypothesized that hyperactivity and/or compromised attentional, cognitive functions may lead to impaired performance in cognitive tasks in Fmr1 knockout mice, the most widely used animal model of fragile X syndrome, and suggested that psychostimulant treatment may improve performance by acting on one or both components. Since hyperactivity and cognitive functions have been suggested to depend on striatal and prefrontal cortex dopaminergic dysfunction, we assessed whether amphetamine produced beneficial, positive effects by acting on dopaminergic corticostriatal systems. Our results show that Fmr1 knockout mice are not able to discriminate between a familiar object and a novel one in the object recognition test, thus showing a clear-cut cognitive impairment that, to date, has been difficult to demonstrate in other cognitive tasks. Amphetamine improved performance of Fmr1 knockout mice, leading to enhanced ability to discriminate novel versus familiar objects, without significantly affecting locomotor activity. In agreement with behavioural data, amphetamine produced a greater increase in dopamine release in the prefrontal cortex of Fmr1 knockout compared with the wild-type mice, while a weak striatal dopaminergic response was observed in Fmr1 knockout mice. Our data support the view that the psychostimulant ameliorates performance in Fmr1 knockout mice by improving merely cognitive functions through its action on prefrontal cortical dopamine, irrespective of its action on motor hyperactivity. These results indicate that prefrontal cortical dopamine plays a major role in cognitive impairments characterizing Fmr1 knockout mice, thus pointing to an important aetiological factor in the fragile X syndrome.

Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

PubMed

Walentiny, D Matthew; Vann, Robert E; Wiley, Jenny L

2015-06-01

A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184). Copyright © 2015 Elsevier Ltd. All rights reserved.

A minor role of WNK3 in regulating phosphorylation of renal NKCC2 and NCC co-transporters in vivo.

PubMed

Oi, Katsuyuki; Sohara, Eisei; Rai, Tatemitsu; Misawa, Moko; Chiga, Motoko; Alessi, Dario R; Sasaki, Sei; Uchida, Shinichi

2012-02-15

Mutations in WNK1 and WNK4 kinase genes have been shown to cause a human hereditary hypertensive disease, pseudohypoaldosteronism type II (PHAII). We previously discovered that WNK kinases phosphorylate and activate OSR1/SPAK kinases that regulate renal SLC12A family transporters such as NKCC2 and NCC, and clarified that the constitutive activation of this cascade causes PHAII. WNK3, another member of the WNK kinase family, was reported to be a strong activator of NCC/NKCC2 when assayed in Xenopus oocytes, suggesting that WNK3 also plays a major role in regulating blood pressure and sodium reabsorption in the kidney. However, it remains to be determined whether WNK3 is in fact involved in the regulation of these transporters in vivo. To clarify this issue, we generated and analyzed WNK3 knockout mice. Surprisingly, phosphorylation and expression of OSR1, SPAK, NKCC2 and NCC did not decrease in knockout mouse kidney under normal and low-salt diets. Similarly, expression of epithelial Na channel and Na/H exchanger 3 were not affected in knockout mice. Na(+) and K(+) excretion in urine in WNK3 knockout mice was not affected under different salt diets. Blood pressure in WNK3 knockout mice was not lower under normal diet. However, lower blood pressure was observed in WNK3 knockout mice fed low-salt diet. WNK4 and WNK1 expression was slightly elevated in the knockout mice under low-salt diet, suggesting compensation for WNK3 knockout by these WNKs. Thus, WNK3 may have some role in the WNK-OSR1/SPAK-NCC/NKCC2 signal cascade in the kidney, but its contribution to total WNK kinase activity may be minimal.

The guinea pig ileum lacks the direct, high-potency, M(2)-muscarinic, contractile mechanism characteristic of the mouse ileum.

PubMed

Griffin, Michael T; Matsui, Minoru; Ostrom, Rennolds S; Ehlert, Frederick J

2009-10-01

We explored whether the M(2) muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M(3) muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M(3) selectivity. Then, we used 4-DAMP mustard to inactivate M(3) responses in the guinea pig ileum to attempt to reveal direct, M(2) receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M(2) receptor knockout, and M(3) receptor knockout mice characterized by negative log EC(50) (pEC (50)) values +/- SEM of 6.75 +/- 0.03, 6.26 +/- 0.05, and 6.99 +/- 0.08, respectively. The corresponding E (max) values in wild-type and M(2) receptor knockout mice were approximately the same, but that in the M(3) receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration-response curve of oxotremorine-M in wild-type ileum resembled that of the M(3) knockout mouse in terms of its pEC (50), E (max), and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M(3) responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M(3) knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M(3) response. The guinea pig ileum, therefore, lacks a direct, highly potent, M(2)-contractile component but may have a direct, lower potency M(2) component.

Myostatin knockout using zinc-finger nucleases promotes proliferation of ovine primary satellite cells in vitro.

PubMed

Salabi, Fatemeh; Nazari, Mahmood; Chen, Qing; Nimal, Jonathan; Tong, Jianming; Cao, Wen G

2014-12-20

Myostatin (MSTN) has previously been shown to negatively regulate the proliferation and differentiation of skeletal muscle cells. Satellite cells are quiescent muscle stem cells that promote muscle growth and repair. Because the mechanism of MSTN in the biology of satellite cells is not well understood, this study was conducted to generate MSTN mono-allelic knockout satellite cells using the zinc-finger nuclease mRNA (MSTN-KO ZFN mRNA) and also to investigate the effect of this disruption on the proliferation and differentiation of sheep primary satellite cells (PSCs). Nineteen biallelic and four mono-allelic knockout cell clones were obtained after sequence analysis. The homologous mono-allelic knockout cells with 5-bp deletion were used to further evaluations. The results demonstrated that mono-allelic knockout of MSTN gene leads to translation inhibition. Real-time quantitative PCR results indicated that knockout of MSTN contributed to an increase in CDK2 and follistatin and a decrease in p21 at the transcript level in proliferation conditions. Moreover, MSTN knockout significantly increased the proliferation of mutant clones (P < 0.01). Consistent with the observed increase in CDK2 and decrease in p21 in cells lacking MSTN, cell cycle analysis showed that MSTN negatively regulated the G1 to S progression. In addition, knockout of myostatin resulted in a remarkable increase in MyoD and MyoG expression under differentiating conditions but had no effect on Myf5 expression. These results expanded our understanding of the regulation mechanism of MSTN. Furthermore, the MSTN-KO ZFN mRNA system in PSCs could be used to generate transgenic sheep in the future.

Differential sensitivity of Pak5, Pak6, and Pak5/Pak6 double-knockout mice to the stimulant effects of amphetamine and exercise-induced alterations in body weight.

PubMed

Furnari, Melody A; Jobes, Michelle L; Nekrasova, Tanya; Minden, Audrey; Wagner, George C

2014-04-01

PAK5 and PAK6 are protein kinases highly expressed in the brain. Previously, we observed that Pak6 knockout mice gained significantly more weight during development than Pak5 knockout mice as well as wild-type controls and double-knockout mice lacking both Pak5 and Pak6. In this study, we assessed the effects of exercise on food intake and weight gain of these mice as well as their sensitivity to the stimulant effects of amphetamine. Mice of each genotype were placed in cages with free access to run wheel exercise or in cages without run wheels for a total of 74 days. Food and fluid intake as well as body weight of each mouse were measured on a weekly basis. Finally, mice were given a high dose of amphetamine and activity levels were observed immediately thereafter for 90 minutes. Brains and testes of mice were assayed for protein levels of the estrogen alpha and progesterone receptors. While run wheel mice consumed significantly more food, they weighed less than non-run wheel mice. In addition, although Pak6 knockout mice consumed the same amount of food as wild-type mice, they were significantly heavier regardless of run wheel condition. Pak5 knockout mice were found to be more active than other genotypes after amphetamine treatment. Finally, protein levels of the progesterone and estrogen alpha receptors were altered in brain and testes of the Pak6 knockout mice. Collectively, these data suggest that PAK6 play a role in weight gain unrelated to exercise and caloric intake and that Pak5 knockout mice are more sensitive to the stimulant effects of amphetamine.

Distinct Roles of Opioid and Dopamine Systems in Lateral Hypothalamic Intracranial Self-Stimulation.

PubMed

Ide, Soichiro; Takahashi, Takehiro; Takamatsu, Yukio; Uhl, George R; Niki, Hiroaki; Sora, Ichiro; Ikeda, Kazutaka

2017-05-01

Opioid and dopamine systems play crucial roles in reward. Similarities and differences in the neural mechanisms of reward that are mediated by these 2 systems have remained largely unknown. Thus, in the present study, we investigated the differences in reward function in both µ-opioid receptor knockout mice and dopamine transporter knockout mice, important molecules in the opioid and dopamine systems. Mice were implanted with electrodes into the right lateral hypothalamus (l hour). Mice were then trained to put their muzzle into the hole in the head-dipping chamber for intracranial electrical stimulation, and the influences of gene knockout were assessed. Significant differences are observed between opioid and dopamine systems in reward function. µ-Opioid receptor knockout mice exhibited enhanced intracranial electrical stimulation, which induced dopamine release. They also exhibited greater motility under conditions of "despair" in both the tail suspension test and water wheel test. In contrast, dopamine transporter knockout mice maintained intracranial electrical stimulation responding even when more active efforts were required to obtain the reward. The absence of µ-opioid receptor or dopamine transporter did not lead to the absence of intracranial electrical stimulation responsiveness but rather differentially altered it. The present results in µ-opioid receptor knockout mice are consistent with the suppressive involvement of µ-opioid receptors in both positive incentive motivation associated with intracranial electrical stimulation and negative incentive motivation associated with depressive states. In contrast, the results in dopamine transporter knockout mice are consistent with the involvement of dopamine transporters in positive incentive motivation, especially its persistence. Differences in intracranial electrical stimulation in µ-opioid receptor and dopamine transporter knockout mice underscore the multidimensional nature of reward. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Enhanced serotonin response in the hippocampus of Galphaz protein knock-out mice.

PubMed

Oleskevich, Sharon; Leck, Kwong-Joo; Matthaei, Klaus; Hendry, Ian A

2005-06-21

The serotonin-1A [5-hydroxytryptamine 1A (5HT1A)] receptor is important for emotional and homeostatic processes in the central nervous system. In the hippocampus, the 5HT1A receptor couples to inhibitory Gi/o proteins to decrease pyramidal cell excitability. Here we investigate the 5HT1A receptor in a mouse deficient in the alpha-subunit of Gz protein (Galphaz knock-out). Behavioural tests showed heightened anxiety and depression-like behaviour in the Galphaz knock-out mice. Whole-cell recording in CA1 pyramidal neurons showed a significantly greater 5HT1A receptor-mediated potassium current in Galphaz knock-out mice. The effect was independent of 5HT4 receptors as the slow after-hyperpolarization was unaffected and a slow depolarization was absent in the Galphaz knock-out mice. Other receptors linked to Gi/o proteins [gamma-aminobutyric acid type B receptor (GABAB), adenosine A1 and muscarinic acetylcholine receptors] were not affected in Galphaz knock-out mice. These results suggest that the 5HT1A receptor may be linked to Galphaz protein, as reported previously in cell culture but shown here in an intact neural network.

Cold Shock as a Screen for Genes Involved in Cold Acclimatization in Neurospora crassa

PubMed Central

Watters, Michael K.; Manzanilla, Victor; Howell, Holly; Mehreteab, Alexander; Rose, Erik; Walters, Nicole; Seitz, Nicholas; Nava, Jacob; Kekelik, Sienna; Knuth, Laura; Scivinsky, Brianna

2018-01-01

When subjected to rapid drops of temperature (cold shock), Neurospora responds with a temporary shift in its morphology. This report is the first to examine this response genetically. We report here the results of a screen of selected mutants from the Neurospora knockout library for alterations in their morphological response to cold shock. Three groups of knockouts were selected to be subject to this screen: genes previously suspected to be involved in hyphal development as well as knockouts resulting in morphological changes; transcription factors; and genes homologous to E. coli genes known to alter their expression in response to cold shock. A total of 344 knockout strains were subjected to cold shock. Of those, 118 strains were identified with altered responses. We report here the cold shock morphologies and GO categorizations of strains subjected to this screen. Of strains with knockouts in genes associated with hyphal growth or morphology, 33 of 131 tested (25%) showed an altered response to cold shock. Of strains with knockouts in transcription factor genes, 30 of 145 (20%) showed an altered response to cold shock. Of strains with knockouts in genes homologous to E. coli genes which display altered levels of transcription in response to cold shock, a total of 55 of 68 tested (81%) showed an altered cold shock response. This suggests that the response to cold shock in these two organisms is largely shared in common. PMID:29563189

A STAT-1 Knockout Mouse Model for Machupo Virus Pathogenesis

DTIC Science & Technology

2011-06-14

hemorrhagic fever viruses, including Ebola, Marburg, Junín, and Crimean - Congo Hemorrhagic Fever viruses [11-14...Akerstrom S, Klingstrom J, Mirazimi A: Crimean - Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice. J...Shieh WJ, Camus G, Stroher U, Zaki S, Jones SM: Pathogenesis and immune response of Crimean - Congo hemorrhagic fever virus in a STAT-1 knockout

Embryonic Lethality Due to Arrested Cardiac Development in Psip1/Hdgfrp2 Double-Deficient Mice.

PubMed

Wang, Hao; Shun, Ming-Chieh; Dickson, Amy K; Engelman, Alan N

2015-01-01

Hepatoma-derived growth factor (HDGF) related protein 2 (HRP2) and lens epithelium-derived growth factor (LEDGF)/p75 are closely related members of the HRP2 protein family. LEDGF/p75 has been implicated in numerous human pathologies including cancer, autoimmunity, and infectious disease. Knockout of the Psip1 gene, which encodes for LEDGF/p75 and the shorter LEDGF/p52 isoform, was previously shown to cause perinatal lethality in mice. The function of HRP2 was by contrast largely unknown. To learn about the role of HRP2 in development, we knocked out the Hdgfrp2 gene, which encodes for HRP2, in both normal and Psip1 knockout mice. Hdgfrp2 knockout mice developed normally and were fertile. By contrast, the double deficient mice died at approximate embryonic day (E) 13.5. Histological examination revealed ventricular septal defect (VSD) associated with E14.5 double knockout embryos. To investigate the underlying molecular mechanism(s), RNA recovered from ventricular tissue was subjected to RNA-sequencing on the Illumina platform. Bioinformatic analysis revealed several genes and biological pathways that were significantly deregulated by the Psip1 knockout and/or Psip1/Hdgfrp2 double knockout. Among the dozen genes known to encode for LEDGF/p75 binding factors, only the expression of Nova1, which encodes an RNA splicing factor, was significantly deregulated by the knockouts. However the expression of other RNA splicing factors, including the LEDGF/p52-interacting protein ASF/SF2, was not significantly altered, indicating that deregulation of global RNA splicing was not a driving factor in the pathology of the VSD. Tumor growth factor (Tgf) β-signaling, which plays a key role in cardiac morphogenesis during development, was the only pathway significantly deregulated by the double knockout as compared to control and Psip1 knockout samples. We accordingly speculate that deregulated Tgf-β signaling was a contributing factor to the VSD and prenatal lethality of Psip1/Hdgfrp2 double-deficient mice.

Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice.

PubMed

Most, D; Efron, D T; Shi, H P; Tantry, U S; Barbul, A

2001-10-01

Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test. Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05). Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.

Unnecessary Disruptions? Implications of the Volatility of Within-School Reassignments on Student Achievement. CEPA Working Paper No. 15-13

ERIC Educational Resources Information Center

Atteberry, Allison; Loeb, Susanna; Wyckoff, James

2015-01-01

Educators raise concerns about what happens to students when they are exposed to new teachers or teachers who are new to a school. These teachers face the challenge of preparing a year's worth of new material, perhaps in an unfamiliar work environment. However, even when teachers remain in the same school they can switch jobs--teaching either a…

The Long Run Impacts of Merit Aid: Evidence from California's Cal Grant. CEPA Working Paper No. 16-13

ERIC Educational Resources Information Center

Bettinger, Eric; Gurantz, Oded; Kawano, Laura; Sacerdote, Bruce

2016-01-01

We examine the impacts of being awarded a Cal Grant, among the most generous state merit aid programs. We exploit variation in eligibility rules using GPA and family income cutoffs that are ex ante unknown to applicants. Cal Grant eligibility increases degree completion by 2 to 5 percentage points in our reduced form estimates. Cal Grant also…

Teacher Workforce Developments: Recent Changes in Academic Competiveness and Job Satisfaction of New Teachers. CEPA Working Paper No. 15-16

ERIC Educational Resources Information Center

Loeb, Susanna; Master, Benjamin; Sun, Min

2015-01-01

The capacity of the nation's public schools to recruit and retain highly skilled teachers is a perennial concern of policy makers and school leaders. Over the past two decades, major policy strategies including the federal No Child Left Behind Act and alternative pathways to teaching, as well as changes in the broader labor market, have altered…

How Much Do Test Scores Vary among School Districts? New Estimates Using Population Data, 2009-2015. CEPA Working Paper No. 17-02

ERIC Educational Resources Information Center

Fahle, Erin M.; Reardon, Sean F.

2017-01-01

This paper provides the first population-based evidence on how much standardized test scores vary among public school districts within each state and how segregation explains that variation. Using roughly 300 million standardized test score records in math and ELA for grades 3 through 8 from every U.S. public school district during the 2008-09 to…

Efficacy of molecular markers jnurf13 and AcPms1 for prediction of genotypes at the nuclear Ms locus in North American open-pollinated populations of onion

USDA-ARS?s Scientific Manuscript database

Seed of hybrid onion (Allium cepa L.) is produced using cytoplasmic male sterility (CMS) and the for most widely used source of onion CMS, male sterility is conditioned by the interaction of male-sterile (S) cytoplasm and the homozygous recessive genotype at the nuclear male-fertility locus Ms. Due ...

Micropropagation of onion (Allium cepa L.) from immature inflorescences.

PubMed

Marinangeli, Pablo

2013-01-01

In vitro plant production by direct organogenesis from immature flower heads is an ideal approach for clonal propagation of onions (Allium cepa L.). This technique ensures genetic stability, high propagation rate, and maintains donor plant of explants with an advantage over other means of in vitro regeneration. Onion micropropagation is usually applied in breeding programs, maintenance, and multiplication of cytoplasmic-male sterile lines for hybrid production, germplasm conservation, and as a tool for the application of other biotechnologies. For in vitro culture, mature onion bulbs are induced to reproductive phase by vernalization and forced to inflorescence initiation. Immature umbels are dissected from bulbs or cut directly when they appear from the pseudostem among the leaves. Disinfected inflorescences are cultivated in BDS basal medium supplemented with 30 g/L sucrose, 0.1 mg/L naphthalene acetic acid, 1 mg/L N (6)-benzyladenine, and 8 g/L agar, pH 5.5, under 16 h photoperiod white fluorescent light (PPD: 50-70 μmol/m(2)s) for 35 days. The regenerated shoot clumps are divided and subculture under the same conditions. For bulbification phase, the individual shoots are cultured in BDS basal medium containing 90 g/L sucrose, without plant growth regulators, pH 5.5, under 16 h photoperiod. Microbulbs can be directly cultivated ex vitro without acclimation.

Assessment of the antiproliferative and antigenotoxic activity and phytochemical screening of aqueous extracts of Sambucus australis Cham. & Schltdl. (ADOXACEAE).

PubMed

Tedesco, Marília; Kuhn, Andrielle W; Frescura, Viviane Dal-Souto; Boligon, Aline A; Athayde, Margareth L; Tedesco, Solange B; Silva, Antonio C F DA

2017-01-01

The purpose of this study was to evaluate the antiproliferative and antigenotoxic activity of Sambucus australis Cham. & Schltdl. aqueous extracts on the cell cycle of Allium cepa L. as well as determine the phenolic compounds in such extracts. S. australis inflorescences and leaves of two accessions were used for aqueous extract preparation at concentrations: 0.003 g/ml and 0.012 g/ml. A. cepa bulbs were rooted in distilled water and, subsequently, placed in treatments for 24 hours. Rootlets were collected and fixed in modified Carnoy's solution for 24 hours and kept. The squash technique was performed for slide preparation. Root tips were smashed and stained with 2% acetic orcein, and a total of 4000 cells per treatment were analyzed. The phenolic compounds were determined using high-performance liquid chromatography and data was analyzed using the Scott-Knott test. The results show that S. australis aqueous extracts have antiproliferative potential. Besides, the extracts prepared from S. australis leaves of both accessions at a concentration of 0.012 g/ml have shown antigenotoxic activity. The phytochemical analysis allowed us to determine the presence of flavonoids and phenolic acids, of which kaempferol and chrologenic acid were the most predominant compounds in the extracts from the inflorescences and leaves, respectively.

Mutagenicity and cytotoxicity of liquid waste, press water and pond water, produced in the cassava flour industry, and of antitoxic sodium thiosulfate.

PubMed

Viana, Lilian Ávila; Düsman, Elisângela; Vicentini, Veronica Elisa Pimenta

2014-02-01

Cassava (Manihot esculenta Crantz), a plant used as food and an ingredient in industry, contains cyanogenic glycosides. The cassava root contains wastewater, popularly known as manipueira, which is a toxic substance. Its ingestion by animals causes poisoning although they react positively to treatment with sodium thiosulfate. The present research evaluates the cytotoxicity and the mutagenicity of liquid waste produced in the process of industrialization of the bitter cassava, olho-junto variety. The liquid wastes are characterized as press water, which is obtained when the cassava roots are pressed; pond water, which is press water stored in impounded ponds; and a solution of sodium thiosulfate, pure and with other waste. The system tests comprised root meristematic cells of Allium cepa L. and bone marrow cells of Rattus norvegicus. Treatment with saline solution was cytotoxic for Allium cepa L. and significantly reduced cell division rate. Although no treatment was cytotoxic in any of the tests with rats, the thiosulfate solution was clastogenic for the chromosomal aberrations test. Since it is harmful to the genetic material submitted within the conditions of current research, sodium thiosulfate should only be used in emergency conditions in which the benefits exceed the risks. © 2013 Society of Chemical Industry.

Phyto-mediated metallic nano-architectures via Melissa officinalis L.: synthesis, characterization and biological properties.

PubMed

Fierascu, Irina; Georgiev, Milen I; Ortan, Alina; Fierascu, Radu Claudiu; Avramescu, Sorin Marius; Ionescu, Daniela; Sutan, Anca; Brinzan, Alexandru; Ditu, Lia Mara

2017-09-29

The development of methods for obtaining new materials with antimicrobial properties, based on green chemistry principles has been a target of research over the past few years. The present paper describes the phyto-mediated synthesis of metallic nano-architectures (gold and silver) via an ethanolic extract of Melissa officinalis L. (obtained by accelerated solvent extraction). Different analytic methods were applied for the evaluation of the extract composition, as well as for the characterization of the phyto-synthesized materials. The cytogenotoxicity of the synthesized materials was evaluated by Allium cepa assay, while the antimicrobial activity was examined by applying both qualitative and quantitative methods. The results demonstrate the synthesis of silver nanoparticles (average diameter 13 nm) and gold nanoparticles (diameter of ca. 10 nm); the bi-metallic nanoparticles proved to have a core-shell flower-like structure, composed of smaller particles (ca. 8 nm). The Ag nanoparticles were found not active on nuclear DNA damage. The Au nanoparticles appeared nucleoprotective, but were aggressive in generating clastogenic aberrations in A. cepa root meristematic cells. Results of the antimicrobial assays show that silver nanoparticles were active against most of the tested strains, as the lowest MIC value being obtained against B. cereus (approx. 0.0015 mM).

Effect of single alien chromosome from shallot (Allium cepa L. Aggregatum group) on carbohydrate production in leaf blade of bunching onion (A. fistulosum L.).

PubMed

Hang, Tran Thi Minh; Shigyo, Masayoshi; Yaguchi, Shigenori; Yamauchi, Naoki; Tashiro, Yosuke

2004-12-01

We used a complete set of Allium fistulosum - shallot (A. cepa Aggregatum group) monosomic addition lines (FF+1A - FF+8A) to identify shallot chromosomes affecting the production of sugars. In the alien addition lines grown over two years in an experimental field at Yamaguchi University (34 degrees N, 131 degrees E), shallot chromosomes 2A and 8A altered sugar contents in leaf-bunching onion (A. fistulosum). Except for FF+2A, every monosomic addition accumulated non-reducing sugars in winter leaf blades. FF+8A caused an increase in the amounts of non-reducing sugars in the winter. FF+2A hardly produced non-reducing sugar throughout the two-year study. These results indicated that genes related to non-reducing sugar metabolism are located on the 2A and 8A chromosomes. The results of regression analyses using 2002 data on A. fistulosum and the monosomic addition set revealed a correlation (r = 0.63 +/- 0.07; mean +/- SE., n = 9) between reducing sugar and monosaccharide (Glc+Fru) contents but no correlation between non-reducing sugar and sucrose contents. This result indicates the existence of other polysaccharides (e.g., scorodose) as non-reducing sugars in the leaf blade.

Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.)

PubMed Central

Kim, Seungill; Kim, Myung-Shin; Kim, Yong-Min; Yeom, Seon-In; Cheong, Kyeongchae; Kim, Ki-Tae; Jeon, Jongbum; Kim, Sunggil; Kim, Do-Sun; Sohn, Seong-Han; Lee, Yong-Hwan; Choi, Doil

2015-01-01

The onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops in the world. Although a considerable amount of onion transcriptome data has been deposited into public databases, the sequences of the protein-coding genes are not accurate enough to be used, owing to non-coding sequences intermixed with the coding sequences. We generated a high-quality, annotated onion transcriptome from de novo sequence assembly and intensive structural annotation using the integrated structural gene annotation pipeline (ISGAP), which identified 54,165 protein-coding genes among 165,179 assembled transcripts totalling 203.0 Mb by eliminating the intron sequences. ISGAP performed reliable annotation, recognizing accurate gene structures based on reference proteins, and ab initio gene models of the assembled transcripts. Integrative functional annotation and gene-based SNP analysis revealed a whole biological repertoire of genes and transcriptomic variation in the onion. The method developed in this study provides a powerful tool for the construction of reference gene sets for organisms based solely on de novo transcriptome data. Furthermore, the reference genes and their variation described here for the onion represent essential tools for molecular breeding and gene cloning in Allium spp. PMID:25362073

Amelioration of oxidative stress by dietary fenugreek (Trigonella foenum-graecum L.) seeds is potentiated by onion (Allium cepa L.) in streptozotocin-induced diabetic rats.

PubMed

Pradeep, Seetur R; Srinivasan, Krishnapura

2017-08-01

Oxidative stress has a crucial role in the progression of diabetes and its complications. Soluble fibre-rich fenugreek seeds (Trigonella foenum-graecum L.) are understood to have a beneficial effect in the management of diabetes. Previously, we have shown that the amelioration of diabetic hyperglycemia and related metabolic abnormalities was potentiated by onion (Allium cepa L.) in experimental rats. The present study evaluated the additive beneficial effect of dietary fenugreek seeds (10%) and onion (3%) on oxidative stress in diabetic rats. These dietary interventions lowered oxidative stress, the combination producing a higher beneficial effect (p < 0.05), although not additive. Dietary fenugreek, onion, or fenugreek+onion countered hypercholesterolemia (p < 0.05), especially from low-density lipoprotein-associated fraction by 43%, 35%, and 54%, respectively. Elevated concentrations of cholesterol and triglycerides in the liver and heart under diabetic conditions were significantly counteracted by these dietary interventions, with the additive combination resulting in greater effect. These findings were also corroborated by restoration of histopathological abnormalities of heart and liver tissues along with lowered heart and liver weights. It is inferred that an alleviation of oxidative stress contributes further to the antidiabetic influence and this nutraceutical potential of fenugreek seeds and onion was higher when consumed together.

Genotoxicity reduction in bagasse waste of sugar industry by earthworm technology.

PubMed

Bhat, Sartaj Ahmad; Singh, Jaswinder; Vig, Adarsh Pal

2016-01-01

The aim of the present study was to assess the genotoxicity reduction in post vermicompost feed mixtures of bagasse (B) waste using earthworm Eisenia fetida. The genotoxicity of bagasse waste was determined by using Allium cepa root chromosomal aberration assay. Bagasse was amended with cattle dung in different proportions [0:100 (B0) 25:75 (B25), 50:50 (B50), 75:25 (B75) and 100:0 (B100)] on dry weight basis. Genotoxic effects of initial and post vermicompost bagasse extracts were analysed on the root tips cells of Allium cepa. Root length and mitotic index (MI) was found to be increased in post vermicompost extracts when compared to initial bagasse waste. The maximum percent increase of root length was observed in the B50 bagasse extract (96.60 %) and the maximum MI was observed in B100 mixture (14.20 ± 0.60) 6 h treatment which was similar to the control. Genotoxicity analysis of post vermicompost extracts of bagasse revealed a 21-44 % decline in the aberration frequencies and the maximum reduction was found in B75 extract (44.50 %). The increase in root length and mitotic index, as well as decrease in chromosomal aberrations indicates that E. fetida has the ability to reduce the genotoxicity of the bagasse waste.

Comparative effect of propolis of honey bee and some herbal extracts on Candida albicans.

PubMed

Gavanji, Shahin; Larki, Behrouz

2017-03-01

To determine the effect of propolis on Candida albicans and to compare it with the effects of some other herbal extracts and antibiotics on this pathogenic fungi. The extracts of propolis, Thymus vulgaris, Caryophillium aromaticus, Echinophora platyloba, Allium cepa and Cinnamomum zeylanicum were prepared and the antifungi effects of the extracts were examined on Candida albicans ATCC10231 using disc-diffusion assay and micro-broth dilution. The minimum fungicidal concentration (MFC) and minimum inhibitory concentrations (MIC) as well as inhibition zone were evaluated and the anti fungi effects of herbal extracts were compared with amphotricin B and nystatin at the times of 24, 48 and 72 h. Data analysis was performed using t test. Obtained results showed that propolis extract with MIC 90 and MFC equal to 39 and 65 μg/mL, respectively, possess the highest antifungal activity when compared with other studied extracts. The extracts of Allium cepa and Thymus vulgaris, with MFC of 169 and 137 μg/mL, respectively, showed the lowest effects on the fungi. Also nystatin and amphotricin B yielded better effects on the tested fungi compared with the effects of all studied extracts on Candida albicans. Propolis extract is effective in controlling Candida albicans. However, the issue requires further investigation on samples in animals and performing toxicological examinations.

Consensus on the Definition of Advanced Parkinson's Disease: A Neurologists-Based Delphi Study (CEPA Study).

PubMed

Luquin, Maria-Rosario; Kulisevsky, Jaime; Martinez-Martin, Pablo; Mir, Pablo; Tolosa, Eduardo S

2017-01-01

To date, no consensus exists on the key factors for diagnosing advanced Parkinson disease (APD). To obtain consensus on the definition of APD, we performed a prospective, multicenter, Spanish nationwide, 3-round Delphi study (CEPA study). An ad hoc questionnaire was designed with 33 questions concerning the relevance of several clinical features for APD diagnosis. In the first-round, 240 neurologists of the Spanish Movement Disorders Group participated in the study. The results obtained were incorporated into the questionnaire and both, results and questionnaire, were sent out to and fulfilled by 26 experts in Movement Disorders. Review of results from the second-round led to a classification of symptoms as indicative of "definitive," "probable," and "possible" APD. This classification was confirmed by 149 previous participating neurologists in a third-round, where 92% completely or very much agreed with the classification. Definitive symptoms of APD included disability requiring help for the activities of daily living, presence of motor fluctuations with limitations to perform basic activities of daily living without help, severe dysphagia, recurrent falls, and dementia. These results will help neurologists to identify some key factors in APD diagnosis, thus allowing users to categorize the patients for a homogeneous recognition of this condition.

Distribution of Nidogen in the Murine Eye and Ocular Phenotype of the Nidogen-1 Knockout Mouse

PubMed Central

May, Christian Albrecht

2012-01-01

Distribution and lack of nidogen-1, part of numerous basement membranes, were studied in the mouse eye. For that purpose, eyes of C57BL/6 and nidogen-1 knockout mice were stained immunohistochemically for nidogen-1, and intraocular pressure measurements and light- and electron microscopy were used to study the nidogen-1 knockout animals. In normal mice, nidogen-1 was present in many basement membranes, but showed irregularities underneath the corneal epithelium, in Bruch's membrane and in the iris. Homozygous knockout of nidogen-1 in the mouse showed only mild pathological changes. In the anterior eye segment, small interruptions were noted in the nonpigmented ciliary epithelium without further consequences. In the posterior eye segment, interruptions of the inner limiting membrane led to small retinal ectopias and subsequent changes in the optic nerve. In summary, the knockout of nidogen-1 showed mild but significant morphological changes pointing to the importance of this protein which can in part, but not completely; be replaced by nidogen-2. PMID:24555126

Dcdc2 knockout mice display exacerbated developmental disruptions following knockdown of Dcx

PubMed Central

Wang, Yu; Yin, Xiuyin; Rosen, Glenn; Gabel, Lisa; Guadiana, Sarah M.; Sarkisian, Matthew R; Galaburda, Albert M.; LoTurco, Joseph J.

2011-01-01

The dyslexia-associated gene DCDC2 is a member of the DCX family of genes known to play roles in neurogenesis, neuronal migration and differentiation. Here we report the first phenotypic analysis of a Dcdc2 knockout mouse. Comparisons between Dcdc2 knockout mice and wild type littermates revealed no significant differences in neuronal migration, neocortical lamination, neuronal cilliogenesis or dendritic differentiation. Considering previous studies showing genetic interactions and potential functional redundancy among members of the DCX family, we tested whether decreasing Dcx expression by RNAi would differentially impair neurodevelopment in Dcdc2 knockouts and wild type mice. Consistent with this hypothesis, we found that deficits in neuronal migration, and dendritic growth caused by RNAi of Dcx were more severe in Dcdc2 knockouts than in wild type mice with the same transfection. These results indicate that Dcdc2 is not required for neurogenesis, neuronal migration or differentiation in mice, but may have partial functional redundancy with Dcx. PMID:21689730

Methionine Sulfoxide Reductase A Knockout Mice Show Progressive Hearing Loss and Sensitivity to Acoustic Trauma.

PubMed

Alqudah, Safa; Chertoff, Mark; Durham, Dianne; Moskovitz, Jackob; Staecker, Hinrich; Peppi, Marcello

2018-06-21

Methionine sulfoxide reductases (MsrA and MsrB) protect the biological activity of proteins from oxidative modifications to methionine residues and are important for protecting against the pathological effects of neurodegenerative diseases. In the current study, we characterized the auditory phenotype of the MsrA knockout mouse. Young MsrA knockout mice showed small high-frequency threshold elevations for auditory brainstem response and distortion product otoacoustic emission compared to those of wild-type mice, which progressively worsened in older MsrA knockout mice. MsrA knockout mice showed an increased sensitivity to noise at young and older ages, suggesting that MsrA is part of a mechanism that protects the cochlea from acoustic damage. MsrA mRNA in the cochlea was increased following acoustic stimulation. Finally, expression of mRNA MsrB1 was compromised at 6 months old, but not in younger MsrA knockout mice (compared to controls). The identification of MsrA in the cochlea as a protective mediator from both early onset hearing loss and acoustic trauma expands our understanding of the pathways that may induce protection from acoustic trauma and foster further studies on how to prevent the damaging effect of noise exposure through Msr-based therapy. © 2018 S. Karger AG, Basel.

Alterations of amino acids and monoamine metabolism in male Fmr1 knockout mice: a putative animal model of the human fragile X mental retardation syndrome.

PubMed

Gruss, M; Braun, K

2001-01-01

The Fragile X syndrome, a common form of mental retardation in humans, is caused by silencing the fragile X mental retardation (FMR1) gene leading to the absence of the encoded fragile X mental retardation protein 1 (FMRP). We describe morphological and behavioral abnormalities for both affected humans and Fmr1 knockout mice, a putative animal model for the human Fragile X syndrome. The aim of the present study was to identify possible neurochemical abnormalities in Fmr1 knockout mice, with particular focus on neurotransmission. Significant region-specific differences of basal neurotransmitter and metabolite levels were found between wildtype and Fmr1 knockout animals, predominantly in juveniles (post-natal days 28 to 31). Adults (postnatal days 209 to 221) showed only few abnormalities as compared with the wildtype. In juvenile knockout mice, aspartate and taurine were especially increased in cortical regions, striatum, hippocampus, cerebellum, and brainstem. In addition, juveniles showed an altered balance between excitatory and inhibitory amino acids in the caudal cortex, hippocampus, and brainstem. We detected very few differences in monoamine turnover in both age stages. The results presented here provide the first evidence that lack of FMRP expression in FMRP knockout mice is accompanied by age-dependent, region-specific alterations in neurotransmission.

Knockouts of high-ranking males have limited impact on baboon social networks.

PubMed

Franz, Mathias; Altmann, Jeanne; Alberts, Susan C

Social network structures can crucially impact complex social processes such as collective behaviour or the transmission of information and diseases. However, currently it is poorly understood how social networks change over time. Previous studies on primates suggest that `knockouts' (due to death or dispersal) of high-ranking individuals might be important drivers for structural changes in animal social networks. Here we test this hypothesis using long-term data on a natural population of baboons, examining the effects of 29 natural knockouts of alpha or beta males on adult female social networks. We investigated whether and how knockouts affected (1) changes in grooming and association rates among adult females, and (2) changes in mean degree and global clustering coefficient in these networks. The only significant effect that we found was a decrease in mean degree in grooming networks in the first month after knockouts, but this decrease was rather small, and grooming networks rebounded to baseline levels by the second month after knockouts. Taken together our results indicate that the removal of high-ranking males has only limited or no lasting effects on social networks of adult female baboons. This finding calls into question the hypothesis that the removal of high-ranking individuals has a destabilizing effect on social network structures in social animals.

A Review of Gene Knockout Strategies for Microbial Cells.

PubMed

Tang, Phooi Wah; Chua, Pooi San; Chong, Shiue Kee; Mohamad, Mohd Saberi; Choon, Yee Wen; Deris, Safaai; Omatu, Sigeru; Corchado, Juan Manuel; Chan, Weng Howe; Rahim, Raha Abdul

2015-01-01

Predicting the effects of genetic modification is difficult due to the complexity of metabolic net- works. Various gene knockout strategies have been utilised to deactivate specific genes in order to determine the effects of these genes on the function of microbes. Deactivation of genes can lead to deletion of certain proteins and functions. Through these strategies, the associated function of a deleted gene can be identified from the metabolic networks. The main aim of this paper is to review the available techniques in gene knockout strategies for microbial cells. The review is done in terms of their methodology, recent applications in microbial cells. In addition, the advantages and disadvantages of the techniques are compared and discuss and the related patents are also listed as well. Traditionally, gene knockout is done through wet lab (in vivo) techniques, which were conducted through laboratory experiments. However, these techniques are costly and time consuming. Hence, various dry lab (in silico) techniques, where are conducted using computational approaches, have been developed to surmount these problem. The development of numerous techniques for gene knockout in microbial cells has brought many advancements in the study of gene functions. Based on the literatures, we found that the gene knockout strategies currently used are sensibly implemented with regard to their benefits.

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis

PubMed Central

Sun, Xingshen; Sui, Hongshu; Fisher, John T.; Yan, Ziying; Liu, Xiaoming; Cho, Hyung-Ju; Joo, Nam Soo; Zhang, Yulong; Zhou, Weihong; Yi, Yaling; Kinyon, Joann M.; Lei-Butters, Diana C.; Griffin, Michelle A.; Naumann, Paul; Luo, Meihui; Ascher, Jill; Wang, Kai; Frana, Timothy; Wine, Jeffrey J.; Meyerholz, David K.; Engelhardt, John F.

2010-01-01

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments. PMID:20739752

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis.

PubMed

Sun, Xingshen; Sui, Hongshu; Fisher, John T; Yan, Ziying; Liu, Xiaoming; Cho, Hyung-Ju; Joo, Nam Soo; Zhang, Yulong; Zhou, Weihong; Yi, Yaling; Kinyon, Joann M; Lei-Butters, Diana C; Griffin, Michelle A; Naumann, Paul; Luo, Meihui; Ascher, Jill; Wang, Kai; Frana, Timothy; Wine, Jeffrey J; Meyerholz, David K; Engelhardt, John F

2010-09-01

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

PubMed Central

Hart, Traver; Tong, Amy Hin Yan; Chan, Katie; Van Leeuwen, Jolanda; Seetharaman, Ashwin; Aregger, Michael; Chandrashekhar, Megha; Hustedt, Nicole; Seth, Sahil; Noonan, Avery; Habsid, Andrea; Sizova, Olga; Nedyalkova, Lyudmila; Climie, Ryan; Tworzyanski, Leanne; Lawson, Keith; Sartori, Maria Augusta; Alibeh, Sabriyeh; Tieu, David; Masud, Sanna; Mero, Patricia; Weiss, Alexander; Brown, Kevin R.; Usaj, Matej; Billmann, Maximilian; Rahman, Mahfuzur; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Durocher, Daniel; Moffat, Jason

2017-01-01

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines. PMID:28655737

Trpc2 Depletion Protects RBC from Oxidative Stress-Induced Hemolysis

PubMed Central

Hirschler-Laszkiewicz, Iwona; Zhang, Wenyi; Keefer, Kerry; Conrad, Kathleen; Tong, Qin; Chen, Shu-jen; Bronson, Sarah; Cheung, Joseph Y.; Miller, Barbara A.

2011-01-01

Transient receptor potential channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca2+]i) through TRPC2 and TRPC3. Because modulation of [Ca2+]i is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild type littermate controls. Although the erythropoietin induced increase in [Ca2+]i was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenyhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. While erythropoietin induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion. PMID:21924222

TRPV2 KNOCKOUT MICE ARE SUSCEPTIBLE TO PERINATAL LETHALITY BUT DISPLAY NORMAL THERMAL AND MECHANICAL NOCICEPTION

PubMed Central

Park, Una; Vastani, Nisha; Guan, Yun; Raja, Srinivasa N.; Koltzenburg, Martin; Caterina, Michael J.

2011-01-01

TRPV2 is a nonselective cation channel expressed prominently in medium- to large-diameter sensory neurons that can be activated by extreme heat (>52°C). These features suggest that TRPV2 might be a transducer of noxious heat in vivo. TRPV2 can also be activated by hypoosmolarity or cell stretch, suggesting potential roles in mechanotransduction. To address the physiological functions of TRPV2 in somatosensation, we generated TRPV2 knockout mice and examined their behavioral and electrophysiological responses to heat and mechanical stimuli. TRPV2 knockout mice showed reduced embryonic weight and perinatal viability. As adults, surviving knockout mice also exhibited a slightly reduced body weight. TRPV2 knockout mice showed normal behavioral responses to noxious heat over a broad range of temperatures and normal responses to punctate mechanical stimuli, both in the basal state and under hyperalgesic conditions such as peripheral inflammation and L5 spinal nerve ligation. Moreover, behavioral assays of TRPV1/TRPV2 double knockout mice or of TRPV2 knockout mice treated with resiniferatoxin to desensitize TRPV1-expressing afferents revealed no thermosensory consequences of TRPV2 absence. In line with behavioral findings, electrophysiological recordings from skin afferents showed that C-fiber responses to heat and C- and Aδ-fiber responses to noxious mechanical stimuli were unimpaired in the absence of TRPV2. The prevalence of thermosensitive Aδ-fibers was too low to permit comparison between genotypes. Thus, TRPV2 is important for perinatal viability but is not essential for heat or mechanical nociception or hypersensitivity in the adult mouse. PMID:21832173

Quality control method to measure predator evasion in wild and mass-reared Mediterranean fruit flies (Diptera: Tephritidae)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hendrichs, M.; Wornoayporn, V.; Hendrichs, J.

Sterile male insects, mass-reared and released as part of sterile insect technique (SIT) programs, must survive long enough in the field to mature sexually and compete effectively with wild males for wild females. An often reported problem in Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) SIT programs is that numbers of released sterile males decrease rapidly in the field for various reasons, including losses to different types of predators. This is a serious issue in view that most operational programs release sterile flies at an age when they are still immature. Previous field and field-cage tests have confirmed that fliesmore » of laboratory strains are less able to evade predators than wild flies. Such tests involve, however, considerable manipulation and observation of predators and are therefore not suitable for routine measurements of predator evasion. Here we describe a simple quality control method with aspirators to measure agility in medflies and show that this parameter is related to the capacity of flies to evade predators. Although further standardization of the test is necessary to allow more accurate inter-strain comparisons, results confirm the relevance of measuring predator evasion in mass-reared medfly strains. Besides being a measure of this sterile male quality parameter, the described method could be used for the systematic selection of strains with a higher capacity for predator evasion. (author) [Spanish] Insectos machos esteriles criados en forma masiva para ser liberados en programas que utilizan la tecnica del insecto esteril (TIE), tienen que tener la capacidad de sobrevivir en el campo el tiempo necesario para poder madurar sexualmente y competir efectivamente con los machos silvestres por hembras silvestres. Un problema frecuentemente reportado por dichos programas de la mosca del Mediterraneo, Ceratitis capitata (Wiedemann), es que el numero de machos esteriles de laboratorio liberados en el campo, decrecen rapidamente por varias razones, incluyendo perdidas debidas a diferentes tipos de depredadores. Estudios anteriores conducidos en el campo, y en jaulas de campo, han confirmado que las cepas de machos de laboratorio tienen menos capacidad de evadir depredadores que los machos silvestres. Estos estudios involucran, sin embargo, una considerable cantidad de manipulacion y observacion de depredadores, por lo que no son adecuados para ser usados como medidas rutinarias en los programas de cria masiva. Aqui describimos un metodo sencillo de control de calidad usando aspiradores para medir agilidad en la mosca del Mediterraneo y mostramos que este parametro esta relacionado a la capacidad de la moscas a evadir a depredadores. Aunque aun es necesario refinar la estandarizacion de este metodo para permitir la comparacion entre cepas, los resultados confirman la importancia de tener un metodo rutinario para medir la capacidad de evasion de depredadores en cepas de cria de laboratorio de la mosca del Mediterraneo. Ademas de medir este parametro de control de calidad de los machos esteriles, el metodo descrito podria tambien ser usado para la seleccion sistematica de cepas con una mayor capacidad de evasion de depredadores. (author)« less

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology

PubMed Central

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J.; Salomons, Gajja S.

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease. PMID:29053735

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

PubMed

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J; Salomons, Gajja S; Mercimek-Andrews, Saadet

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

Decreased APOE-containing HDL subfractions and cholesterol efflux capacity of serum in mice lacking Pcsk9

PubMed Central

2013-01-01

Background Studies in animals showed that PCSK9 is involved in HDL metabolism. We investigated the molecular mechanism by which PCSK9 regulates HDL cholesterol concentration and also whether Pcsk9 inactivation might affect cholesterol efflux capacity of serum and atherosclerotic fatty streak volume. Methods Mass spectrometry and western blot were used to analyze the level of apolipoprotein E (APOE) and A1 (APOA1). A mouse model overexpressing human LDLR was used to test the effect of high levels of liver LDLR on the concentration of HDL cholesterol and APOE-containing HDL subfractions. Pcsk9 knockout males lacking LDLR and APOE were used to test whether LDLR and APOE are necessary for PCSK9-mediated HDL cholesterol regulation. We also investigated the effects of Pcsk9 inactivation on cholesterol efflux capacity of serum using THP-1 and J774.A1 macrophage foam cells and atherosclerotic fatty streak volume in the aortic sinus of Pcsk9 knockout males fed an atherogenic diet. Results APOE and APOA1 were reduced in the same HDL subfractions of Pcsk9 knockout and human LDLR transgenic male mice. In Pcsk9/Ldlr double-knockout mice, HDL cholesterol concentration was lower than in Ldlr knockout mice and higher than in wild-type controls. In Pcsk9/Apoe double-knockout mice, HDL cholesterol concentration was similar to that of Apoe knockout males. In Pcsk9 knockout males, THP-1 macrophage cholesterol efflux capacity of serum was reduced and the fatty streak lesion volume was similar to wild-type controls. Conclusions In mice, LDLR and APOE are important factors for PCSK9-mediated HDL regulation. Our data suggest that, although LDLR plays a major role in PCSK9-mediated regulation of HDL cholesterol concentration, it is not the only mechanism and that, regardless of mechanism, APOE is essential. Pcsk9 inactivation decreases the HDL cholesterol concentration and cholesterol efflux capacity in serum, but does not increase atherosclerotic fatty streak volume. PMID:23883163

What We're Missing: A Descriptive Analysis of Part-Day Absenteeism in Secondary School. CEPA Working Paper No. 16-16

ERIC Educational Resources Information Center

Whitney, Camille R.; Liu, Jing

2016-01-01

For schools and teachers to help students develop knowledge and skills, students need to show up to class. Yet absenteeism is high, especially in high schools. This study uses a rich dataset tracking class attendance by day for over 50,000 middle and high school students from an urban district in Academic Years 2007-'08 through 2012-'13. Our…

[Upregulation of P2X3 receptors in dorsal root ganglion of TRPV1 knockout female mice].

PubMed

Fang, Xiao; Shi, Xiao-Han; Huang, Li-Bin; Rong, Wei-Fang; Ma, Bei

2014-08-25

The study was aimed to investigate the changes in mechanical pain threshold in the condition of chronic inflammatory pain after transient receptor potential vanilloid 1 (TRPV1) gene was knockout. Hind-paw intraplantar injection of complete freund's adjuvant (CFA, 20 μL) produced peripheral inflammation in wild-type and TRPV1 knockout female mice. The mechanical pain thresholds were measured during the 8 days after injection and pre-injection by using Von-Frey hair. Nine days after injection, mice were killed and the differences of expression of c-Fos and P2X3 receptor in the dorsal root ganglia (DRG) and spinal cord dorsal horn were examined by Western blotting between the two groups. Compared with that in wild-type mice, the mechanical pain threshold was increased significantly in TRPV1 knockout mice (P < 0.05); 3 days after CFA injection, the baseline mechanical pain threshold in the TRPV1 knockout mice group was significantly higher than that in the wild-type mice group (P < 0.05); The result of Western blotting showed that the expression of c-Fos protein both in DRG and spinal cord dorsal horn of TRPV1 knockout mice group was decreased significantly compared with that in wild-type mice group (P < 0.01, P < 0.05), while the expression of P2X3 receptor in DRG of TRPV1 knockout mice group was increased significantly compared with that in wild-type mice group (P < 0.05). Our findings indicate that TRPV1 may influence the peripheral mechanical pain threshold by mediating the expression of c-Fos protein both in DRG and spinal cord dorsal horn and changing the expression of P2X3 receptor in DRG.

Glutaminyl Cyclase Knock-out Mice Exhibit Slight Hypothyroidism but No Hypogonadism

PubMed Central

Schilling, Stephan; Kohlmann, Stephanie; Bäuscher, Christoph; Sedlmeier, Reinhard; Koch, Birgit; Eichentopf, Rico; Becker, Andreas; Cynis, Holger; Hoffmann, Torsten; Berg, Sabine; Freyse, Ernst-Joachim; von Hörsten, Stephan; Rossner, Steffen; Graubner, Sigrid; Demuth, Hans-Ulrich

2011-01-01

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development. PMID:21330373

Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive.

PubMed

Williams, Michael R; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T; Luikart, Bryan W

2015-01-21

Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either "birthdate" or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons. Copyright © 2015 the authors 0270-6474/15/350943-17$15.00/0.

The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

PubMed

Miyamoto, Kei; Suzuki, Ken-Ichi T; Suzuki, Miyuki; Sakane, Yuto; Sakuma, Tetsushi; Herberg, Sarah; Simeone, Angela; Simpson, David; Jullien, Jerome; Yamamoto, Takashi; Gurdon, J B

2015-01-01

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

Behavioral characterization of mice deficient in the phosphodiesterase-10A (PDE10A) enzyme on a C57/Bl6N congenic background.

PubMed

Siuciak, Judith A; McCarthy, Sheryl A; Chapin, Douglas S; Martin, Ashley N; Harms, John F; Schmidt, Christopher J

2008-02-01

The phenotype of genetically modified animals is strongly influenced by both the genetic background of the animal as well as environmental factors. We have previously reported the behavioral and neurochemical characterization of PDE10A knockout mice maintained on a DBA1LacJ (PDE10A(DBA)) genetic background. The aim of the present studies was to assess the behavioral and neurochemical phenotype of PDE10A knockout mice on an alternative congenic C57BL/6N (PDE10A(C57)) genetic background. Consistent with our previous results, PDE10A(C57) knockout mice showed a decrease in exploratory locomotor activity and a delay in the acquisition of conditioned avoidance responding. Also consistent with previous studies, the elimination of PDE10A did not alter basal levels of striatal cGMP or cAMP or affect behavior in several other well-characterized behavioral assays. PDE10A(C57) knockout mice showed a blunted response to MK-801, although to a lesser degree than previously observed in the PDE10A(DBA) knockout mice, and no differences were observed following a PCP challenge. PDE10A(C57) knockout mice showed a significant change in striatal dopamine turnover, which was accompanied by an enhanced locomotor response to AMPH, These studies demonstrate that while many of the behavioral effects of the PDE10A gene deletion appear to be independent of genetic background, the impact of the deletion on behavior can vary in magnitude. Furthermore, the effects on the dopaminergic system appear to be background-dependent, with significant effects observed only in knockout mice on the C57BL6N genetic background.

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

PubMed Central

Huang, Yi; Askew, Emily B.; Knudson, Cheryl B.; Knudson, Warren

2016-01-01

Hyaluronan (HA) plays an essential role in cartilage where it functions to retain aggrecan. Previous studies have suggested that aggrecan is anchored indirectly to the plasma membrane of chondrocytes via its binding to cell-associated HA. However, reagents used to test these observations such as hyaluronidase and HA oligosaccharides are short term and may have side activities that complicate interpretation. Using the CRISPR/Cas9 gene editing approach, a model system was developed by generating HA-deficient chondrocyte cell lines. HA synthase-2 (Has2)-specific single guide RNA was introduced into two different variant lines of rat chondrosarcoma chondrocytes; knockout clones were isolated and characterized. Two other members of the HA synthase gene family were expressed at very low relative copy number but showed no compensatory response in the Has2 knockouts. Wild type chondrocytes of both variants exhibited large pericellular matrices or coats extending from the plasma membrane. Addition of purified aggrecan monomer expanded the size of these coats as the proteoglycan became retained within the pericellular matrix. Has2 knockout chondrocytes lost all capacity to assemble a particle-excluding pericellular matrix and more importantly, no matrices formed around the knockout cells following the addition of purified aggrecan. When grown as pellet cultures so as to generate a bioengineered neocartilage tissue, the Has2 knockout chondrocytes assumed a tightly-compacted morphology as compared to the wild type cells. When knockout chondrocytes were transduced with Adeno-ZsGreen1-mycHas2, the cell-associated pericellular matrices were restored including the capacity to bind and incorporate additional exogenous aggrecan into the matrix. These results suggest that HA is essential for aggrecan retention and maintaining cell separation during tissue formation. PMID:27094859

Vasoactive intestinal peptide-null mice demonstrate enhanced sweet taste preference, dysglycemia, and reduced taste bud leptin receptor expression.

PubMed

Martin, Bronwen; Shin, Yu-Kyong; White, Caitlin M; Ji, Sunggoan; Kim, Wook; Carlson, Olga D; Napora, Joshua K; Chadwick, Wayne; Chapter, Megan; Waschek, James A; Mattson, Mark P; Maudsley, Stuart; Egan, Josephine M

2010-05-01

It is becoming apparent that there is a strong link between taste perception and energy homeostasis. Recent evidence implicates gut-related hormones in taste perception, including glucagon-like peptide 1 and vasoactive intestinal peptide (VIP). We used VIP knockout mice to investigate VIP's specific role in taste perception and connection to energy regulation. Body weight, food intake, and plasma levels of multiple energy-regulating hormones were measured and pancreatic morphology was determined. In addition, the immunocytochemical profile of taste cells and gustatory behavior were examined in wild-type and VIP knockout mice. VIP knockout mice demonstrate elevated plasma glucose, insulin, and leptin levels, with no islet beta-cell number/topography alteration. VIP and its receptors (VPAC1, VPAC2) were identified in type II taste cells of the taste bud, and VIP knockout mice exhibit enhanced taste preference to sweet tastants. VIP knockout mouse taste cells show a significant decrease in leptin receptor expression and elevated expression of glucagon-like peptide 1, which may explain sweet taste preference of VIP knockout mice. This study suggests that the tongue can play a direct role in modulating energy intake to correct peripheral glycemic imbalances. In this way, we could view the tongue as a sensory mechanism that is bidirectionally regulated and thus forms a bridge between available foodstuffs and the intricate hormonal balance in the animal itself.

A mental retardation gene, motopsin/neurotrypsin/prss12, modulates hippocampal function and social interaction

PubMed Central

Mitsui, Shinichi; Osako, Yoji; Yokoi, Fumiaki; Dang, Mai T.; Yuri, Kazunari; Li, Yuqing; Yamaguchi, Nozomi

2010-01-01

Motopsin is a mosaic serine protease secreted from neuronal cells in various brain regions including the hippocampus. The loss of motopsin function causes nonsyndromic mental retardation in humans and impairs long-term memory formation in Drosophila. To understand motopsin’s function in the mammalian brain, motopsin knockout mice were generated. Motopsin knockout mice did not have significant deficit in memory formation, as was tested using in the Morris water maze, passive avoidance, and Y-maze tests. A social recognition test showed that the motopsin knockout mice had the ability to recognize two stimulator mice, suggesting normal social memory. In a social novelty test, motopsin knockout mice spent a longer time investigating a familiar mouse than wild-type mice did. In a resident-intruder test, motopsin knockout mice showed prolonged social interaction compared to wild-type mice. Consistent with the behavioral deficit, spine density was significantly decreased on apical dendrites, but not on basal dendrites, of hippocampal pyramidal neurons of motopsin knockout mice. In contrast, pyramidal neurons at the cingulate cortex showed normal spine density. Spatial learning and social interaction induced the phosphorylation of cAMP responsive element binding protein (CREB) in hippocampal neurons of wild-type mice, whereas the phosphorylation of CREB was markedly decreased in mutant mouse brains. Our results indicate that an extracellular protease, motopsin, preferentially affects social behaviors, and modulates the functions of hippocampal neurons. PMID:20092579

Studies of UCP2 transgenic and knockout mice reveal that liver UCP2 is not essential for the antiobesity effects of fish oil.

PubMed

Tsuboyama-Kasaoka, Nobuyo; Sano, Kayo; Shozawa, Chikako; Osaka, Toshimasa; Ezaki, Osamu

2008-03-01

Uncoupling protein 2 (UCP2) is a possible target molecule for energy dissipation. Many dietary fats, including safflower oil and lard, induce obesity in C57BL/6 mice, whereas fish oil does not. Fish oil increases UCP2 expression in hepatocytes and may enhance UCP2 activity by activating the UCP2 molecule or altering the lipid bilayer environment. To examine the role of liver UCP2 in obesity, we created transgenic mice that overexpressed human UCP2 in hepatocytes and examined whether UCP2 transgenic mice showed less obesity when fed a high-fat diet (safflower oil or lard). In addition, we examined whether fish oil had antiobesity effects in UCP2 knockout mice. UCP2 transgenic and wild-type mice fed a high-fat diet (safflower oil or lard) developed obesity to a similar degree. UCP2 knockout and wild-type mice fed fish oil had lower rates of obesity than mice fed safflower oil. Remarkably, safflower oil did not induce obesity in female UCP2 knockout mice, an unexpected phenotype for which we presently have no explanation. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. These data indicate that liver UCP2 is not essential for fish oil-induced decreases in body fat.

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.

PubMed

Merenda, Alessandra; Andersson-Rolf, Amanda; Mustata, Roxana C; Li, Taibo; Kim, Hyunki; Koo, Bon-Kyoung

2017-07-12

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

Abrogation of both short and long forms of latent transforming growth factor-β binding protein-1 causes defective cardiovascular development and is perinatally lethal.

PubMed

Horiguchi, Masahito; Todorovic, Vesna; Hadjiolova, Krassimira; Weiskirchen, Ralf; Rifkin, Daniel B

2015-04-01

Latent transforming growth factor-β binding protein-1 (LTBP-1) is an extracellular protein that is structurally similar to fibrillin and has an important role in controlling transforming growth factor-β (TGF-β) signaling by storing the cytokine in the extracellular matrix and by being involved in the conversion of the latent growth factor to its active form. LTBP-1 is found as both short (LTBP-1S) and long (LTBP-1L) forms, which are derived through the use of separate promoters. There is controversy regarding the importance of LTBP-1L, as Ltbp1L knockout mice showed multiple cardiovascular defects but the complete null mice did not. Here, we describe a third line of Ltbp1 knockout mice generated utilizing a conditional knockout strategy that ablated expression of both L and S forms of LTBP-1. These mice show severe developmental cardiovascular abnormalities and die perinatally; thus these animals display a phenotype similar to previously reported Ltbp1L knockout mice. We reinvestigated the other "complete" knockout line and found that these mice express a splice variant of LTBP-1L and, therefore, are not complete Ltbp1 knockouts. Our results clarify the phenotypes of Ltbp1 null mice and re-emphasize the importance of LTBP-1 in vivo. Copyright © 2015. Published by Elsevier B.V.

Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice.

PubMed

Leon, L R; Kozak, W; Peschon, J; Kluger, M J

1997-02-01

We examined the effects of injections of systemic [lipopolysaccharide (LPS), 2.5 mg/kg or 50 pg/kg ip] or local (turpentine, 100 microl sc) inflammatory stimuli on fever, motor activity, body weight, and food intake in tumor necrosis factor (TNF) double receptor (TNFR)-knockout mice. A high dose of LPS resulted in exacerbated fevers in TNFR-knockout mice compared with wild-type mice for the early phase of fever (3-15 h); the late phase of fever (16-24 h) and fevers to a low dose of LPS were similar in both groups. Motor activity, body weight, and food intake were similarly reduced in both groups of mice after LPS administration. In response to turpentine, TNFR-knockout and wild-type mice developed virtually identical responses to all variables monitored. These results suggest that 1) TNF modulates fevers to LPS dose dependently, 2) TNF does not modulate fevers to a subcutaneous injection of turpentine, and 3) knockout mice may develop cytokine redundancy in the regulation of the acute phase response to intraperitoneally injected LPS or subcutaneously injected turpentine.

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype

PubMed Central

BOOT-HANDFORD, R. P.; MICHAELIDIS, T. M.; HILLARBY, M. C.; ZAMBELLI, A.; DENTON, J.; HOYLAND, J. A.; FREEMONT, A. J.; GRANT, M. E.; WALLIS, G. A.

1998-01-01

Histological examination of long bones from 1-day-old bcl-2 knockout and age-matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35-day-old bcl-2 knockout mice that are growth retarded or ‘dwarfed’, the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age-matched wild-type controls. The most marked histological effects of bcl-2 ablation were on osteoblasts and bone. 35-day-old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl-2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo-woven appearance. The results suggest an important role for Bcl-2 in controlling osteoblast phenotype and bone deposition in vivo. PMID:10193316

CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.

PubMed

Chu, H W; Rios, C; Huang, C; Wesolowska-Andersen, A; Burchard, E G; O'Connor, B P; Fingerlin, T E; Nichols, D; Reynolds, S D; Seibold, M A

2015-10-01

Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.

MONOAMINE OXIDASE: From Genes to Behavior

PubMed Central

Shih, J. C.; Chen, K.; Ridd, M. J.

2010-01-01

Cloning of MAO (monoamine oxidase) A and B has demonstrated unequivocally that these enzymes are made up of different polypeptides, and our understanding of MAO structure, regulation, and function has been significantly advanced by studies using their cDNA. MAO A and B genes are located on the X-chromosome (Xp11.23) and comprise 15 exons with identical intron-exon organization, which suggests that they are derived from the same ancestral gene. MAO A and B knockout mice exhibit distinct differences in neurotransmitter metabolism and behavior. MAO A knock-out mice have elevated brain levels of serotonin, norephinephrine, and dopamine and manifest aggressive behavior similar to human males with a deletion of MAO A. In contrast, MAO B knock-out mice do not exhibit aggression and only levels of phenylethylamine are increased. Mice lacking MAO B are resistant to the Parkinsongenic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine. Both MAO A and B knock-out mice show increased reactivity to stress. These knock-out mice are valuable models for investigating the role of monoamines in psychoses and neurodegenerative and stress-related disorders. PMID:10202537

Population of positive-parity states in {sup 53}Sc through one-proton knockout.

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDaniel, S.; Gade, A.; Janssens, R. V. F.

2010-02-01

The one-proton knockout reaction {sup 9}Be({sup 54}Ti,{sup 53}Sc+{gamma})X at 72 MeV/nucleon has been measured. The location of the first 3/2{sup -} state at 2110(3) keV was confirmed, and new {gamma}-ray transitions were observed at 1111(2), 1273(2), 1539(4), and 2495(5) keV. Large spectroscopic strength to excited states in {sup 53}Sc was found and attributed to the knockout of sd-shell protons.

Population of positive-parity states in {sup 53}Sc through one-proton knockout

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDaniel, S.; Gade, A.; Brown, B. A.

2010-02-15

The one-proton knockout reaction {sup 9}Be({sup 54}Ti,{sup 53}Sc+{gamma})X at 72 MeV/nucleon has been measured. The location of the first 3/2{sup -} state at 2110(3) keV was confirmed, and new {gamma}-ray transitions were observed at 1111(2), 1273(2), 1539(4), and 2495(5) keV. Large spectroscopic strength to excited states in {sup 53}Sc was found and attributed to the knockout of sd-shell protons.

Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

PubMed Central

Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G.; Ronimus, Ron S.; McGee, Sean L.; Wilson, William R.

2013-01-01

Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines. PMID:23799003

Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor) in experimental autoimmune encephalomyelitis models of multiple sclerosis.

PubMed

Sisay, Sofia; Pryce, Gareth; Jackson, Samuel J; Tanner, Carolyn; Ross, Ruth A; Michael, Gregory J; Selwood, David L; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PubMed Central

Jackson, Samuel J.; Tanner, Carolyn; Ross, Ruth A.; Michael, Gregory J.; Selwood, David L.; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 tm1Zim) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility. PMID:24130809

Identification of bacteria pathogenic to or associated with onion (Allium cepa) based on sequence differences in a portion of the conserved gyrase B gene.

PubMed

Bonasera, Jean M; Asselin, Jo Ann E; Beer, Steven V

2014-08-01

We have developed a method for the identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae, based on sequence variation in a portion of the gyrB gene. Thus, we identified, in most cases to species level, over 1000 isolates from onion bulbs and leaves and soil in which onions were grown. Copyright © 2014 Elsevier B.V. All rights reserved.

Quercetin and isorhamnetin glycosides in onion (Allium cepa L.): varietal comparison, physical distribution, coproduct evaluation, and long-term storage stability.

PubMed

Lee, Jihyun; Mitchell, Alyson E

2011-02-09

During onion processing, the outer dried protective layer (outer paper layer) and first two fleshy leaf layers are removed. This coproduct material is a potential commercial source of flavonoids especially quercetin. In the following study, the flavonoid composition was determined in coproduct materials and the press cake (material generated after juice extraction) in several commercially important onion varieties grown in California. Flavonoids were characterized and quantified using LC-(ESI)MS/MS and HPLC. The long-term stability of quercetin glycosides was assessed in dried coproduct materials stored at 4 and 22 °C over a 12 month period. In all varieties, the predominant forms of quercetin were the quercetin 3,4'-O-glucoside and 4'-O-glucoside. The first layer had significantly higher levels of flavonoids than the outer paper, second, and inner flesh layers on a DW basis (p < 0.05). Allium cepa "Milestone" contained the highest levels (p < 0.05) of flavonoids (1703 mg/100 g on a dry weight basis (DW). Onion press cake had significantly higher levels of total quercetin as compared with fresh onions (p < 0.05). The levels of 4'-O-glucoside significantly decreased during the first month of storage and remained stable for 12 months of storage at either 4 or 22 °C (p < 0.05).

Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.

PubMed Central

Serrano, A.; Cordoba, F.; Gonzalez-Reyes, J. A.; Navas, P.; Villalba, J. M.

1994-01-01

Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate. PMID:12232306

Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.).

PubMed

Kim, Seungill; Kim, Myung-Shin; Kim, Yong-Min; Yeom, Seon-In; Cheong, Kyeongchae; Kim, Ki-Tae; Jeon, Jongbum; Kim, Sunggil; Kim, Do-Sun; Sohn, Seong-Han; Lee, Yong-Hwan; Choi, Doil

2015-02-01

The onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops in the world. Although a considerable amount of onion transcriptome data has been deposited into public databases, the sequences of the protein-coding genes are not accurate enough to be used, owing to non-coding sequences intermixed with the coding sequences. We generated a high-quality, annotated onion transcriptome from de novo sequence assembly and intensive structural annotation using the integrated structural gene annotation pipeline (ISGAP), which identified 54,165 protein-coding genes among 165,179 assembled transcripts totalling 203.0 Mb by eliminating the intron sequences. ISGAP performed reliable annotation, recognizing accurate gene structures based on reference proteins, and ab initio gene models of the assembled transcripts. Integrative functional annotation and gene-based SNP analysis revealed a whole biological repertoire of genes and transcriptomic variation in the onion. The method developed in this study provides a powerful tool for the construction of reference gene sets for organisms based solely on de novo transcriptome data. Furthermore, the reference genes and their variation described here for the onion represent essential tools for molecular breeding and gene cloning in Allium spp. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

De Novo Transcriptome Analysis of Allium cepa L. (Onion) Bulb to Identify Allergens and Epitopes.

PubMed

Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman

2015-01-01

Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37-100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins.

Amelioration of hyperglycemia and associated metabolic abnormalities by a combination of fenugreek (Trigonella foenum-graecum) seeds and onion (Allium cepa) in experimental diabetes.

PubMed

Pradeep, Seetur R; Srinivasan, Krishnapura

2017-09-26

Fenugreek (Trigonella foenum-graecum) seeds and onion (Allium cepa) are independently known to have antidiabetic effects through different mechanisms. The beeneficial influence of a combination of dietary fenugreek seeds and onion on hyperglycemia and its associated metabolic abnormalities were evaluated in streptozotocin-induced diabetic rats. Diabetes was experimentally induced with streptozotocin and diabetic rats were fed with 10% fenugreek or 3% onion or their combination for 6 weeks. These dietary interventions significantly countered hyperglycemia, partially improved peripheral insulin resistance and impaired insulin secretion, reduced β-cell mass and markedly reversed the abnormalities in plasma albumin, urea, creatinine, glycated hemoglobin and advanced glycation end products in diabetic rats. These beneficial effects were highest in the fenugreek+onion group. Diabetic rats with these dietary interventions excreted lesser glucose, albumin, urea and creatinine, which were accompanied by improved body weights compared with the diabetic controls. These dietary interventions produced ameliorative effects on pancreatic pathology as reflected by near-normal islet cells, restored glycogen and collagen fiber deposition in diabetic rats. This study documented the hypoglycemic and insulinotropic effects of dietary fenugreek and onion, which were associated with countering of metabolic abnormalities and pancreatic pathology. It may be strategic to derive maximum nutraceutical antidiabetic benefits from these functional food ingredients by consuming them together.

Toxicity and genotoxicity of hospital laundry wastewaters treated with photocatalytic ozonation.

PubMed

Kern, Deivid I; Schwaickhardt, Rômulo de O; Mohr, Geane; Lobo, Eduardo A; Kist, Lourdes T; Machado, Ênio L

2013-01-15

The aim of the present study was to assess the efficiency of advanced oxidative processes based on photocatalytic ozonation (O(3), UV, UV/O(3), UV/O(3)/Fe(2+) 50 mg L(-1) and 150 mg L(-1)) in the treatment of hospital laundry wastewaters. The analysis of the investigated wastewater revealed high chemical oxygen demand (COD - 3343.8 mg L(-1)), biochemical oxygen demand (BOD(5) - 1906.4 mg L(-1)), total Kjeldahl nitrogen (TKN - 79.8 mg L(-1)) and Daphnia magna toxicity (EC50=1.73). Genotoxic effects were also detected for Allium cepa. Reductions of some parameters occurred after photocatalytic ozonation. The UV/O(3)/Fe(2+) 150 mg L(-1) method was more efficient in reducing COD (59.1%), BOD(5) (50.3%) and TKN (86.8%). There was significant reduction (p<0.05) in D. magna toxicity, O(3) (EC50=47.3%), UV (EC50=50.6%) and UV/O(3)/Fe(2+) 150 mg L(-1) (EC50=45.4%) processes. Normalization of the mitotic index and reduction of micronucleated cells were observed in A. cepa after the treatments. Results demonstrate that these methods were efficient in the degradation of hospital laundry wastewaters, representing a thriving alternative for the removal of pollutants that cause toxicity and genotoxicity. Copyright © 2012 Elsevier B.V. All rights reserved.

The phosphatase JKAP/DUSP22 inhibits T-cell receptor signalling and autoimmunity by inactivating Lck.

PubMed

Li, Ju-Pi; Yang, Chia-Yu; Chuang, Huai-Chia; Lan, Joung-Liang; Chen, Der-Yuan; Chen, Yi-Ming; Wang, Xiaohong; Chen, Alice J; Belmont, John W; Tan, Tse-Hua

2014-04-09

JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knockout T cells display enhanced cell proliferation and cytokine production. JKAP-knockout mice show enhanced T-cell-mediated immune responses and are more susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, the recipient mice that are adoptively transferred with JKAP-knockout T cells show exacerbated EAE symptoms. Aged JKAP-knockout mice spontaneously develop inflammation and autoimmunity. Thus, our results indicate that JKAP is an important phosphatase that inactivates Lck in the TCR signalling turn-off stage, leading to suppression of T-cell-mediated immunity and autoimmunity.

IL-6-Type Cytokine Signaling in Adipocytes Induces Intestinal GLP-1 Secretion.

PubMed

Wueest, Stephan; Laesser, Céline I; Böni-Schnetzler, Marianne; Item, Flurin; Lucchini, Fabrizio C; Borsigova, Marcela; Müller, Werner; Donath, Marc Y; Konrad, Daniel

2018-01-01

We recently showed that interleukin (IL)-6-type cytokine signaling in adipocytes induces free fatty acid release from visceral adipocytes, thereby promoting obesity-induced hepatic insulin resistance and steatosis. In addition, IL-6-type cytokines may increase the release of leptin from adipocytes and by those means induce glucagon-like peptide 1 (GLP-1) secretion. We thus hypothesized that IL-6-type cytokine signaling in adipocytes may regulate insulin secretion. To this end, mice with adipocyte-specific knockout of gp130, the signal transducer protein of IL-6, were fed a high-fat diet for 12 weeks. Compared with control littermates, knockout mice showed impaired glucose tolerance and circulating leptin, GLP-1, and insulin levels were reduced. In line, leptin release from isolated adipocytes was reduced, and intestinal proprotein convertase subtilisin/kexin type 1 ( Pcsk1 ) expression, the gene encoding PC1/3, which controls GLP-1 production, was decreased in knockout mice. Importantly, treatment with the GLP-1 receptor antagonist exendin 9-39 abolished the observed difference in glucose tolerance between control and knockout mice. Ex vivo, supernatant collected from isolated adipocytes of gp130 knockout mice blunted Pcsk1 expression and GLP-1 release from GLUTag cells. In contrast, glucose- and GLP-1-stimulated insulin secretion was not affected in islets of knockout mice. In conclusion, adipocyte-specific IL-6 signaling induces intestinal GLP-1 release to enhance insulin secretion, thereby counteracting insulin resistance in obesity. © 2017 by the American Diabetes Association.

Knockout mutations of insulin-like peptide genes enhance sexual receptivity in Drosophila virgin females.

PubMed

Watanabe, Kazuki; Sakai, Takaomi

2016-01-01

In the fruitfly Drosophila melanogaster, females take the initiative to mate successfully because they decide whether to mate or not. However, little is known about the molecular and neuronal mechanisms regulating sexual receptivity in virgin females. Genetic tools available in Drosophila are useful for identifying molecules and neural circuits involved in the regulation of sexual receptivity. We previously demonstrated that insulin-producing cells (IPCs) in the female brain are critical to the regulation of female sexual receptivity. Ablation and inactivation of IPCs enhance female sexual receptivity, suggesting that neurosecretion from IPCs inhibits female sexual receptivity. IPCs produce and release insulin-like peptides (Ilps) that modulate various biological processes such as metabolism, growth, lifespan and behaviors. Here, we report a novel role of the Ilps in sexual behavior in Drosophila virgin females. Compared with wild-type females, females with knockout mutations of Ilps showed a high mating success rate toward wild-type males, whereas wild-type males courted wild-type and Ilp-knockout females to the same extent. Wild-type receptive females retard their movement during male courtship and this reduced female mobility allows males to copulate. Thus, it was anticipated that knockout mutations of Ilps would reduce general locomotion. However, the locomotor activity in Ilp-knockout females was significantly higher than that in wild-type females. Thus, our findings indicate that the high mating success rate in Ilp-knockout females is caused by their enhanced sexual receptivity, but not by improvement of their sex appeal or by general sluggishness.

[Quantitative changes of main components of erythrocyte membranes which define architectonics of cells under pttg gene knockout].

PubMed

Kaniuka, O P; Filiak, Ie Z; Kulachkovs'kyĭ, O R; Osyp, Iu L; Sybirna, N O

2014-01-01

A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and increasing of polyunsaturated fatty acids amount by 18%. Analyzing the erythrocyte surface architectonics of mice under pttg gene knockout, it was found that on the background of reduction of the functionally complete biconcave discs population one could observe an increase of the number of transformed cells at different degeneration stages. Researches have shown that in mice with a pttg gene knockout compared with a control group of animals cytoskeletal protein--beta-spectrin was reduced by 17.03%. However, there is a reduction of membrane protein band 3 by 33.04%, simultaneously the content of anion transport protein band 4.5 increases by 35.2% and protein band 4.2 by 32.1%. The lectin blot analysis has helped to reveal changes in the structure of the carbohydrate determinants of erythrocyte membrane glycoproteins under conditions of directed pttg gene inactivation, accompanied by changes in the type of communication, which joins the terminal residue in carbohydrate determinant of glycoproteins. Thus, a significant redistribution of protein and fatty acids contents in erythrocyte membranes that manifested in the increase of the deformed shape of red blood cells is observed underpttg gene knockout.

Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9.

PubMed

Matsunaga, Taichi; Yamashita, Jun K

2014-02-07

Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments. Copyright © 2014 Elsevier Inc. All rights reserved.

The role of system Xc- in methamphetamine-induced dopaminergic neurotoxicity in mice.

PubMed

Dang, Duy-Khanh; Shin, Eun-Joo; Tran, Hai-Quyen; Kim, Dae-Joong; Jeong, Ji Hoon; Jang, Choon-Gon; Nah, Seung-Yeol; Sato, Hideyo; Nabeshima, Toshitaka; Yoneda, Yukio; Kim, Hyoung-Chun

2017-09-01

The cystine/glutamate antiporter (system Xc - , Sxc) transports cystine into cell in exchange for glutamate. Since xCT is a specific subunit of Sxc, we employed xCT knockout mice and investigated whether this antiporter affected methamphetamine (MA)-induced dopaminergic neurotoxicity. MA treatment significantly increased striatal oxidative burdens in wild type mice. xCT inhibitor [i.e., S-4-carboxy-phenylglycine (CPG), sulfasalazine] or an xCT knockout significantly protected against these oxidative burdens. MA-induced increases in Iba-1 expression and Iba-1-labeled microglial immunoreactivity (Iba-1-IR) were significantly attenuated by CPG or sulfasalazine administration or xCT knockout. CPG or sulfasalazine significantly attenuated MA-induced TUNEL-positive cell populations in the striatum of Taconic ICR mice. The decrease in excitatory amino acid transporter-2 (or glutamate transporter-1) expression and increase in glutamate release were attenuated by CPG, sulfasalazine or xCT knockout. In addition, CPG, sulfasalazine or xCT knockout significantly protected against dopaminergic loss (i.e., decreases in tyrosine hydroxylase expression and immunoreactivity, and an increase in dopamine turnover rate) induced by MA. However, CPG, sulfasalazine or xCT knockout did not significantly affect the impaired glutathione system [i.e., decrease in reduced glutathione (GSH) and increase in oxidized glutathione (GSSG)] induced by MA. Our results suggest that Sxc mediates MA-induced neurotoxicity via facilitating oxidative stress, microgliosis, proapoptosis, and glutamate-related toxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

PubMed Central

Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

2012-01-01

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

Cathepsin K knockout alleviates aging-induced cardiac dysfunction

PubMed Central

Hua, Yinan; Robinson, Timothy J; Cao, Yongtao; Shi, Guo-Ping; Ren, Jun; Nair, Sreejayan

2015-01-01

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis. PMID:25692548

The joint in vitro action of polymyxin B and miconazole against pathogens associated with canine otitis externa from three European countries

PubMed Central

Pietschmann, Silvia; Meyer, Michael; Voget, Michael; Cieslicki, Michael

2013-01-01

Background Canine otitis externa, an inflammation of the external ear canal, can be maintained and worsened by bacterial or fungal infections. For topical treatment, combinations of anti-inflammatory and antimicrobial ingredients are mainly used. Hypothesis/Objectives This study was conducted to elucidate the in vitro activity of polymyxin B and miconazole against clinical bacterial isolates from three European countries, to investigate possible differences in sensitivity and to assess drug interactions. Animals Seventeen strains of Escherichia coli, 24 strains of Pseudomonas aeruginosa, 24 strains of Proteus mirabilis and 25 strains of Staphylococcus pseudintermedius from dogs with diagnosed otitis externa had been isolated in Germany, France and Italy. Methods Drug activities were evaluated by minimal inhibitory concentration (MIC) and minimal bactericidal concentration. The potentiation of polymyxin B plus miconazole was calculated using the fractional inhibitory concentration index (FICI). An FICI ≤0.5 defined synergy. Furthermore, geographical variations in the FICI and MIC were assessed by statistical analysis. Results Bacterial susceptibilities were comparable in different European countries, because there were no significant MIC and FICI variations (P > 0.05). As a single agent, polymyxin B had bactericidal activity against most E. coli and P. aeruginosa strains and, in higher concentrations, against S. pseudintermedius strains. Miconazole was bactericidal against all Staphylococcus strains. Synergy was demonstrated against strains of E. coli and P. aeruginosa (FICI = 0.25 and 0.50, respectively), whereas overall there was no interaction against S. pseudintermedius strains (FICI = 1.25). Proteus mirabilis strains were not inhibited by each of the drugs individually or by their combination. Conclusions and clinical importance In vitro synergy of polymyxin B and miconazole against E. coli and P. aeruginosa isolates indicates a rationale for applying both agents in combination to treat otitis externa when infected with these types of bacteria. Résumé Contexte L'otite externe canine, une inflammation du conduit auriculaire externe, peut être entretenue et aggravée par les infections bactériennes ou fongiques. Pour le traitement topique, les associations d'anti-inflammatoires et d'antimicrobiens sont principalement utilisées. Hypothèses/Objectifs Cette étude a été menée pour déterminer l'activité in vitro de la polymyxine B et du miconazole contre les souches bactériennes cliniques isolées dans trois pays européens, d'étudier les différences possibles de sensibilité et de déterminer les interactions médicamenteuses. Sujets Dix-sept souches d'Escherichia coli, 24 souches de Pseudomonas aeruginosa, 24 souches de Proteus mirabilis et 25 souches de Staphylococcus pseudintermedius ont été isolées de chiens atteints d'otite externe en Allemagne, France et Italie. Résultats L'activité des molécules a été évaluée par la concentration minimale inhibitrice (CMI) et la concentration minimale bactéricide. La potentialisation de la polymyxine B et du miconazole a été calculée par l'indice de concentration inhibitrice fractionnaire (FICI). Un FICI ≤ 0.5 définissait la synergie. En outre, les variations géographiques dans le FICI et la CMI étaient évaluées par analyses statistiques. Résultats Les sensibilités bactériennes étaient comparables dans les différents pays européens parce qu'aucune différence significative n'a été mise en évidence entre les variations de CMI et de FICI (P > 0.05). La polymyxine B en tant que seul agent avait une activité bactéricide contre la plupart des souches de E. coli et P. aeruginosa, et, à plus forte concentration, contre les souches de S. pseudintermedius. Le miconazole était bactéricide contre toutes les souches de S. pseudintermedius. Une synergie a été mise en évidence contre les souches de E. coli et P. aeruginosa (FICI = 0.25 et 0.50, respectivement), alors qu'aucune interaction n'a été mise en évidence contre les souches de S. pseudintermedius (FICI = 1.25). Les souches de Proteus mirabilis n'ont été inhibées par aucune des molécules, individuellement ou en association. Conclusions et importance clinique La synergie in vitro de la polymyxine B et du miconazole contre les souches d'E. coli et de P. aeruginosa justifie l'application de la combinaison des deux agents dans le traitement de l'otite externe lors d'infection par ces bactéries. Resumen Introducción la otitis externa canina, inflamación del canal auditivo externo, puede perpetuarse y empeorar debido a la presencia de infecciones bacterianas o fúngicas. Para el tratamiento tópico se utilizan fundamentalmente combinaciones de ingredientes antiinflamatorios y antimicrobianos. Hipótesis/objetivos este estudio se condujo para elucidar la actividad in vitro de polimixina B y miconazol frente a aislados clínicos bacterianos de tres países europeos, investigar posibles diferencias en sensibilidad y analizar interacciones de fármacos. Animales diecisiete cepas de Escherichia coli, 24 cepas de Pseudomonas aeruginosa, 24 cepas de Proteus mirabilis y 25 cepas de Staphylococcus pseudintermedius de perros diagnosticados con otitis externa asilados de Alemania, Francia e Italia. Métodos se evaluó la actividad de los fármacos mediante la concentración inhibitoria minima (MIC) y la concentración bactericida minima. La potenciación de polimixina B y miconazol se calculó usando el índice de concentración fraccional inhibitoria (FICI). Un FICI≤ 0,5 definía sinergismo. Además se analizaron estadísticamente las variaciones en la FICI y MIC dependiendo de la región de origen. Resultados la susceptibilidad bacteriana fue comparable en los diferentes países europeos ya que no hubo diferencias significativas en MIC y FICI (P > 0,05). Como agente único la polimixina B tuvo actividad antimicrobiana frente a la mayoría de cepas de E. coli y P. aeruginosa, y a mayores concentraciones frente a cepas de S. pseudintermedius. El miconazol fue bactericida frente a todas las cepas de Staphylococcus. Se observó sinergismo frente a cepas de E. coli y P. aeruginosa (FICI = 0.25 y 0,50, respectivamente), mientras en general no hubo sinergismo frente a las cepas de S. pseudintermedius (FICI = 1.25). Cepas de Proteus mirabilis no fueron inhibidas por los fármacos individualmente ni en combinación. Conclusiones e importancia clínica el sinergismo in vitro de la polimixina B y el miconazol frente a aislados de E. coli y P. aeruginosa indica un motivo para utilizar ambos agentes en combinación para tratar casos de otitis externa producidos por infecciones con estas bacterias. Zusammenfassung Hintergrund Die canine Otitis externa, eine Entzündung des äußeren Ohrkanals, kann durch eine bakterielle Infektion oder durch eine Infektion mit Hefepilzen aufrechterhalten bzw. verschlimmert werden. Zur topischen Behandlung werden hauptsächlich Kombinationen aus entzündungshemmenden und antimikrobiellen Wirkstoffen verwendet. Hypothese/Ziele Diese Studie wurde durchgeführt, um die in vitro Aktivität von Polymyxin B und Mikonazol gegenüber klinischen Bakterienisolaten aus drei europäischen Ländern zu beleuchten und um mögliche Unterschiede in der Sensibilität zu untersuchen und um Interaktionen von Medikamenten zu beurteilen. Tiere Siebzehn Stämme von Escherichia coli, 24 Stämme von Pseudomonas aeruginosa, 24 Stämme von Proteus mirabilis und 25 Stämme von Staphylokokkus pseudintermedius von Hunden mit einer diagnostizierten Otitis externa waren in Deutschland, Frankreich und Italien isoliert worden. Methoden Die Wirkstoffaktivitäten wurden mittels minimaler inhibitorischer Konzentration (MIC) und minimaler bakterizider Konzentration evaluiert. Die Potenzierung von Polymyxin B plus Mikonazol wurde mittels „Fractional Inhibitory Concentration Index” (FICI) kalkuliert. Ein FICI ≤ 0,5 definierte eine Synergie. Weiters wurden geographische Variationen des FICI und der MIC mittels statistischer Analyse beurteilt. Ergebnisse Die bakteriellen Empfindlichkeiten waren in den verschiedenen europäischen Ländern vergleichbar, da keine signifikanten Unterschiede bei MIC und FICI bestanden (P > 0,05). Als alleiniger Wirkstoff zeigte Polymyxin B eine bakterizide Wirkung gegenüber den meisten E. coli und P. aeruginosa Stämmen und, in höheren Konzentrationen, gegenüber S. pseudintermedius Stämmen. Eine Synergie wurde gegen E. coli und P. aeruginosa Stämme (FICI = 0.25 bzw. 0,50) demonstriert, während insgesamt keine Interaktion gegen S. pseudintermedius Stämme (FICI = 1.25) bestand. Proteus mirabilis Stämme wurden von keinem dieser Wirkstoffe, weder individuell noch in Kombination, inhibiert. Schlussfolgerungen und klinische Bedeutung Eine in vitro Synergie von Polymyxin B und Mikonazol gegenüber E. coli und P. aeruginosa Isolaten bekräftigt die Argumentation dafür, beide Wirkstoffe in Kombination zu verwenden, um eine Otitis externa, bei der diese Bakterien vorkommen, zu behandeln. PMID:23721182

Manifestation of α clustering in 10Be via α -knockout reaction

NASA Astrophysics Data System (ADS)

Lyu, Mengjiao; Yoshida, Kazuki; Kanada-En'yo, Yoshiko; Ogata, Kazuyuki

2018-04-01

Background: Proton-induced α -knockout reactions may allow direct experimental observation of α clustering in nuclei. This is obtained by relating the theoretical descriptions of clustering states to the experimental reaction observables. It is desired to introduce microscopic structure models into the theoretical frameworks for α -knockout reactions. Purpose: Our goal is to probe the α clustering in the 10Be nucleus by proton-induced α -knockout reaction observables. Method: We adopt an extended version of the Tohsaki-Horiuchi-Schuck-Röpke wave function of 10Be and integrate it with the distorted-wave impulse approximation framework for the calculation of (p ,p α ) -knockout reactions. Results: We make the first calculation for the 10Be(p ,p α )6He reaction at 250 MeV by implementing a microscopic α -cluster wave function, and we predict the triple-differential cross section (TDX). Furthermore, by constructing artificial states of the target nucleus 10Be with compact or dilute spatial distributions, the TDX is found to be highly sensitive to the extent of clustering in the target nuclei. Conclusions: These results provide reliable manifestation of α clustering in 10Be.

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

PubMed

Menegola, Milena; Clark, Eliana; Trimmer, James S

2012-06-01

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

Different roles of axon guidance cues and patterned spontaneous activity in establishing receptive fields in the mouse superior colliculus.

PubMed

Liu, Mingna; Wang, Lupeng; Cang, Jianhua

2014-01-01

Visual neurons in the superior colliculus (SC) respond to both bright (On) and dark (Off) stimuli in their receptive fields. This receptive field property is due to proper convergence of On- and Off-centered retinal ganglion cells to their target cells in the SC. In this study, we have compared the receptive field structure of individual SC neurons in two lines of mutant mice that are deficient in retinotopic mapping: the ephrin-A knockouts that lack important retinocollicular axonal guidance cues and the nAChR-β2 knockouts that have altered activity-dependent refinement of retinocollicular projections. We find that even though the receptive fields are much larger in the ephrin-A knockouts, their On-Off overlap remains unchanged. These neurons also display normal level of selectivity for stimulus direction and orientation. In contrast, the On-Off overlap is disrupted in the β2 knockouts. Together with the previous finding of disrupted direction and orientation selectivity in the β2 knockout mice, our results indicate that molecular guidance cues and activity-dependent processes play different roles in the development of receptive field properties in the SC.

Olivocochlear neuron central anatomy is normal in alpha 9 knockout mice.

PubMed

Brown, M Christian; Vetter, Douglas E

2009-03-01

Olivocochlear (OC) neurons were studied in a transgenic mouse with deletion of the alpha 9 nicotinic acetylcholine receptor subunit. In this alpha 9 knockout mouse, the peripheral effects of OC stimulation are lacking and the peripheral terminals of OC neurons under outer hair cells have abnormal morphology. To account for this mouse's apparently normal hearing, it has been proposed to have central compensation via collateral branches to the cochlear nucleus. We tested this idea by staining OC neurons for acetylcholinesterase and examining their morphology in knockout mice, wild-type mice of the same background strain, and CBA/CaJ mice. Knockout mice had normal OC systems in terms of numbers of OC neurons, dendritic patterns, and numbers of branches to the cochlear nucleus. The branch terminations were mainly to edge regions and to a lesser extent the core of the cochlear nucleus, and were similar among the strains in terms of the distribution and staining density. These data demonstrate that there are no obvious changes in the central morphology of the OC neurons in alpha 9 knockout mice and make less attractive the idea that there is central compensation for deletion of the peripheral receptor in these mice.

Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells.

PubMed

Korge, Sandra; Grudziecki, Astrid; Kramer, Achim

2015-10-01

Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms. © 2015 The Author(s).

Sequence determinants of improved CRISPR sgRNA design.

PubMed

Xu, Han; Xiao, Tengfei; Chen, Chen-Hao; Li, Wei; Meyer, Clifford A; Wu, Qiu; Wu, Di; Cong, Le; Zhang, Feng; Liu, Jun S; Brown, Myles; Liu, X Shirley

2015-08-01

The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies. © 2015 Xu et al.; Published by Cold Spring Harbor Laboratory Press.

Knockout of Eva1a leads to rapid development of heart failure by impairing autophagy

PubMed Central

Zhang, Shu; Lin, Xin; Li, Ge; Shen, Xue; Niu, Di; Lu, Guang; Fu, Xin; Chen, Yingyu; Cui, Ming; Bai, Yun

2017-01-01

EVA1A (Eva-1 homologue A) is a novel lysosome and endoplasmic reticulum-associated protein that can regulate cell autophagy and apoptosis. Eva1a is expressed in the myocardium, but its function in myocytes has not yet been investigated. Therefore, we generated inducible, cardiomyocyte-specific Eva1a knockout mice with an aim to determine the role of Eva1a in cardiac remodelling in the adult heart. Data from experiments showed that loss of Eva1a in the adult heart increased cardiac fibrosis, promoted cardiac hypertrophy, and led to cardiomyopathy and death. Further investigation suggested that this effect was associated with impaired autophagy and increased apoptosis in Eva1a knockout hearts. Moreover, knockout of Eva1a activated Mtor signalling and the subsequent inhibition of autophagy. In addition, Eva1a knockout hearts showed disorganized sarcomere structure and mitochondrial misalignment and aggregation, leading to the lack of ATP generation. Collectively, these data demonstrated that Eva1a improves cardiac function and inhibits cardiac hypertrophy and fibrosis by increasing autophagy. In conclusion, our results demonstrated that Eva1a may have an important role in maintaining cardiac homeostasis. PMID:28151473

Novel mTORC1 and 2 Signaling Pathways in Polycystic Kidney Disease (PKD)

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-16-1-0172 TITLE: Novel mTORC1 and 2 Signaling Pathways in Polycystic Kidney Disease (PKD) PRINCIPAL INVESTIGATOR: Charles...TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-16-1-0172 Novel mTORC1 and 2 Signaling Pathways in Polycystic Kidney Disease (PKD) 5b. GRANT NUMBER 5c...investigate the effects of mTORC1 (Raptor) knockout, mTORC2 (Rictor) knockout or combined mTORC1 and 2 knockout on cyst growth and kidney function. The

Novel Therapeutic Targets to Inhibit Tumor Microenvironment-Induced Castration Resistant Prostate Cancer

DTIC Science & Technology

2016-10-01

MAPK4. We are also in the process of generating MAPK4-knockout LNCaP cells using the CRISPR /Cas9 system. Altogether, we hope that we can generate the...engineered LNCaP cells that are stable for either total loss of MAPK4 ( CRISPR /Cas9 knockout) or with inducible knockdown of MAPK4 (Dox-inducible...knockdown or CRISPR /Cas9 mediated knockout of MAPK4 (we are working on them). Major Task 2: Determine whether inhibition of MAPK4 (in PCa) and TGF-β

Proton Knock-Out in Hall A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kees de Jager

Proton knock-out is studied in a broad program in Hall A at Jefferson Lab. The first experiment performed in Hall A studied the {sup 16}O(e,e'p) reaction. Since then proton knock-out experiments have studied a variety of aspects of that reaction, from single-nucleon properties to its mechanism, such as final-state interactions and two-body currents, in nuclei from {sup 2}H to {sup 16}O. In this review the results of this program will be summarized and an outlook given of future accomplishments.

Effect of knockout of α2δ-1 on action potentials in mouse sensory neurons.

PubMed

Margas, Wojciech; Ferron, Laurent; Nieto-Rostro, Manuela; Schwartz, Arnold; Dolphin, Annette C

2016-08-05

Gene deletion of the voltage-gated calcium channel auxiliary subunit α2δ-1 has been shown previously to have a cardiovascular phenotype, and a reduction in mechano- and cold sensitivity, coupled with delayed development of neuropathic allodynia. We have also previously shown that dorsal root ganglion (DRG) neuron calcium channel currents were significantly reduced in α2δ-1 knockout mice. To extend our findings in these sensory neurons, we have examined here the properties of action potentials (APs) in DRG neurons from α2δ-1 knockout mice in comparison to their wild-type (WT) littermates, in order to dissect how the calcium channels that are affected by α2δ-1 knockout are involved in setting the duration of individual APs and their firing frequency. Our main findings are that there is reduced Ca(2+) entry on single AP stimulation, particularly in the axon proximal segment, reduced AP duration and reduced firing frequency to a 400 ms stimulation in α2δ-1 knockout neurons, consistent with the expected role of voltage-gated calcium channels in these events. Furthermore, lower intracellular Ca(2+) buffering also resulted in reduced AP duration, and a lower frequency of AP firing in WT neurons, mimicking the effect of α2δ-1 knockout. By contrast, we did not obtain any consistent evidence for the involvement of Ca(2+)-activation of large conductance calcium-activated potassium (BK) and small conductance calcium-activated potassium (SK) channels in these events. In conclusion, the reduced Ca(2+) elevation as a result of single AP stimulation is likely to result from the reduced duration of the AP in α2δ-1 knockout sensory neurons.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. © 2016 The Authors.

GLT-1: The elusive presynaptic glutamate transporter

PubMed Central

Rimmele, Theresa S.; Rosenberg, Paul A.

2016-01-01

Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiologial significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (Vmax) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5–10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate homeostasis associated with normal functions, neurodegeneration, and response to drugs. PMID:27129805

Changes in the expression of neurotransmitter receptors in Parkin and DJ-1 knockout mice--A quantitative multireceptor study.

PubMed

Cremer, J N; Amunts, K; Schleicher, A; Palomero-Gallagher, N; Piel, M; Rösch, F; Zilles, K

2015-12-17

Parkinson's disease (PD) is a well-characterized neurological disorder with regard to its neuropathological and symptomatic appearance. At the genetic level, mutations of particular genes, e.g. Parkin and DJ-1, were found in human hereditary PD with early onset. Neurotransmitter receptors constitute decisive elements in neural signal transduction. Furthermore, since they are often altered in neurological and psychiatric diseases, receptors have been successful targets for pharmacological agents. However, the consequences of PD-associated gene mutations on the expression of transmitter receptors are largely unknown. Therefore, we studied the expression of 16 different receptor binding sites of the neurotransmitters glutamate, GABA, acetylcholine, adrenaline, serotonin, dopamine and adenosine by means of quantitative receptor autoradiography in Parkin and DJ-1 knockout mice. These knockout mice exhibit electrophysiological and behavioral deficits, but do not show the typical dopaminergic cell loss. We demonstrated differential changes of binding site densities in eleven brain regions. Most prominently, we found an up-regulation of GABA(B) and kainate receptor densities in numerous cortical areas of Parkin and DJ-1 knockout mice, as well as increased NMDA but decreased AMPA receptor densities in different brain regions of the Parkin knockout mice. The alterations of three different glutamate receptor types may indicate the potential relevance of the glutamatergic system in the pathogenesis of PD. Furthermore, the cholinergic M1, M2 and nicotinic receptors as well as the adrenergic α2 and the adenosine A(2A) receptors showed differentially increased densities in Parkin and DJ-1 knockout mice. Taken together, knockout of the PD-associated genes Parkin or DJ-1 results in differential changes of neurotransmitter receptor densities, highlighting a possible role of altered non-dopaminergic, and in particular of glutamatergic neurotransmission in PD pathogenesis. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Increased Contextual Fear Conditioning in iNOS Knockout Mice: Additional Evidence for the Involvement of Nitric Oxide in Stress-Related Disorders and Contribution of the Endocannabinoid System

PubMed Central

Gomes, Felipe V.; Silva, Andréia L.; Uliana, Daniela L.; Camargo, Laura H. A.; Guimarães, Francisco S.; Cunha, Fernando Q.; Joca, Sâmia R. L.; Resstel, Leonardo B. M.

2015-01-01

Background: Inducible or neuronal nitric oxide synthase gene deletion increases or decreases anxiety-like behavior in mice, respectively. Since nitric oxide and endocannabinoids interact to modulate defensive behavior, the former effect could involve a compensatory increase in basal brain nitric oxide synthase activity and/or changes in the endocannabinoid system. Thus, we investigated the expression and extinction of contextual fear conditioning of inducible nitric oxide knockout mice and possible involvement of endocannabinoids in these responses. Methods: We evaluated the effects of a preferential neuronal nitric oxide synthase inhibitor, 7-nitroindazol, nitric oxide synthase activity, and mRNA changes of nitrergic and endocannabinoid systems components in the medial prefrontal cortex and hippocampus of wild-type and knockout mice. The effects of URB597, an inhibitor of the fatty acid amide hydrolase enzyme, which metabolizes the endocannabinoid anandamide, WIN55,212-2, a nonselective cannabinoid agonist, and AM281, a selective CB1 antagonist, on contextual fear conditioning were also evaluated. Results: Contextual fear conditioning expression was similar in wild-type and knockout mice, but the latter presented extinction deficits and increased basal nitric oxide synthase activity in the medial prefrontal cortex. 7-Nitroindazol decreased fear expression and facilitated extinction in wild-type and knockout mice. URB597 decreased fear expression in wild-type and facilitated extinction in knockout mice, whereas WIN55,212-2 and AM281 increased it in wild-type mice. Nonconditioned knockout mice showed changes in the mRNA expression of nitrergic and endocannabinoid system components in the medial prefrontal cortex and hippocampus that were modified by fear conditioning. Conclusion: These data reinforce the involvement of the nitric oxide and endocannabinoids (anandamide) in stress-related disorders and point to a deregulation of the endocannabinoid system in situations where nitric oxide signaling is increased. PMID:25618404

Increased Contextual Fear Conditioning in iNOS Knockout Mice: Additional Evidence for the Involvement of Nitric Oxide in Stress-Related Disorders and Contribution of the Endocannabinoid System.

PubMed

Lisboa, Sabrina F; Gomes, Felipe V; Silva, Andréia L; Uliana, Daniela L; Camargo, Laura H A; Guimarães, Francisco S; Cunha, Fernando Q; Joca, Sâmia R L; Resstel, Leonardo B M

2015-01-24

Inducible or neuronal nitric oxide synthase gene deletion increases or decreases anxiety-like behavior in mice, respectively. Since nitric oxide and endocannabinoids interact to modulate defensive behavior, the former effect could involve a compensatory increase in basal brain nitric oxide synthase activity and/or changes in the endocannabinoid system. Thus, we investigated the expression and extinction of contextual fear conditioning of inducible nitric oxide knockout mice and possible involvement of endocannabinoids in these responses. We evaluated the effects of a preferential neuronal nitric oxide synthase inhibitor, 7-nitroindazol, nitric oxide synthase activity, and mRNA changes of nitrergic and endocannabinoid systems components in the medial prefrontal cortex and hippocampus of wild-type and knockout mice. The effects of URB597, an inhibitor of the fatty acid amide hydrolase enzyme, which metabolizes the endocannabinoid anandamide, WIN55,212-2, a nonselective cannabinoid agonist, and AM281, a selective CB1 antagonist, on contextual fear conditioning were also evaluated. Contextual fear conditioning expression was similar in wild-type and knockout mice, but the latter presented extinction deficits and increased basal nitric oxide synthase activity in the medial prefrontal cortex. 7-Nitroindazol decreased fear expression and facilitated extinction in wild-type and knockout mice. URB597 decreased fear expression in wild-type and facilitated extinction in knockout mice, whereas WIN55,212-2 and AM281 increased it in wild-type mice. Nonconditioned knockout mice showed changes in the mRNA expression of nitrergic and endocannabinoid system components in the medial prefrontal cortex and hippocampus that were modified by fear conditioning. These data reinforce the involvement of the nitric oxide and endocannabinoids (anandamide) in stress-related disorders and point to a deregulation of the endocannabinoid system in situations where nitric oxide signaling is increased. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Altered Sleep and Affect in the Neurotensin Receptor 1 Knockout Mouse

PubMed Central

Fitzpatrick, Karrie; Winrow, Christopher J.; Gotter, Anthony L.; Millstein, Joshua; Arbuzova, Janna; Brunner, Joseph; Kasarskis, Andrew; Vitaterna, Martha H.; Renger, John J.; Turek, Fred W.

2012-01-01

Study Objective: Sleep and mood disorders have long been understood to have strong genetic components, and there is considerable comorbidity of sleep abnormalities and mood disorders, suggesting the involvement of common genetic pathways. Here, we examine a candidate gene implicated in the regulation of both sleep and affective behavior using a knockout mouse model. Design: Previously, we identified a quantitative trait locus (QTL) for REM sleep amount, REM sleep bout number, and wake amount in a genetically segregating population of mice. Here, we show that traits mapping to this QTL correlated with an expression QTL for neurotensin receptor 1 (Ntsr1), a receptor for neurotensin, a ligand known to be involved in several psychiatric disorders. We examined sleep as well as behaviors indicative of anxiety and depression in the NTSR1 knockout mouse. Measurements and Results: NTSR1 knockouts had a lower percentage of sleep time spent in REM sleep in the dark phase and a larger diurnal variation in REM sleep duration than wild types under baseline conditions. Following sleep deprivation, NTSR1 knockouts exhibited more wake and less NREM rebound sleep. NTSR1 knockouts also showed increased anxious and despair behaviors. Conclusions: Here we illustrate a link between expression of the Ntsr1 gene and sleep traits previously associated with a particular QTL. We also demonstrate a relationship between Ntsr1 and anxiety and despair behaviors. Given the considerable evidence that anxiety and depression are closely linked with abnormalities in sleep, the data presented here provide further evidence that neurotensin and Ntsr1 may be a component of a pathway involved in both sleep and mood disorders. Citation: Fitzpatrick K; Winrow CJ; Gotter AL; Millstein J; Arbuzova J; Brunner J; Kasarskis A; Vitaterna MH; Renger JJ; Turek FW. Altered sleep and affect in the neurotensin receptor 1 knockout mouse. SLEEP 2012;35(7):949-956. PMID:22754041

Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes.

PubMed Central

Weinstock, P H; Bisgaier, C L; Aalto-Setälä, K; Radner, H; Ramakrishnan, R; Levak-Frank, S; Essenburg, A D; Zechner, R; Breslow, J L

1995-01-01

Lipoprotein lipase (LPL)-deficient mice have been created by gene targeting in embryonic stem cells. At birth, homozygous knockout pups have threefold higher triglycerides and sevenfold higher VLDL cholesterol levels than controls. When permitted to suckle, LPL-deficient mice become pale, then cyanotic, and finally die at approximately 18 h of age. Before death, triglyceride levels are severely elevated (15,087 +/- 3,805 vs 188 +/- 71 mg/dl in controls). Capillaries in tissues of homozygous knockout mice are engorged with chylomicrons. This is especially significant in the lung where marginated chylomicrons prevent red cell contact with the endothelium, a phenomenon which is presumably the cause of cyanosis and death in these mice. Homozygous knockout mice also have diminished adipose tissue stores as well as decreased intracellular fat droplets. By crossbreeding with transgenic mice expressing human LPL driven by a muscle-specific promoter, mouse lines were generated that express LPL exclusively in muscle but not in any other tissue. This tissue-specific LPL expression rescued the LPL knockout mice and normalized their lipoprotein pattern. This supports the contention that hypertriglyceridemia caused the death of these mice and that LPL expression in a single tissue was sufficient for rescue. Heterozygous LPL knockout mice survive to adulthood and have mild hypertriglyceridemia, with 1.5-2-fold elevated triglyceride levels compared with controls in both the fed and fasted states on chow, Western-type, or 10% sucrose diets. In vivo turnover studies revealed that heterozygous knockout mice had impaired VLDL clearance (fractional catabolic rate) but no increase in transport rate. In summary, total LPL deficiency in the mouse prevents triglyceride removal from plasma, causing death in the neonatal period, and expression of LPL in a single tissue alleviates this problem. Furthermore, half-normal levels of LPL cause a decrease in VLDL fractional catabolic rate and mild hypertriglyceridemia, implying that partial LPL deficiency has physiological consequences. Images PMID:8675619

Knock-Outs, Stick-Outs, Cut-Outs: Clipping Paths Separate Objects from Background.

ERIC Educational Resources Information Center

Wilson, Bradley

1998-01-01

Outlines a six-step process that allows computer operators, using Photoshop software, to create "knock-outs" to precisely define the path that will serve to separate the object from the background. (SR)

Generating gene knockout rats by homologous recombination in embryonic stem cells

PubMed Central

Tong, Chang; Huang, Guanyi; Ashton, Charles; Li, Ping; Ying, Qi-Long

2013-01-01

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats. PMID:21637202

CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells.

PubMed

Kawamura, Norihiko; Nimura, Keisuke; Nagano, Hiromichi; Yamaguchi, Sohei; Nonomura, Norio; Kaneda, Yasufumi

2015-09-08

NANOG expression in prostate cancer is highly correlated with cancer stem cell characteristics and resistance to androgen deprivation. However, it is not clear whether NANOG or its pseudogenes contribute to the malignant potential of cancer. We established NANOG- and NANOGP8-knockout DU145 prostate cancer cell lines using the CRISPR/Cas9 system. Knockouts of NANOG and NANOGP8 significantly attenuated malignant potential, including sphere formation, anchorage-independent growth, migration capability, and drug resistance, compared to parental DU145 cells. NANOG and NANOGP8 knockout did not inhibit in vitro cell proliferation, but in vivo tumorigenic potential decreased significantly. These phenotypes were recovered in NANOG- and NANOGP8-rescued cell lines. These results indicate that NANOG and NANOGP8 proteins are expressed in prostate cancer cell lines, and NANOG and NANOGP8 equally contribute to the high malignant potential of prostate cancer.

Mapping pathological phenotypes in a mouse model of CDKL5 disorder.

PubMed

Amendola, Elena; Zhan, Yang; Mattucci, Camilla; Castroflorio, Enrico; Calcagno, Eleonora; Fuchs, Claudia; Lonetti, Giuseppina; Silingardi, Davide; Vyssotski, Alexei L; Farley, Dominika; Ciani, Elisabetta; Pizzorusso, Tommaso; Giustetto, Maurizio; Gross, Cornelius T

2014-01-01

Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder.

Skeletal muscle-specific HMG-CoA reductase knockout mice exhibit rhabdomyolysis: A model for statin-induced myopathy.

PubMed

Osaki, Yoshinori; Nakagawa, Yoshimi; Miyahara, Shoko; Iwasaki, Hitoshi; Ishii, Akiko; Matsuzaka, Takashi; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Yahagi, Naoya; Suzuki, Hiroaki; Sone, Hirohito; Ohashi, Ken; Ishibashi, Shun; Yamada, Nobuhiro; Shimano, Hitoshi

2015-10-23

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonic acid (MVA); this is the rate-limiting enzyme of the mevalonate pathway that synthesizes cholesterol. Statins, HMGCR inhibitors, are widely used as cholesterol-reducing drugs. However, statin-induced myopathy is the most adverse side effect of statins. To eludicate the mechanisms underlying statin the myotoxicity and HMGCR function in the skeletal muscle, we developed the skeletal muscle-specific HMGCR knockout mice. Knockout mice exhibited postnatal myopathy with elevated serum creatine kinase levels and necrosis. Myopathy in knockout mice was completely rescued by the oral administration of MVA. These results suggest that skeletal muscle toxicity caused by statins is dependent on the deficiencies of HMGCR enzyme activity and downstream metabolites of the mevalonate pathway in skeletal muscles rather than the liver or other organs. Copyright © 2015 Elsevier Inc. All rights reserved.

Transgenic and gene knockout mice in gastric cancer research

PubMed Central

Jiang, Yannan; Yu, Yingyan

2017-01-01

Mouse models are useful tool for carcinogenic study. They will greatly enrich the understanding of pathogenesis and molecular mechanisms for gastric cancer. However, only few of mice could develop gastric cancer spontaneously. With the development and improvement of gene transfer technology, investigators created a variety of transgenic and knockout/knockin mouse models of gastric cancer, such as INS-GAS mice and gastrin knockout mice. Combined with helicobacter infection and carcinogens treatment, these transgenic/knockout/knockin mice developed precancerous or cancerous lesions, which are proper for gene function study or experimental therapy. Here we review the progression of genetically engineered mouse models on gastric cancer research, and emphasize the effects of chemical carcinogens or infectious factors on carcinogenesis of genetically modified mouse. We also emphasize the histological examination on mouse stomach. We expect to provide researchers with some inspirations on this field. PMID:27713138

Biochemistry and Chemotherapy of Leishmaniasis and Malaria

DTIC Science & Technology

1995-12-01

Allium cepa) and licorice ( Glycyrrhiza glabra). Growth of cells of Leishmania chagasi 13 and Leishmania mexicana 227 was monitored after 72 hr at 590...Leishmania chagasi. Protein Conc. Percent Range Inhibition IC25 IC50 Natural Product Solvent (4g/ml) Range (4g/ml) (0g/ml) Glycyrrhiza AQ 15-150 7.3...jig/ml) Range (jg/ml) (jIg/ml) Glycyrrhiza AQ 15-150 26.4-41.7 6* none glabra ETOH 0.26-268 No Inhibition none none (licorice) Hydrastis AQ 5-15 0.0

An interferometer for high-resolution optical surveillance from GEO - internal metrology breadboard

NASA Astrophysics Data System (ADS)

Bonino, L.; Bresciani, F.; Piasini, G.; Pisani, M.; Cabral, A.; Rebordão, J.; Musso, F.

2017-11-01

This paper describes the internal metrology breadboard development activities performed in the frame of the EUCLID CEPA 9 RTP 9.9 "High Resolution Optical Satellite Sensor" project of the WEAO Research Cell by AAS-I and INETI. The Michelson Interferometer Testbed demonstrates the possibility of achieving a cophasing condition between two arms of the optical interferometer starting from a large initial white light Optical Path Difference (OPD) unbalance and of maintaining the fringe pattern stabilized in presence of disturbances.

Deficiency of eNOS exacerbates early-stage NAFLD pathogenesis by changing the fat distribution.

PubMed

Nozaki, Yuichi; Fujita, Koji; Wada, Koichiro; Yoneda, Masato; Shinohara, Yoshiyasu; Imajo, Kento; Ogawa, Yuji; Kessoku, Takaomi; Nakamuta, Makoto; Saito, Satoru; Masaki, Naohiko; Nagashima, Yoji; Terauchi, Yasuo; Nakajima, Atsushi

2015-12-17

Although many factors and molecules that are closely associated with non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) have been reported, the role of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) in the pathogenesis of NAFLD/NASH remains unclear. We therefore investigated the role of eNOS-derived NO in NAFLD pathogenesis using systemic eNOS-knockout mice fed a high-fat diet. eNOS-knockout and wild-type mice were fed a basal diet or a high-fat diet for 12 weeks. Lipid accumulation and inflammation were evaluated in the liver, and various factors that are closely associated with NAFLD/NASH and hepatic tissue blood flow were analyzed. Lipid accumulation and inflammation were more extensive in the liver and lipid accumulation was less extensive in the visceral fat tissue in eNOS-knockout mice, compared with wild-type mice, after 12 weeks of being fed a high-fat diet. While systemic insulin resistance was comparable between the eNOS-knockout and wild-type mice fed a high-fat diet, hepatic tissue blood flow was significantly suppressed in the eNOS-knockout mice, compared with the wild-type mice, in mice fed a high-fat diet. The microsomal triglyceride transfer protein activity was down-regulated in eNOS-knockout mice, compared with wild-type mice, in mice fed a high-fat diet. A deficiency of eNOS-derived NO may exacerbate the early-stage of NASH pathogenesis by changing the fat distribution in a mouse model via the regulation of hepatic tissue blood flow.

Decreased consumption of sweet fluids in mu opioid receptor knockout mice: a microstructural analysis of licking behavior

PubMed Central

Ostlund, Sean B.; Kosheleff, Alisa; Maidment, Nigel T.; Murphy, Niall P.

2013-01-01

Summary Rationale Evidence suggests that the palatability of food (i.e., the hedonic impact produced by its sensory features) can promote feeding and may underlie compulsive eating, leading to obesity. Pharmacological studies implicate opioid transmission in the hedonic control of feeding, though these studies often rely on agents lacking specificity for particular opioid receptors. Objectives Here, we investigated the role of mu opioid receptors (MORs) specifically in determining hedonic responses to palatable sweet stimuli. Methods In Experiment 1, licking microstructure when consuming sucrose solution (2 to 20 %) was compared in MOR knockout and wildtype mice as a function of sucrose concentration and level of food deprivation. In Experiment 2, a similar examination was conducted using the palatable but calorie-free stimulus sucralose (0.001 to 1%), allowing study of licking behavior independent of homeostatic variables. Results In Experiment 1, MOR knockout mice exhibited several alterations in sucrose licking. Although wildtype mice exhibited a two-fold increase in the burst length when food deprived, relative to the nondeprived test, this aspect of sucrose licking was generally insensitive to manipulations of food deprivation for MOR knockout mice. Furthermore, during concentration testing, their rate of sucrose licking was less than half that of wildtype mice. During sucralose testing (Experiment 2), MOR knockout mice licked at approximately half the wildtype rate, providing more direct evidence that MOR knockout mice were impaired in processing stimulus palatability. Conclusions These results suggest that transmission through MORs mediates hedonic responses to palatable stimuli, and therefore likely contributes to normal and pathological eating. PMID:23568577

Age- and region-specific imbalances of basal amino acids and monoamine metabolism in limbic regions of female Fmr1 knock-out mice.

PubMed

Gruss, Michael; Braun, Katharina

2004-07-01

The Fragile X syndrome, a common form of mental retardation in humans, originates from the loss of expression of the Fragile X mental retardation gene leading to the absence of the encoded Fragile X mental retardation protein 1 (FMRP). A broad pattern of morphological and behavioral abnormalities is well described for affected humans as well as Fmr1 knock-out mice, a transgenic animal model for the human Fragile X syndrome. In the present study, we examined neurochemical differences between female Fmr1 knock-out and wildtype mice with particular focus on neurotransmission. Significant age- and region-specific differences of basal tissue neurotransmitter and metabolite levels measured by high performance liquid chromatography were found. Those differences were more numerous in juvenile animals (postnatal day (PND) 28-31) compared to adults (postnatal day 209-221). In juvenile female knock-out mice, especially aspartate and taurine were increased in cortical regions, striatum, cerebellum, and brainstem. Furthermore, compared to the wildtype animals, the juvenile knock-out mice displayed an increased level of neuronal inhibition in the hippocampus and brainstem reflected by decreased ratios of (aspartate + glutamate)/(taurine + GABA), as well as an increased dopamine (DA) turnover in cortical regions, striatum, and hippocampus. These results provide the first evidence that the lack of FMRP expression in female Fmr1 knock-out mice is accompanied by age-dependent, region-specific alterations in brain amino acids, and monoamine turnover, which might be related to the reported synaptical and behavioural alterations in these animals.

Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK.

PubMed

Chae, Yun Jeong; Zhang, Jianan; Au, Paul; Sabbadini, Marta; Xie, Guo-Xi; Yost, C Spencer

2010-12-01

We investigated the role of tandem pore potassium ion channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice. Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wild type, knockout, or heterozygote) was determined using polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared with wild-type littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane, and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype. With the exception of decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test), TRESK knockout mice had healthy development and behavior. TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared with wild-type littermates. Sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice after initial anesthesia testing was nearly threefold higher than that of wild-type littermates. TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogeneous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the theory that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver*

PubMed Central

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S.; Sun, Baodong

2016-01-01

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. PMID:27358407

Global Nav1.7 Knockout Mice Recapitulate the Phenotype of Human Congenital Indifference to Pain

PubMed Central

Gingras, Jacinthe; Smith, Sarah; Matson, David J.; Johnson, Danielle; Nye, Kim; Couture, Lauren; Feric, Elma; Yin, Ruoyuan; Moyer, Bryan D.; Peterson, Matthew L.; Rottman, James B.; Beiler, Rudolph J.; Malmberg, Annika B.; McDonough, Stefan I.

2014-01-01

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain. PMID:25188265

Altered sleep and affect in the neurotensin receptor 1 knockout mouse.

PubMed

Fitzpatrick, Karrie; Winrow, Christopher J; Gotter, Anthony L; Millstein, Joshua; Arbuzova, Janna; Brunner, Joseph; Kasarskis, Andrew; Vitaterna, Martha H; Renger, John J; Turek, Fred W

2012-07-01

Sleep and mood disorders have long been understood to have strong genetic components, and there is considerable comorbidity of sleep abnormalities and mood disorders, suggesting the involvement of common genetic pathways. Here, we examine a candidate gene implicated in the regulation of both sleep and affective behavior using a knockout mouse model. Previously, we identified a quantitative trait locus (QTL) for REM sleep amount, REM sleep bout number, and wake amount in a genetically segregating population of mice. Here, we show that traits mapping to this QTL correlated with an expression QTL for neurotensin receptor 1 (Ntsr1), a receptor for neurotensin, a ligand known to be involved in several psychiatric disorders. We examined sleep as well as behaviors indicative of anxiety and depression in the NTSR1 knockout mouse. NTSR1 knockouts had a lower percentage of sleep time spent in REM sleep in the dark phase and a larger diurnal variation in REM sleep duration than wild types under baseline conditions. Following sleep deprivation, NTSR1 knockouts exhibited more wake and less NREM rebound sleep. NTSR1 knockouts also showed increased anxious and despair behaviors. Here we illustrate a link between expression of the Ntsr1 gene and sleep traits previously associated with a particular QTL. We also demonstrate a relationship between Ntsr1 and anxiety and despair behaviors. Given the considerable evidence that anxiety and depression are closely linked with abnormalities in sleep, the data presented here provide further evidence that neurotensin and Ntsr1 may be a component of a pathway involved in both sleep and mood disorders.

UMA/GAN network architecture analysis

NASA Astrophysics Data System (ADS)

Yang, Liang; Li, Wensheng; Deng, Chunjian; Lv, Yi

2009-07-01

This paper is to critically analyze the architecture of UMA which is one of Fix Mobile Convergence (FMC) solutions, and also included by the third generation partnership project(3GPP). In UMA/GAN network architecture, UMA Network Controller (UNC) is the key equipment which connects with cellular core network and mobile station (MS). UMA network could be easily integrated into the existing cellular networks without influencing mobile core network, and could provides high-quality mobile services with preferentially priced indoor voice and data usage. This helps to improve subscriber's experience. On the other hand, UMA/GAN architecture helps to integrate other radio technique into cellular network which includes WiFi, Bluetooth, and WiMax and so on. This offers the traditional mobile operators an opportunity to integrate WiMax technique into cellular network. In the end of this article, we also give an analysis of potential influence on the cellular core networks ,which is pulled by UMA network.

A Student Profile.

ERIC Educational Resources Information Center

Maine Univ., Augusta.

A survey of graduates and students from the University of Maine at Augusta (UMA) was conducted in Spring 1980 to provide a profile of new and continuing students and to assess the vocational success of recent UMA graduates. The survey sample included: (1) 461 students selected from the entire UMA population; (2) 49 students enrolled in UMA's…

Identification of essential genes and synthetic lethal gene combinations in Escherichia coli K-12.

PubMed

Mori, Hirotada; Baba, Tomoya; Yokoyama, Katsushi; Takeuchi, Rikiya; Nomura, Wataru; Makishi, Kazuichi; Otsuka, Yuta; Dose, Hitomi; Wanner, Barry L

2015-01-01

Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein.

The groEL2 gene, but not groEL1, is required for biosynthesis of the secondary metabolite myxovirescin in Myxococcus xanthus DK1622.

PubMed

Wang, Yan; Li, Xi; Zhang, Wenyan; Zhou, Xiuwen; Li, Yue-zhong

2014-03-01

Myxococcus xanthus DK1622 possesses two copies of the groEL gene: groEL1, which participates in development, and groEL2, which is involved in the predatory ability of cells. In this study, we determined that the groEL2 gene is required for the biosynthesis of the secondary metabolite myxovirescin (TA), which plays essential roles in predation. The groEL2-knockout mutant strain was defective in producing a zone of inhibition and displayed decreased killing ability against Escherichia coli, while the groEL1-knockout mutant strain exhibited little difference from the wild-type strain DK1622. HPLC revealed that deletion of the groEL2 gene blocked the production of TA, which was present in the groEL1-knockout mutant. The addition of exogenous TA rescued the inhibition and killing abilities of the groEL2-knockout mutant against E. coli. Analysis of GroEL domain-swapping mutants indicated that the C-terminal equatorial domain of GroEL2 was essential for TA production, while the N-terminal equatorial or apical domains of GroEL2 were not sufficient to rescue TA production of the groEL2 knockout.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study.

PubMed

Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

2016-09-09

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. Copyright © 2016 Elsevier Inc. All rights reserved.

The Role of ARX in Human Pancreatic Endocrine Specification

PubMed Central

Gage, Blair K.; Asadi, Ali; Baker, Robert K.; Webber, Travis D.; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J.

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs. PMID:26633894

The Role of ARX in Human Pancreatic Endocrine Specification.

PubMed

Gage, Blair K; Asadi, Ali; Baker, Robert K; Webber, Travis D; Wang, Rennian; Itoh, Masayuki; Hayashi, Masaharu; Miyata, Rie; Akashi, Takumi; Kieffer, Timothy J

2015-01-01

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.

Quasi-free Proton Knockout Reactions on the Oxygen Isotopic Chain

NASA Astrophysics Data System (ADS)

Atar, Leyla; Aumann, Thomas; Bertulani, Carlos; Paschalis, Stefanos; R3B Collaboration

2017-09-01

It is well known from electron-induced knockout data that the single-particle (SP) strength is reduced to about 60-70% for stable nuclei in comparison to the independent particle model due to the presence of short- and long-range correlations. This finding has been confirmed by nuclear knockout reactions using stable and exotic beams, however, with a strong dependency on the proton-neutron asymmetry. The observed strong reduction of SP cross sections for the deeply bound valence nucleons in asymmetric nuclei is theoretically not understood. To understand this dependency quantitatively a complementary approach, quasi-free (QF) knockout reactions in inverse kinematics, is introduced. We have performed a systematic study of spectroscopic strength of oxygen isotopes using QF (p,2p) knockout reactions in complete kinematics at the R3B/LAND setup at GSI with secondary beams containing 13-24O. The oxygen isotopic chain covers a large variation of separ ation energies, which allow a systematic study of SF with respect to isospin asymmetry. We will present results on the (p,2p) cross sections for the entire oxygen isotopic chain obtained from a single experiment. By comparison with the Eikonal reaction theory the SF and reduction factors will be presented. The work is supported by GSI-TU Darmstadt cooperation and BMBF project 05P15RDFN1.

Cystatin C deficiency suppresses tumor growth in a breast cancer model through decreased proliferation of tumor cells.

PubMed

Završnik, Janja; Butinar, Miha; Prebanda, Mojca Trstenjak; Krajnc, Aleksander; Vidmar, Robert; Fonović, Marko; Grubb, Anders; Turk, Vito; Turk, Boris; Vasiljeva, Olga

2017-09-26

Cysteine cathepsins are proteases that, in addition to their important physiological functions, have been associated with multiple pathologies, including cancer. Cystatin C (CstC) is a major endogenous inhibitor that regulates the extracellular activity of cysteine cathepsins. We investigated the role of cystatin C in mammary cancer using CstC knockout mice and a mouse model of breast cancer induced by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium. We showed that the ablation of CstC reduced the rate of mammary tumor growth. Notably, a decrease in the proliferation of CstC knockout PyMT tumor cells was demonstrated ex vivo and in vitro , indicating a role for this protease inhibitor in signaling pathways that control cell proliferation. An increase in phosphorylated p-38 was observed in CstC knockout tumors, suggesting a novel function for cystatin C in cancer development, independent of the TGF-β pathway. Moreover, proteomic analysis of the CstC wild-type and knockout PyMT primary cell secretomes revealed a decrease in the levels of 14-3-3 proteins in the secretome of knock-out cells, suggesting a novel link between cysteine cathepsins, cystatin C and 14-3-3 proteins in tumorigenesis, calling for further investigations.

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model.

PubMed

Jobst-Schwan, Tilman; Schmidt, Johanna Magdalena; Schneider, Ronen; Hoogstraten, Charlotte A; Ullmann, Jeremy F P; Schapiro, David; Majmundar, Amar J; Kolb, Amy; Eddy, Kaitlyn; Shril, Shirlee; Braun, Daniela A; Poduri, Annapurna; Hildebrandt, Friedhelm

2018-01-01

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.

Neutral endopeptidase knockout induces hyperalgesia in a model of visceral pain, an effect related to bradykinin and nitric oxide.

PubMed

Fischer, Hanspeter S; Zernig, Gerald; Hauser, Kurt F; Gerard, Craig; Hersh, Louis B; Saria, Alois

2002-01-01

Neutral endopeptidase (EC3.4.24.11, NEP, enkephalinase) is a zinc-metalloendopeptidase, cleaving a variety of substrates like enkephalins, substance P, and bradykinin. In the brain, NEP is a key enzyme in the degradation of enkephalins. Pharmacological inhibition of NEP-activity causes analgesia resulting from enhanced extracellular enkephalin concentrations. Recently, transgenic mice lacking the enzyme NEP have been developed (Lu, 1995). The present study was designed to investigate the nociceptive behavior of these NEP-knockout mice. Interestingly, NEP-deficient mice did not respond with decreased pain perception, but exhibited hyperalgesia in the hot-plate jump, warm-water tail-withdrawal, and mostnotablyin theacetic-acid writhing test. Inhibition of aminopeptidase N by bestatin reduced writhing in both strains, whereas NEP-inhibition by thiorphan reduced writhing selectively in wild-type mice. Naloxone increased writhing in wild-type but not in knockouts, whereas the bradykinin B2-receptor antagonist HOE140 reduced writhing selectively in NEP-knockouts. Similarly, the nitric oxide synthase inhibitor L-NAME reduced writhing in NEP-knockouts. These results indicate that genetic elimination of NEP, in contrast to pharmacological inhibition, leads to bradykinin-induced hyperalgesia instead of enkephalin-mediated analgesia. Nitric oxide (NO) is suggested to be involved in this process.

Target sequencing and CRISPR/Cas editing reveal simultaneous loss of UTX and UTY in urothelial bladder cancer.

PubMed

Ahn, Jinwoo; Kim, Kwang Hyun; Park, Sanghui; Ahn, Young-Ho; Kim, Ha Young; Yoon, Hana; Lee, Ji Hyun; Bang, Duhee; Lee, Dong Hyeon

2016-09-27

UTX is a histone demethylase gene located on the X chromosome and is a frequently mutated gene in urothelial bladder cancer (UBC). UTY is a paralog of UTX located on the Y chromosome. We performed target capture sequencing on 128 genes in 40 non-metastatic UBC patients. UTX was the most frequently mutated gene (30%, 12/40). Of the genetic alterations identified, 75% were truncating mutations. UTY copy number loss was detected in 8 male patients (22.8%, 8/35). Of the 9 male patients with UTX mutations, 6 also had copy number loss (66.7%). To evaluate the functional roles of UTX and UTY in tumor progression, we designed UTX and UTY single knockout and UTX-UTY double knockout experiments using a CRISPR/Cas9 lentiviral system, and compared the proliferative capacities of two UBC cell lines in vitro. Single UTX or UTY knockout increased cell proliferation as compared to UTX-UTY wild-type cells. UTX-UTY double knockout cells exhibited greater proliferation than single knockout cells. These findings suggest both UTX and UTY function as dose-dependent suppressors of UBC development. While UTX escapes X chromosome inactivation in females, UTY may function as a male homologue of UTX, which could compensate for dosage imbalances.

Cathepsin K Knockout Alleviates Pressure Overload–Induced Cardiac Hypertrophy

PubMed Central

Hua, Yinan; Xu, Xihui; Shi, Guo-Ping; Chicco, Adam J.; Ren, Jun; Nair, Sreejayan

2014-01-01

Evidence from human and animal studies has documented elevated levels of lysosomal cysteine protease cathepsin K in failing hearts. Here, we hypothesized that ablation of cathepsin K mitigates pressure overload–induced cardiac hypertrophy. Cathepsin K knockout mice and their wild-type littermates were subjected to abdominal aortic constriction, resulting in cardiac remodeling (heart weight, cardiomyocyte size, left ventricular wall thickness, and end diastolic and end systolic dimensions) and decreased fractional shortening, the effects of which were significantly attenuated or ablated by cathepsin K knockout. Pressure overload dampened cardiomyocyte contractile function along with decreased resting Ca2+ levels and delayed Ca2+ clearance, which were partly resolved by cathepsin K knockout. Cardiac mammalian target of rapamycin and extracellular signal-regulated kinases (ERK) signaling cascades were upregulated by pressure overload, the effects of which were attenuated by cathepsin K knockout. In cultured H9c2 myoblast cells, silencing of cathepsin K blunted, whereas cathepsin K transfection mimicked phenylephrine–induced hypertrophic response, along with elevated phosphorylation of mammalian target of rapamycin and ERK. In addition, cathepsin K protein levels were markedly elevated in human hearts of end-stage dilated cardiomyopathy. Collectively, our data suggest that cathepsin K ablation mitigates pressure overload–induced hypertrophy, possibly via inhibition of the mammalian target of rapamycin and ERK pathways. PMID:23529168

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

PubMed

Autio, Kaija J; Schmitz, Werner; Nair, Remya R; Selkälä, Eija M; Sormunen, Raija T; Miinalainen, Ilkka J; Crick, Peter J; Wang, Yuqin; Griffiths, William J; Reddy, Janardan K; Baes, Myriam; Hiltunen, J Kalervo

2014-07-01

Cholesterol is catabolized to bile acids by peroxisomal β-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this β-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

EPA Science Inventory

Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

Vermistabilization of sugar beet (Beta vulgaris L) waste produced from sugar factory using earthworm Eisenia fetida: Genotoxic assessment by Allium cepa test.

PubMed

Bhat, Sartaj Ahmad; Singh, Jaswinder; Vig, Adarsh Pal

2015-08-01

In the present study, sugar beet mud (SBM) and pulp (SBP) produced as a waste by-products of the sugar industry were mixed with cattle dung (CD) at different ratios on dry weight basis for vermicomposting with Eisenia fetida. Minimum mortality and highest population of worms were observed in 20:80 (SBM20) mixture of SBM and 10:90 (SBP10) ratios. However, increased percentages of wastes significantly affected the growth and fecundity of worms. Nutrients like nitrogen, phosphorus, sodium, increased from initial feed mixture to final products (i.e., vermicompost), while organic carbon (OC), C:N ratio and electrical conductivity (EC) declined in all the products of vermicomposting. Although there was an increase in the contents of all the heavy metals except copper, chromium, and iron in SBM, the contents were less than the international standards for compost which indicates that the vermicompost can be used in the fields without any ill effects on the soil. Allium cepa root chromosomal aberration assay was used to evaluate the genotoxicity of pre- and post-vermicomposted SBM to understand the effect of vermicomposting on the reduction of toxicity. Genotoxicity analysis of post-vermicomposted samples of SBM revealed 18-75% decline in the aberration frequencies. Scanning electron microscopy (SEM) was recorded to identify the changes in texture in the control and vermicomposted samples. The vermicomposted mixtures in the presence of earthworms confirm more numerous surface irregularities that prove to be good manure.

Anti-Parasitic Activities of Allium sativum and Allium cepa against Trypanosoma b. brucei and Leishmania tarentolae.

PubMed

Krstin, Sonja; Sobeh, Mansour; Braun, Markus Santhosh; Wink, Michael

2018-04-21

Background: Garlics and onions have been used for the treatment of diseases caused by parasites and microbes since ancient times. Trypanosomiasis and leishmaniasis are a concern in many areas of the world, especially in poor countries. Methods: Trypanosoma brucei brucei and Leishmania tarentolae were used to investigate the anti-parasitic effects of dichloromethane extracts of Allium sativum (garlic) and Allium cepa (onion) bulbs. As a confirmation of known antimicrobial activities, they were studied against a selection of G-negative, G-positive bacteria and two fungi. Chemical analyses were performed using high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (LC-ESI-MS/MS). Results: Chemical analyses confirmed the abundance of several sulfur secondary metabolites in garlic and one (zwiebelane) in the onion extract. Both extracts killed both types of parasites efficiently and inhibited the Trypanosoma brucei trypanothione reductase irreversibly. In addition, garlic extract decreased the mitochondrial membrane potential in trypanosomes. Garlic killed the fungi C. albicans and C. parapsilosis more effectively than the positive control. The combinations of garlic and onion with common trypanocidal and leishmanicidal drugs resulted in a synergistic or additive effect in 50% of cases. Conclusion: The mechanism for biological activity of garlic and onion appears to be related to the amount and the profile of sulfur-containing compounds. It is most likely that vital substances inside the parasitic cell, like trypanothione reductase, are inhibited through disulfide bond formation between SH groups of vital redox compounds and sulfur-containing secondary metabolites.

Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin-like proteins.

PubMed

Pérez-Munive, Clara; Blumenthal, Sonal S D; de la Espina, Susana Moreno Díaz

2012-01-01

Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.

Ameliorating effects of ethyl acetate fraction from onion (Allium cepa L.) flesh and peel in mice following trimethyltin-induced learning and memory impairment.

PubMed

Park, Seon Kyeong; Jin, Dong Eun; Park, Chang Hyeon; Seung, Tae Wan; Guo, Tian Jiao; Song, Jong Wook; Kim, Jong Hwan; Kim, Dae Ok; Heo, Ho Jin

2015-09-01

The anti-amnesic effects of onion (Allium cepa L.) flesh (OF) 1 and peel (OP) 2 on trimethyltin (TMT) 3 -induced learning and memory dysfunction were investigated to confirm learning and memory function. The inhibitory effect against cellular acetylcholinesterase (AChE) 4 showed that the EtOAc fraction of OP (EOP 5 , IC 50 value=37.11μg/mL) was higher than the EtOAc fraction of OF (EOF 6 , IC 50 value=433.34μg/mL). The cognitive effects in ICR mice were also evaluated using Y-maze, passive avoidance, and Morris water maze tests. After the behavioral tests, AChE activity (control=100%, TMT=128%, EOF 20=108%, EOP 10=104%, and EOP 20=98%), superoxide dismutase (SOD) 7 activity, oxidized glutathione (GSSG) 8 /total glutathione (GSH) 9 and malondialdehyde (MDA) 10 production were examined. These results indicate that both EOF and EOP improved learning and memory function. The main compounds of the EOF and EOP were analyzed by Q-TOF UPLC/MS, and the results were as follows: The EOF (quercetin and quercetin-4'-glucoside) and the EOP (quercetin-4'-glucoside and isorhamnetin-4'-glucoside). Consequently, our results suggest that both EOF and EOP could be efficacious in improving cognitive function through AChE inhibition and antioxidant activity in mice brains. Copyright © 2015 Elsevier Ltd. All rights reserved.

De Novo Transcriptome Analysis of Allium cepa L. (Onion) Bulb to Identify Allergens and Epitopes

PubMed Central

Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman

2015-01-01

Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37–100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins. PMID:26284934

The role of gamma irradiation on the extraction of phenolic compounds in onion (Allium cepa L.)

NASA Astrophysics Data System (ADS)

Yang, Eun In; Lee, Eun Mi; Kim, Young Soo; Chung, Byung Yeoup

2012-08-01

The effect of gamma irradiation on the content of total phenolic compounds, especially quercetin (Q), in onion (Allium cepa L.) skin was investigated. Onion skin extracts contained two predominant flavonoid compounds, Q and quercetin-4'-glucoside (Q4'G). After 10 kGy gamma irradiation, the yield of Q in the extracts increased significantly from 36.8 to 153.9 μg/ml of the extract, and the Q4'G content decreased slightly from 165.0 to 134.1 μg/ml. In addition, the total phenolic compound content also increased after irradiation at 10 kGy, from 228.0 μg/g of fresh weight to 346.6 μg/g; negligible changes (237.1-256.7 μg/g) occurred at doses of up to 5 kGy. As we expected, radical-scavenging activity was enhanced remarkably (by 88.8%) in the 10 kGy irradiated sample. A dose-dependent increase in the peak intensity of the electron paramagnetic resonance (EPR) spectra was observed in all irradiated samples, with a maximum increase at 10 kGy. The intensity relative to that of the control was 0.15, and it increased to 1.10 in 10 kGy irradiated samples. The optimum gamma irradiation dose, which is sufficient to break the chemical or physical bonds and release soluble phenols of low molecular weight in onion skin, is about 10 kGy.

Cytotoxicity and genotoxicity of Guaribas river water (Piauí, Brazil), influenced by anthropogenic action.

PubMed

de Castro E Sousa, João Marcelo; Peron, Ana Paula; da Silva E Sousa, Louridânya; de Moura Holanda, Mércia; de Macedo Vieira Lima, Ataíde; de Oliveira, Vitor Alves; da Silva, Felipe Cavalcanti Carneiro; de Morais Lima, Leonardo Henrique Guedes; Matos, Leomá Albuquerque; de Moura Dantas, Sandra Maria Mendes; de Aguiar, Raí Pablo Sousa; Islam, Muhammad Torequl; de Carvalho Melo-Cavalcante, Ana Amélia; Bonecker, Cláudia Costa; Junior, Horácio Ferreira Júlio

2017-06-01

In general, tropical rivers have a great impact on human activities. Bioaccumulation of toxins is a worldwide problem nowadays and has been, historically, overlooked by the supervisory authorities. This study evaluated cytogenotoxic effects of Guaribas river (a Brazilian river) water during dry and rainy seasons of 2014 by using the Allium cepa test system. The toxicogenetic variables, including root growth, mitotic index, and chromosomal aberrations, were analyzed in meristematic cells of A. cepa exposed to water samples taken from the up-, within, and downstream of the city Picos (state: Piauí). The physical-chemical parameters were also analyzed to explain water quality and possible anthropogenic action. Additionally, the presence of heavy metals was also analyzed to explain water quality and possible damaging effects on eukaryotic cells. The results suggest that the river water exerted cytotoxic, mutagenic, and genotoxic effects, regardless of the seasons. In addition, Guaribas river presented physico-chemical values outside the Brazilian laws, which can be a characteristic of human pollution (domestic sewage, industrial, and local agriculture). The genetic damage was positively correlated with higher levels of heavy metals. The pollution of the Guaribas river water may link to the chemical contamination, including the action of heavy metals and their impacts on genetic instability in the aquatic ecosystem. In conclusion, necessary steps should be taken into account for further toxicogenetic studies of the Guaribas river water, as it has an influence in human health of the same region of Brazil.

Effects of onion (Allium cepa L.) and garlic (Allium sativum L.) essential oils on the Aspergillus versicolor growth and sterigmatocystin production.

PubMed

Kocić-Tanackov, Sunčica; Dimić, Gordana; Lević, Jelena; Tanackov, Ilija; Tepić, Aleksandra; Vujičić, Biserka; Gvozdanović-Varga, Jelica

2012-05-01

In the present study the effects of individual and combined essential oils (EOs) extracted from onion (Allium cepa L.) bulb and garlic (Allium sativum L.) clove on the growth of Aspergillus versicolor and sterigmatocystin (STC) production were investigated. The EOs obtained by hydrodistillation were analyzed by GC/MS. Twenty one compounds were identified in onion EO. The major components were: dimethyl-trisulfide (16.64%), methyl-propyl-trisulfide (14.21%), dietil-1,2,4-tritiolan (3R,5S-, 3S,5S- and 3R,5R- isomers) (13.71%), methyl-(1-propenyl)-disulfide (13.14%), and methyl-(1-propenyl)-trisulfide (13.02%). The major components of garlic EO were diallyl-trisulfide (33.55%), and diallyl-disulfide (28.05%). The mycelial growth and the STC production were recorded after 7, 14, and 21 d of the A. versicolor growth in Yeast extract sucrose (YES) broth containing different EOs concentrations. Compared to the garlic EO, the onion EO showed a stronger inhibitory effect on the A. versicolor mycelial growth and STC production. After a 21-d incubation of fungi 0.05 and 0.11 μg/mL of onion EO and 0.11 μg/mL of garlic EO completely inhibited the A. versicolor mycelial growth and mycotoxins biosynthesis. The combination of EOs of onion (75%) and garlic (25%) had a synergistic effect on growth inhibition of A. versicolor and STC production. © 2012 Institute of Food Technologists®

Deletion of Gαq in the telencephalon alters specific neurobehavioral outcomes.

PubMed

Graham, Devon L; Buendia, Matthew A; Chapman, Michelle A; Durai, Heather H; Stanwood, Gregg D

2015-09-01

G(αq) -coupled receptors are ubiquitously expressed throughout the brain and body, and it has been shown that these receptors and associated signaling cascades are involved in a number of functional outputs, including motor function and learning and memory. Genetic alterations to G(αq) have been implicated in neurodevelopmental disorders such as Sturge-Weber syndrome. Some of these associated disease outcomes have been modeled in laboratory animals, but as G(αq) is expressed in all cell types, it is difficult to differentiate the underlying circuitry or causative neuronal population. To begin to address neuronal cell type diversity in G(αq) function, we utilized a conditional knockout mouse whereby G(αq) was eliminated from telencephalic glutamatergic neurons. Unlike the global G(αq) knockout mouse, we found that these conditional knockout mice were not physically different from control mice, nor did they exhibit any gross motor abnormalities. However, similarly to the constitutive knockout animal, G(αq) conditional knockout mice demonstrated apparent deficits in spatial working memory. Loss of G(αq) from glutamatergic neurons also produced enhanced sensitivity to cocaine-induced locomotion, suggesting that cortical G(αq) signaling may limit behavioral responses to psychostimulants. Screening for a variety of markers of forebrain neuronal architecture revealed no obvious differences in the conditional knockouts, suggesting that the loss of G(αq) in telencephalic excitatory neurons does not result in major alterations in brain structure or neuronal differentiation. Taken together, our results define specific modulation of spatial working memory and psychostimulant responses through disruptions in G(αq) signaling within cerebral cortical glutamatergic neurons. © 2015 Wiley Periodicals, Inc.

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice

PubMed Central

Gerard-O'Riley, Rita L.; Acton, Dena; McQueen, Amie K.; Strobel, Isabel E.; Witcher, Phillip C.; Feng, Jian Q.; Econs, Michael J.

2017-01-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. PMID:28005411

Dietary phosphate restriction normalizes biochemical and skeletal abnormalities in a murine model of tumoral calcinosis.

PubMed

Ichikawa, Shoji; Austin, Anthony M; Gray, Amie K; Allen, Matthew R; Econs, Michael J

2011-12-01

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis.

Dietary Phosphate Restriction Normalizes Biochemical and Skeletal Abnormalities in a Murine Model of Tumoral Calcinosis

PubMed Central

Austin, Anthony M.; Gray, Amie K.; Allen, Matthew R.; Econs, Michael J.

2011-01-01

Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis. PMID:22009723

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice.

PubMed

Ichikawa, Shoji; Gerard-O'Riley, Rita L; Acton, Dena; McQueen, Amie K; Strobel, Isabel E; Witcher, Phillip C; Feng, Jian Q; Econs, Michael J

2017-03-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. Copyright © 2017 by the Endocrine Society.

Protein arginine methyltransferase 1 modulates innate immune responses through regulation of peroxisome proliferator-activated receptor γ-dependent macrophage differentiation.

PubMed

Tikhanovich, Irina; Zhao, Jie; Olson, Jody; Adams, Abby; Taylor, Ryan; Bridges, Brian; Marshall, Laurie; Roberts, Benjamin; Weinman, Steven A

2017-04-28

Arginine methylation is a common posttranslational modification that has been shown to regulate both gene expression and extranuclear signaling events. We recently reported defects in protein arginine methyltransferase 1 (PRMT1) activity and arginine methylation in the livers of cirrhosis patients with a history of recurrent infections. To examine the role of PRMT1 in innate immune responses in vivo , we created a cell type-specific knock-out mouse model. We showed that myeloid-specific PRMT1 knock-out mice demonstrate higher proinflammatory cytokine production and a lower survival rate after cecal ligation and puncture. We found that this defect is because of defective peroxisome proliferator-activated receptor γ (PPARγ)-dependent M2 macrophage differentiation. PPARγ is one of the key transcription factors regulating macrophage polarization toward a more anti-inflammatory and pro-resolving phenotype. We found that PRMT1 knock-out macrophages failed to up-regulate PPARγ expression in response to IL4 treatment resulting in 4-fold lower PPARγ expression in knock-out cells than in wild-type cells. Detailed study of the mechanism revealed that PRMT1 regulates PPARγ gene expression through histone H4R3me2a methylation at the PPARγ promoter. Supplementing with PPARγ agonists rosiglitazone and GW1929 was sufficient to restore M2 differentiation in vivo and in vitro and abrogated the difference in survival between wild-type and PRMT1 knock-out mice. Taken together these data suggest that PRMT1-dependent regulation of macrophage PPARγ expression contributes to the infection susceptibility in PRMT1 knock-out mice. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

PubMed

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

2016-08-05

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Lithium ameliorates open-field and elevated plus maze behaviors, and brain phospho-glycogen synthase kinase 3-beta expression in fragile X syndrome model mice.

PubMed

Chen, Xi; Sun, Weiwen; Pan, Ying; Yang, Quan; Cao, Kaiyi; Zhang, Jin; Zhang, Yizhi; Chen, Mincong; Chen, Feidi; Huang, Yueling; Dai, Lijun; Chen, Shengqiang

2013-10-01

To investigate whether lithium modifies open-field and elevated plus maze behavior, and brain phospho-glycogen synthase kinase 3 (P-GSK3beta) expression in Fmr1 knockout mice. One hundred and eighty FVB mice, including knockout and wild type, with an age of 30 days were used. An open-field and elevated plus maze was utilized to test behavior, while western blot was used to measure the P-GSK3beta expression. Six groups were formed: control (saline), lithium chloride 30, 60, 90, 120, and 200 mg/kg. The experiments were carried out in the Institute of Neuroscience, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China between January and June 2012. Lithium significantly decreased total distance, crossing, central area time, and center entry in the open-field test (p<0.05), and significantly reduced open-arm tracking, open-arm entry, and open-arm time in the elevated plus maze (p<0.05) in knockout mice. In wild type mice, significant changes were observed in both behavior tests in some treatment groups. Lithium ameliorated P-GSK3beta expression in the hippocampus of all the treatment groups in knockout mice (p<0.05). However, lithium did not modify either GSK3beta expression in tissues of knockout mice, or P-GSK3beta or GSK3beta expression in tissues of wild type mice. Lithium ameliorated open-field and elevated plus maze behaviors of Fmr1 knockout mice. This effect may be related to its enhancement of P-GSK3beta expression. Our findings suggest that lithium might have a therapeutic effect in fragile X syndrome.

Effects of HAb18G/CD147 knockout on hepatocellular carcinoma cells in vitro using a novel zinc-finger nuclease-targeted gene knockout approach.

PubMed

Li, Hong-Wei; Yang, Xiang-Min; Tang, Juan; Wang, Shi-Jie; Chen, Zhi-Nan; Jiang, Jian-Li

2015-03-01

HAb18G/CD147 belongs to the immunoglobulin superfamily and predominantly functions as an inducer of matrix metalloproteinase secretion for tumor invasion and metastasis. This study was designed to investigate the effects of HAb18G/CD147 knockout on hepatocellular carcinoma cells using zinc-finger nuclease (ZFNs)-targeted gene knockout approach. The HCC cell line SMMC-7721 was used for ZFNs-targeted cleavage of the HAb18G/CD147 gene. RT-PCR and Western blot assays were used to detect HAb18G/CD147 expression. HAb18G phenotypic changes following HAb18G/CD147 knockout in SMMC-K7721 cells were assessed using tumor cell adhesion, invasion, migration and colony formation and flow cytometric assays. These data demonstrated that tumor cell adhesion, invasion, migration, and colony formation capabilities of SMMC-K7721 were significantly reduced compared to parental cells or SMMC-7721 with re-expression of HAb18G/CD147 protein transfected with HAb18G/CD147 cDNA. Moreover, knockout of HAb18G/CD147 expression also induced SMMC-K7721 cells to undergo apoptosis compared to SMMC-7721 and SMMC-R7721 (P < 0.01). Molecularly, protein expression of p53 was induced in these cells, but re-expression of HAb18G/CD147 reduced p53 levels in SMMC-R7721 cells, possibly through inhibition of the PI3K-Akt-MDM2 signaling pathway. The findings provide a novel insight into the mechanisms underlying HAb18G/CD147-induced progression of HCC cells.

Akt2 Knockout Alleviates Prolonged Caloric Restriction-Induced Change in Cardiac Contractile Function through Regulation of Autophagy

PubMed Central

Zhang, Yingmei; Han, Xuefeng; Hu, Nan; Huff, Anna F.; Gao, Feng; Ren, Jun

2014-01-01

Caloric restriction leads to changes in heart geometry and function although the underlying mechanism remains elusive. Autophagy, a conserved pathway for degradation of intracellular proteins and organelles, preserves energy and nutrient in the face of caloric insufficiency. This study was designed to examine the role of Akt2 in prolonged caloric restriction-induced change in cardiac homeostasis and the underlying mechanism(s) involved. Wild-type (WT) and Akt2 knockout mice were caloric restricted (by 40%) for 30 weeks. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties, autophagy and its regulatory proteins were evaluated. Caloric restriction compromised echocardiographic indices (decreased left ventricular mass, left ventricular diameters and cardiac output), cardiomyocyte contractile and intracellular Ca2+ properties associated with dampened SERCA2a phosphorylation, upregulated phospholamban and autophagy (Beclin-1, Atg7, LC3BII-to-LC3BI ratio), increased autophagy adaptor protein p62, elevated phosphorylation of AMPK, Akt2 and the Akt downstream signal molecule TSC2, the effects of which with the exception of autophagy protein markers (Beclin-1, Atg7, LC3B) and AMPK were mitigated or significantly alleviated by Akt2 knockout. Lysosomal inhibition using bafilomycin A1 negated Akt2 knockout-induced protective effect on p62. Evaluation of downstream signaling molecules of Akt and AMPK including mTOR and ULK1 revealed that caloric restriction suppressed and promoted phosphorylation of mTOR and ULK1, respectively, without affecting total mTOR and ULK1 expression. Akt2 knockout significantly augmented caloric restriction-induced responses on mTOR and ULK1. Taken together, these data suggest a beneficial role of Akt2 knockout in preservation of cardiac homeostasis against prolonged caloric restriction-induced pathological changes possibly through facilitating autophagy. PMID:24368095

Protection of the Villus Epithelial Cells of the Small Intestine from Rotavirus Infection Does Not Require Immunoglobulin A

PubMed Central

O'Neal, Christine M.; Harriman, Gregory R.; Conner, Margaret E.

2000-01-01

Immunoglobulin A (IgA) is the primary immune response induced in the intestine by rotavirus infection, but vaccination with virus-like particles induces predominantly IgG, not IgA. To definitively assess the role of IgA in protection from rotavirus infection, IgA knockout mice, which are devoid of serum and secretory IgA, were infected and then rechallenged with murine rotavirus at either 6 weeks or 10 months. Following primary rotavirus infection, IgA knockout mice cleared virus as effectively as IgA normal control mice. Rotavirus-infected IgA knockout mice produced no serum or fecal IgA but did have high levels of antirotavirus serum IgG and IgM and fecal IgG, whereas IgA normal control mice made both serum IgA and IgG and fecal IgA. Both IgA normal and IgA knockout mice were totally protected from rotavirus challenge at 42 days. Ten months following a primary infection, both IgA normal and knockout mice still had high levels of serum and fecal antirotavirus antibody and were totally protected from rotavirus challenge. To determine if compensatory mechanisms other than IgG were responsible for protection from rotavirus infection in IgA knockout mice, mice were depleted of CD4+ T cells or CD8+ T cells. No changes in the level of protection were seen in depleted mice. These data show that fecal or systemic IgA is not essential for protection from rotavirus infection and suggest that in the absence of IgA, IgG may play a significant role in protection from mucosal pathogens. PMID:10756022

Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

PubMed

Baym, Michael; Shaket, Lev; Anzai, Isao A; Adesina, Oluwakemi; Barstow, Buz

2016-11-10

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs.

PubMed

Zuo, Erwei; Cai, Yi-Jun; Li, Kui; Wei, Yu; Wang, Bang-An; Sun, Yidi; Liu, Zhen; Liu, Jiwei; Hu, Xinde; Wei, Wei; Huo, Xiaona; Shi, Linyu; Tang, Cheng; Liang, Dan; Wang, Yan; Nie, Yan-Hong; Zhang, Chen-Chen; Yao, Xuan; Wang, Xing; Zhou, Changyang; Ying, Wenqin; Wang, Qifang; Chen, Ren-Chao; Shen, Qi; Xu, Guo-Liang; Li, Jinsong; Sun, Qiang; Xiong, Zhi-Qi; Yang, Hui

2017-07-01

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

Genetic cathepsin B deficiency reduces beta-amyloid in transgenic mice expressing human wild-type amyloid precursor protein.

PubMed

Hook, Vivian Y H; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

2009-08-21

Neurotoxic beta-amyloid (Abeta) peptides participate in Alzheimer's disease (AD); therefore, reduction of Abeta generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Abeta may identify targets for reducing Abeta. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decreases in brain Abeta40 and Abeta42 by 67% and decreases in levels of the C-terminal beta-secretase fragment (CTFbeta) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Abeta. The difference in reduction of Abeta in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Abeta in AD.

Mapping Pathological Phenotypes in a Mouse Model of CDKL5 Disorder

PubMed Central

Amendola, Elena; Zhan, Yang; Mattucci, Camilla; Castroflorio, Enrico; Calcagno, Eleonora; Fuchs, Claudia; Lonetti, Giuseppina; Silingardi, Davide; Vyssotski, Alexei L.; Farley, Dominika; Ciani, Elisabetta; Pizzorusso, Tommaso; Giustetto, Maurizio; Gross, Cornelius T.

2014-01-01

Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder. PMID:24838000

Impaired fear extinction learning in adult heterozygous BDNF knock-out mice.

PubMed

Psotta, Laura; Lessmann, Volkmar; Endres, Thomas

2013-07-01

Brain-derived neurotrophic factor (BDNF) is a crucial regulator of neuroplasticity, which underlies learning and memory processes in different brain areas. To investigate the role of BDNF in the extinction of amygdala-dependent cued fear memories, we analyzed fear extinction learning in heterozygous BDNF knock-out mice, which possess a reduction of endogenous BDNF protein levels to ~50% of wild-type animals. Since BDNF expression has been shown to decline with aging of animals, we tested the performance in extinction learning of these mice at 2 months (young adults) and 7 months (older adults) of age. The present study shows that older adult heterozygous BDNF knock-out mice, which have a chronic 50% lack of BDNF, also possess a deficit in the acquisition of extinction memory, while extinction learning remains unaffected in young adult heterozygous BDNF knock-out mice. This deficit in extinction learning is accompanied by a reduction of BDNF protein in the hippocampus, amygdala and the prefrontal cortex. Copyright © 2013 Elsevier Inc. All rights reserved.

The effect of PDIA3 gene knockout on the mucosal immune function in IBS rats.

PubMed

Zhuang, Zhao-Meng; Wang, Xiao-Teng; Zhang, Lu; Tao, Li-Yuan; Lv, Bin

2015-01-01

To observe the changes of intestinal inflammation on PDIA3 gene knockout IBS rats and its effect on immune function. 36 SD rats were randomly divided into four groups: the control group (n = 8); IBS- empty virus group (IBS-GFP, which); IBS-PDIA3 knockout group (n = 12); IBS- the control group (n = 12). After modeling, colon and ileocecal tissue pathology in each group were observed separately. Changes of immune and inflammatory markers were measured. At the same time, ultrastructural changes in each group were observed by electron microscopy. Compared with the IBS control group, inflammation was reduced significantly in IBS-PDIA3 knockout group. IgE, IL-4 and IL-9 and the level of intestinal trypsin type were decreased significantly. Furthermore, mast cell degranulation and PAR 2 receptor reduced significantly. PDIA3 may play an important role in the development of IBS by mediating through immune responses of mucosal abnormalities. However, the mechanism needs to be confirmed in further study.

Effect of spices formulations on the physicochemical and sensory properties of Nnam gon, a Cameroonian dish prepared with cucurbitaceae seeds.

PubMed

Manejo Djiogue, Josiane E; Nguimbou, Richard M; Tatsadjieu, Leopold N; Mang, Yannick D; Njintang, Nicolas Y

2017-05-01

Nnam gon , a cake made by steam cooking a mixture of Cucurbitaceae seeds paste and others ingredients specially spices, is a highly prized dish in Central African region. A preliminary investigation conducted as part of this study highlighted that formulations used in the processing of Nnam gon vary according to the spices used. This study was carried out to determine the best formulation for the preparation of this dish. For this purpose, Nnam gon samples were produced from four formulations which differ according to the number of spices used: F0 (no spices); F1 (0.91 g of Allium cepa paste); F2 (0.88 g of A. cepa paste, 0.35 g of Allium sativum paste, 0.41 g of fresh Officinale zingiber paste, 0.41 g of fresh Petroselinum crispum paste, 0.33 g of Monodora myristica , 0.48 g of fresh Celery graveolens paste, 1.19 g of fresh Allium porrum paste, 0.13 g of Allium lepidophyllus powder, and 0.13 g of Piper nigrum ), and F3 (0.90 g of A. cepa paste, 0.35 g of A. sativum paste, and 0.42 g of fresh O. zingiber paste). The samples were evaluated for their physicochemical characteristics and sensory profile (Quantitative Descriptive Analysis). The results revealed that proteins (16.56-17.38%), carbohydrates (4.71-5.10%), lipids (23.14-24.25%), ash (4.03-5.92%), and fibers (2.17-2.68%) increased significantly ( p  < .05) with spices adding. The increase in polyphenols (310.55-592.80 mg/100 g FM) and phytates (2.23-12.49 mg/100 g FM) contents was positively correlated with antioxidant properties of Nnam gon which also increased with spices adding. Significant differences were observed between the samples for all attributes generated (appearance, odor, taste, flavor, texture, and oral texture). Spices adding induced a decrease in hardness, cohesivity, elasticity, and granulous of cake but enhanced oily. Nnam gon produced with spicy formulation (F2 and F3) had higher mean score for general acceptance which was highly correlated ( p  < .05) with spice odor ( r  = .99), spice taste ( r  = .92), and color ( r  = .84). From this study, it is suggested that spicy Cucurbitaceae paste could improve nutritional value, antioxidant properties, and general acceptance of Nnam gon .

Effect of Allium cepa and Allium sativum on some immunological cells in rats.

PubMed

Mirabeau, Tatfeng Y; Samson, Enitan S

2012-01-01

Extracts of some spices have been reported to play a contributory role in enhancing immune function. We evaluated and compared the effect(s) of single and combined oral administration of fresh aqueous onion (Allium cepa) and garlic (Allium sativum) extracts at different concentrations on some immunological determinants in rats. CD₄ cells of the rats were estimated using Partec flow cytometric technique, while total and differential white blood cell (WBC) counts were estimated using the Sysmsex® automated haematology analyzing technique. Our findings revealed that, CD4 and total WBC counts were significantly increased (P≤0.05) in a dose-dependent manner in both onion (250mg/Kg/d: 349±11cell/ul and 2.75±0.15X10³cell/l; 500mg/Kg/d: 389±10cells/µl and 3.05±0.05 X10³cell/l; 750mg/Kg/d: 600±11cell/µl and 3.25±0.05X10³cells/l) and garlic (250mg/Kg/d: 410±10cell/ul and 2.85±0.15X10³cell/l; 500mg/Kg/d: 494±32cells/µl and 3.30±0.10 X10³cell/l; 750mg/Kg/d: 684±11cell/µl and 3.55±0.05X10³cells/l) treated rats when compared to the zero control (200±11cells/µl and 1.55±0.05X10³cells/l, respectively). Extract of garlic at 750mg/Kg/d had significantly increased the CD4 cells and total white cell count when compared to other concentrations (P≤0.05). However, no significant effect was observed on these parameters when extracts were combined (250mg/Kg/d: 252±21cell/µl and 1.80±0.10X10³cells/l; 500mg/Kg/d: 315±21cells/ul and 2.10±0.10X10³cells/l; 750mg/Kg/d: 368±10cells/µl and 2.35±0.05X10³cells/l, respectively), the differential WBC count showed a significant increase in the proportion of cell types (lymphocytes, neutophils and monocytes) (P≤0.05). The results from this study revealed the immune boosting capabilities of Allium cepa and Allium sativum, but underscored their synergistic activities.

Deletion of protein tyrosine phosphatase 1B rescues against myocardial anomalies in high fat diet-induced obesity: Role of AMPK-dependent autophagy.

PubMed

Kandadi, Machender R; Panzhinskiy, Evgeniy; Roe, Nathan D; Nair, Sreejayan; Hu, Dahai; Sun, Aijun

2015-02-01

Obesity-induced cardiomyopathy may be mediated by alterations in multiple signaling cascades involved in glucose and lipid metabolism. Protein tyrosine phosphatase-1B (PTP1B) is an important negative regulator of insulin signaling. This study was designed to evaluate the role of PTP1B in high fat diet-induced cardiac contractile anomalies. Wild-type and PTP1B knockout mice were fed normal (10%) or high (45%) fat diet for 5months prior to evaluation of cardiac function. Myocardial function was assessed using echocardiography and an Ion-Optix MyoCam system. Western blot analysis was employed to evaluate levels of AMPK, mTOR, raptor, Beclin-1, p62 and LC3-II. RT-PCR technique was employed to assess genes involved in hypertrophy and lipid metabolism. Our data revealed increased LV thickness and LV chamber size as well as decreased fractional shortening following high fat diet intake, the effect was nullified by PTP1B knockout. High fat diet intake compromised cardiomyocyte contractile function as evidenced by decreased peak shortening, maximal velocity of shortening/relengthening, intracellular Ca²⁺ release as well as prolonged duration of relengthening and intracellular Ca²⁺ decay, the effects of which were alleviated by PTP1B knockout. High fat diet resulted in enlarged cardiomyocyte area and increased lipid accumulation, which were attenuated by PTP1B knockout. High fat diet intake dampened myocardial autophagy as evidenced by decreased LC3-II conversion and Beclin-1, increased p62 levels as well as decreased phosphorylation of AMPK and raptor, the effects of which were significantly alleviated by PTP1B knockout. Pharmacological inhibition of AMPK using compound C disengaged PTP1B knockout-conferred protection against fatty acid-induced cardiomyocyte contractile anomalies. Taken together, our results suggest that PTP1B knockout offers cardioprotection against high fat diet intake through activation of AMPK. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of melanopsin in circadian responses to light.

PubMed

Ruby, Norman F; Brennan, Thomas J; Xie, Xinmin; Cao, Vinh; Franken, Paul; Heller, H Craig; O'Hara, Bruce F

2002-12-13

Melanopsin has been proposed as an important photoreceptive molecule for the mammalian circadian system. Its importance in this role was tested in melanopsin knockout mice. These mice entrained to a light/dark cycle, phase-shifted after a light pulse, and increased circadian period when light intensity increased. Induction of the immediate-early gene c-fos was observed after a nighttime light pulse in both wild-type and knockout mice. However, the magnitude of these behavioral responses in knockout mice was 40% lower than in wild-type mice. Although melanopsin is not essential for the circadian clock to receive photic input, it contributes significantly to the magnitude of photic responses.

Serotonin Transporter Knockout Rats Show Improved Strategy Set-Shifting and Reduced Latent Inhibition

ERIC Educational Resources Information Center

Nonkes, Lourens J. P.; van de Vondervoort, Ilse I. G. M.; de Leeuw, Mark J. C.; Wijlaars, Linda P.; Maes, Joseph H. R.; Homberg, Judith R.

2012-01-01

Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT[superscript -/-]) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility, extradimensional strategy set-shifting…

CB2 Cannabinoid Receptor Knockout in Mice Impairs Contextual Long-Term Memory and Enhances Spatial Working Memory

PubMed Central

Li, Yong; Kim, Jimok

2016-01-01

Neurocognitive effects of cannabinoids have been extensively studied with a focus on CB1 cannabinoid receptors because CB1 receptors have been considered the major cannabinoid receptor in the nervous system. However, recent discoveries of CB2 cannabinoid receptors in the brain demand accurate determination of whether and how CB2 receptors are involved in the cognitive effects of cannabinoids. CB2 cannabinoid receptors are primarily involved in immune functions, but also implicated in psychiatric disorders such as schizophrenia and depression. Here, we examined the effects of CB2 receptor knockout in mice on memory to determine the roles of CB2 receptors in modulating cognitive function. Behavioral assays revealed that hippocampus-dependent, long-term contextual fear memory was impaired whereas hippocampus-independent, cued fear memory was normal in CB2 receptor knockout mice. These mice also displayed enhanced spatial working memory when tested in a Y-maze. Motor activity and anxiety of CB2 receptor knockout mice were intact when assessed in an open field arena and an elevated zero maze. In contrast to the knockout of CB2 receptors, acute blockade of CB2 receptors by AM603 in C57BL/6J mice had no effect on memory, motor activity, or anxiety. Our results suggest that CB2 cannabinoid receptors play diverse roles in regulating memory depending on memory types and/or brain areas. PMID:26819779

Critical role of FcR gamma-chain, LAT, PLCgamma2 and thrombin in arteriolar thrombus formation upon mild, laser-induced endothelial injury in vivo.

PubMed

Kalia, Neena; Auger, Jocelyn M; Atkinson, Ben; Watson, Steve P

2008-05-01

The role of collagen receptor complex GPVI-FcR gamma-chain, PLCgamma2 and LAT in laser-induced thrombosis is unclear. Controversy surrounds whether collagen is exposed in this model or whether thrombosis is dependent on thrombin. This study hypothesized that collagen exposure plays a critical role in thrombus formation in this model, which was tested by investigating contributions of FcR gamma-chain, LAT, PLCgamma2 and thrombin. Thrombi were monitored using intravital microscopy in anesthetized wild-type and FcR gamma-chain, LAT and PLCgamma2 knockout mice. Hirudin (thrombin inhibitor) was administered to wild-type and FcR gamma-chain knockout mice. Significantly reduced thrombus formation was observed in FcR gamma-chain and PLCgamma2 knockouts with a greater decrease observed in LAT knockouts. Dramatic reduction was observed in wild-types treated with hirudin, with abolished thrombus formation only observed in FcR gamma-chain knockouts treated with hirudin. GPVI-FcR gamma-chain, LAT and PLCgamma2 are essential for thrombus generation and stability in this laser-induced model of injury. More importantly, a greater role for LAT was identified, which may reflect a role for it downstream of a second matrix protein receptor. However, inhibition of platelet activation by matrix proteins and thrombin generation are both required to maximally prevent thrombus formation.

Ontogeny of brain and blood serotonin levels in 5-HT receptor knockout mice: potential relevance to the neurobiology of autism.

PubMed

Janusonis, Skirmantas; Anderson, George M; Shifrovich, Ilya; Rakic, Pasko

2006-11-01

The most consistent neurochemical finding in autism has been elevated group mean levels of blood platelet 5-hydroxytryptamine (5-HT, serotonin). The origin and significance of this platelet hyperserotonemia remain poorly understood. The 5-HT(1A) receptor plays important roles in the developing brain and is also expressed in the gut, the main source of platelet 5-HT. Post-natal tissue levels of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and tryptophan were examined in the brain, duodenum and blood of 5-HT(1A) receptor-knockout and wild-type mice. At 3 days after birth, the knockout mice had lower mean brain 5-HT levels and normal mean platelet 5-HT levels. Also, at 3 days after birth, the mean tryptophan levels in the brain, duodenum and blood of the knockout mice were around 30% lower than those of the wild-type mice. By 2 weeks after birth, the mean brain 5-HT levels of the knockout mice normalized, but their mean platelet 5-HT levels became 24% higher than normal. The possible causes of these dynamic shifts were explored by examining correlations between central and peripheral levels of 5-HT, 5-HIAA and tryptophan. The results are discussed in relation to the possible role of 5-HT in the ontogeny of autism.

A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of human genes.

PubMed

Bisht, Kamlesh; Grill, Sherilyn; Graniel, Jacqueline; Nandakumar, Jayakrishnan

2017-08-01

CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model

PubMed Central

Schneider, Ronen; Hoogstraten, Charlotte A.; Schapiro, David; Majmundar, Amar J.; Kolb, Amy; Eddy, Kaitlyn; Shril, Shirlee; Braun, Daniela A.; Poduri, Annapurna

2018-01-01

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest. PMID:29346415

Macrophage Migration Inhibitory Factor (MIF) Deficiency Exacerbates Aging-Induced Cardiac Remodeling and Dysfunction Despite Improved Inflammation: Role of Autophagy Regulation.

PubMed

Xu, Xihui; Pang, Jiaojiao; Chen, Yuguo; Bucala, Richard; Zhang, Yingmei; Ren, Jun

2016-03-04

Aging leads to unfavorable geometric and functional sequelae in the heart. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) plays a role in the maintenance of cardiac homeostasis under stress conditions although its impact in cardiac aging remains elusive. This study was designed to evaluate the role of MIF in aging-induced cardiac anomalies and the underlying mechanism involved. Cardiac geometry, contractile and intracellular Ca(2+) properties were examined in young (3-4 mo) or old (24 mo) wild type and MIF knockout (MIF(-/-)) mice. Our data revealed that MIF knockout exacerbated aging-induced unfavorable structural and functional changes in the heart. The detrimental effect of MIF knockout was associated with accentuated loss in cardiac autophagy with aging. Aging promoted cardiac inflammation, the effect was attenuated by MIF knockout. Intriguingly, aging-induced unfavorable responses were reversed by treatment with the autophagy inducer rapamycin, with improved myocardial ATP availability in aged WT and MIF(-/-) mice. Using an in vitro model of senescence, MIF knockdown exacerbated doxorubicin-induced premature senescence in H9C2 myoblasts, the effect was ablated by MIF replenishment. Our data indicated that MIF knockout exacerbates aging-induced cardiac remodeling and functional anomalies despite improved inflammation, probably through attenuating loss of autophagy and ATP availability in the heart.

The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis.

PubMed

Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

2016-09-01

The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710.

Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans.

PubMed

Sangaletti, Rachele; D'Amico, Massimo; Grant, Jeff; Della-Morte, David; Bianchi, Laura

2017-08-01

Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions.

[Establishment of L-periaxin gene knock-out RSC96 cell line].

PubMed

Liang, Min; Peng, Tingting; Shi, Yawei

2016-12-25

Periaxin, a protein of noncompact myelin, is specifically expressed in the peripheral nervous system (PNS). There are two protein isoform L-periaxin and S-Periaxin by alternative splicing of periaxin gene, playing an important role in the initiation of myelin formation. So far, 18 different mutation sites in L-periaxin gene have been found to induce the peripheral demyelinating neurological charcot-marie-tooth diseases subtype 4F (CMT4F). The technique of activation of transcription activator-like effector nucleases (TALENS) was used to knock out the L-periaxin gene in RSC 96 cell line of Rattus. According to the design principle, the knock-out site of L-periaxin was assured to NLS domain of L-periaxin, which is target sequence of left and right arms of TALEN. The knock-out vectors of TALEN-L and TALEN-R were established and transfected into RSC96 cell. After puromycin screening, L-periaxin was knocked out successfully in RSC96 cell, which is confirmed by DNA sequence. The mutation efficiency is 21.6%. S-periaxin, not L-periaxin can be detected by Western blotting in L-periaxin gene knock-out RSC96 cell. The cell growth rate was decreased and the number of cells in G1 increased and decreased in S phase in L-periaxin gene knock-out RSC96 cell by flow cytometry and MTT assay.

Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis.

PubMed

Mou, Haiwei; Moore, Jill; Malonia, Sunil K; Li, Yingxiang; Ozata, Deniz M; Hough, Soren; Song, Chun-Qing; Smith, Jordan L; Fischer, Andrew; Weng, Zhiping; Green, Michael R; Xue, Wen

2017-04-04

Genetic lesions that activate KRAS account for ∼30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras Here, we show that tumor cells can survive knockout of oncogenic Kras , indicating the existence of Kras -independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras -independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras -expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles' heel in tumors initiated by oncogenic Kras.

Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis

PubMed Central

Mou, Haiwei; Moore, Jill; Malonia, Sunil K.; Li, Yingxiang; Ozata, Deniz M.; Hough, Soren; Song, Chun-Qing; Smith, Jordan L.; Fischer, Andrew; Weng, Zhiping; Xue, Wen

2017-01-01

Genetic lesions that activate KRAS account for ∼30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras. Here, we show that tumor cells can survive knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras-expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles’ heel in tumors initiated by oncogenic Kras. PMID:28320962

Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

PubMed

Bracht, Thilo; Hagemann, Sascha; Loscha, Marius; Megger, Dominik A; Padden, Juliet; Eisenacher, Martin; Kuhlmann, Katja; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

2014-06-06

The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

PubMed

Gao, Hanchao; Zhao, Chengjiang; Xiang, Xi; Li, Yong; Zhao, Yanli; Li, Zesong; Pan, Dengke; Dai, Yifan; Hara, Hidetaka; Cooper, David K C; Cai, Zhiming; Mou, Lisha

2017-02-16

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Interrater agreement of an observational tool to code knockouts and technical knockouts in mixed martial arts.

PubMed

Lawrence, David W; Hutchison, Michael G; Cusimano, Michael D; Singh, Tanveer; Li, Luke

2014-09-01

Interrater agreement evaluation of a tool to document and code the situational factors and mechanisms of knockouts (KOs) and technical knockouts (TKOs) in mixed martial arts (MMA). Retrospective case series. Professional MMA matches from the Ultimate Fighting Championship-2006-2012. Two nonmedically trained independent raters. The MMA Knockout Tool (MMA-KT) consists of 20 factors and captures and codes information on match characteristics, situational context preceding KOs and TKOs, as well as describing competitor states during these outcomes. The MMA-KT also evaluates the mechanism of action and subsequent events surrounding a KO. The 2 raters coded 125 unique events for a total of 250 events. The 8 factors of Part A had an average κ of 0.87 (SD = 0.10; range = 0.65-0.98); 7 were considered "substantial" agreement and 1 "moderate." Part B consists of 12 factors with an average κ of 0.84 (SD = 0.16; range = 0.59-1.0); 7 classified as "substantial" agreement, 4 "moderate," and 1 "fair." The majority of the factors in the MMA-KT demonstrated substantial interrater agreement, with an average κ of 0.86 (SD = 0.13; range = 0.59-1.0). The MMA-KT is a reliable tool to extract and code relevant information to investigate the situational factors and mechanism of KOs and TKOs in MMA competitions.

Optimal knockout strategies in genome-scale metabolic networks using particle swarm optimization.

PubMed

Nair, Govind; Jungreuthmayer, Christian; Zanghellini, Jürgen

2017-02-01

Knockout strategies, particularly the concept of constrained minimal cut sets (cMCSs), are an important part of the arsenal of tools used in manipulating metabolic networks. Given a specific design, cMCSs can be calculated even in genome-scale networks. We would however like to find not only the optimal intervention strategy for a given design but the best possible design too. Our solution (PSOMCS) is to use particle swarm optimization (PSO) along with the direct calculation of cMCSs from the stoichiometric matrix to obtain optimal designs satisfying multiple objectives. To illustrate the working of PSOMCS, we apply it to a toy network. Next we show its superiority by comparing its performance against other comparable methods on a medium sized E. coli core metabolic network. PSOMCS not only finds solutions comparable to previously published results but also it is orders of magnitude faster. Finally, we use PSOMCS to predict knockouts satisfying multiple objectives in a genome-scale metabolic model of E. coli and compare it with OptKnock and RobustKnock. PSOMCS finds competitive knockout strategies and designs compared to other current methods and is in some cases significantly faster. It can be used in identifying knockouts which will force optimal desired behaviors in large and genome scale metabolic networks. It will be even more useful as larger metabolic models of industrially relevant organisms become available.

In vivo imaging of the mouse model of X-linked juvenile retinoschisis with fourier domain optical coherence tomography.

PubMed

Xu, Jing; Molday, Laurie L; Molday, Robert S; Sarunic, Marinko V

2009-06-01

The purpose of this study was to investigate Fourier domain optical coherence tomography (FD OCT) as a noninvasive tool for retinal imaging in the Rs1h-knockout mouse (model for X-linked juvenile retinoschisis). A prototype spectrometer-based FD OCT system was used in combination with a custom optical beam-scanning platform. Images of the retinas from wild-type and Rs1h-knockout mice were acquired noninvasively with FD OCT with the specimen anesthetized. At the completion of the noninvasive FD OCT imaging, invasive retinal cross-sectional images (histology) were acquired from a nearby region for comparison to the FD OCT images. The retinal layers were identifiable in the FD OCT images, permitting delineation and thickness measurement of the outer nuclear layer (ONL). During FD OCT in vivo imaging of the Rs1h-knockout mouse, holes were observed in the inner nuclear layer (INL), and retinal cell disorganization was observed as a change in the backscattering intensity profile. Comparison of the ONL measurements acquired noninvasively with FD OCT to measurements taken using histology at nearby locations showed a degeneration of roughly 30% of the ONL by the age of 2 months in Rs1h-knockout mice relative to wild-type. FD OCT was demonstrated to be effective for noninvasive imaging of retinal degeneration and observation of retinal holes in Rs1h-knockout mice.

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology

PubMed Central

Hüttemann, Maik; Lee, Icksoo; Gao, Xiufeng; Pecina, Petr; Pecinova, Alena; Liu, Jenney; Aras, Siddhesh; Sommer, Natascha; Sanderson, Thomas H.; Tost, Monica; Neff, Frauke; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Naton, Beatrix; Rathkolb, Birgit; Rozman, Jan; Favor, Jack; Hans, Wolfgang; Prehn, Cornelia; Puk, Oliver; Schrewe, Anja; Sun, Minxuan; Höfler, Heinz; Adamski, Jerzy; Bekeredjian, Raffi; Graw, Jochen; Adler, Thure; Busch, Dirk H.; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Weissmann, Norbert; Doan, Jeffrey W.; Bassett, David J. P.; Grossman, Lawrence I.

2012-01-01

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (−50 and −29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (−12%), reduced total oxygen consumption rate (−8%), improved glucose tolerance, and reduced grip force (−14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.—Hüttemann, M., Lee, I., Gao, X., Pecina, P., Pecinova, A., Liu, J., Aras, S., Sommer, N., Sanderson, T. H., Tost, M., Neff, F., Aguilar-Pimentel, J. A., Becker, L., Naton, B., Rathkolb, B., Rozman, J., Favor, J., Hans, W., Prehn, C., Puk, O., Schrewe, A., Sun, M., Höfler, H., Adamski, J., Bekeredjian, R., Graw, J., Adler, T., Busch, D. H., Klingenspor, M., Klopstock, T., Ollert, M., Wolf, E., Fuchs, H., Gailus-Durner, V., Hrabě de Angelis, M., Weissmann, N., Doan, J. W., Bassett, D. J. P., Grossman, L. I. Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology. PMID:22730437

Blueberries reduce lipid peroxidation and boost antioxidant enzymes in apoe knockout mice

USDA-ARS?s Scientific Manuscript database

ApoE knockout (ApoE-/-) mice fed AIN-93G diet (CD) formulated to contain 1 % freeze-dried whole wild blueberries (CD1 percent BB) were found to have significantly less atherosclerotic lesions in aorta. Biomarkers of lipid peroxidation, including F2-isoprostanes, hydroxyoctadecadienoic acids (HODEs) ...

An interferometer for high-resolution optical surveillance from geostationary orbit

NASA Astrophysics Data System (ADS)

Bonino, L.; Bresciani, F.; Piasini, G.; Flebus, C.; Lecat, J.-H.; Roose, S.; Pisani, M.; Cabral, A.; Rebordão, J.; Proença, C.; Costal, J.; Lima, P. U.; Loix, N.; Musso, F.

2017-11-01

The activities described in this paper have been developed in the frame of the EUCLID CEPA 9 RTP 9.9 "High Resolution Optical Satellite Sensor" project of the WEAO Research Cell. They have been focused on the definition of an interferometric instrument optimised for the high-resolution optical surveillance from geostationary orbit (GEO) by means of the synthetic aperture technique, and on the definition and development of the related enabling technologies. In this paper we describe the industrial team, the selected mission specifications and overview of the whole design and manufacturing activities performed.

Response of animal and vegetative cells to the effect of a typical magnetic storm

NASA Astrophysics Data System (ADS)

Talikina, M. G.; Izyumov, Yu. G.; Krylov, V. V.

2013-12-01

Experimentally reproduced fluctuations of a low-frequency magnetic field in a nanotesla range (magnetic storm) affect the mitosis of animals and vegetative cells. Action of this factor during twenty four hours leads to a significant increase in the proliferative activity of embryo cells in roach ( Rutilus rutilus L.) and meristem cells of onion rootlets ( Allium cepa). The clastogenic effect statistically confirmed only in the Allium test seems to reflect the species specificity of the response and higher sensitivity of the cell association of the onion meristem to magnetic storm.

Orbital period variations of two W UMa-type binaries: UY UMa and EF Boo

NASA Astrophysics Data System (ADS)

Yu, Yun-Xia; Zhang, Xu-Dong; Hu, Ke; Xiang, Fu-Yuan

2017-08-01

The orbital period variations of two W UMa-type contact binaries, UY UMa and EF Boo, are analyzed by using all available times of light minimum. It is detected that the general trends of their (O - C) curves show an upward parabolic variation, which reveals their continuous period increases at the rates of dP / dt = 2.545 ×10-7 days yr-1 and dP / dt = 2.623 ×10-7 days yr-1 , respectively. Meanwhile, UY UMa also shows a cyclic period variation with a small amplitude of A = 0.0026 days superposed on the long-term increase. Due to their contact configurations, the secular period increases are interpreted as a result of mass transfer from the less massive component to the more massive one. The cyclic period variation of UY UMa may be interpreted in terms of either the magnetic activity or the light time effect.

Lymphocyte signaling: beyond knockouts.

PubMed

Saveliev, Alexander; Tybulewicz, Victor L J

2009-04-01

The analysis of lymphocyte signaling was greatly enhanced by the advent of gene targeting, which allows the selective inactivation of a single gene. Although this gene 'knockout' approach is often informative, in many cases, the phenotype resulting from gene ablation might not provide a complete picture of the function of the corresponding protein. If a protein has multiple functions within a single or several signaling pathways, or stabilizes other proteins in a complex, the phenotypic consequences of a gene knockout may manifest as a combination of several different perturbations. In these cases, gene targeting to 'knock in' subtle point mutations might provide more accurate insight into protein function. However, to be informative, such mutations must be carefully based on structural and biophysical data.

Urea transporter knockout mice and their renal phenotypes.

PubMed

Fenton, Robert A; Yang, Baoxue

2014-01-01

Urea transporter gene knockout mice have been created for the study of the urine-concentrating mechanism. The major findings in studies of the renal phenotype of these mice are as follows: (1) Urea accumulation in the inner medullary interstitium is dependent on intrarenal urea recycling mediated by urea transporters; (2) urea transporters are essential for preventing urea-induced osmotic diuresis and thus for water conservation; (3) NaCl concentration in the inner medullary interstitium is not significantly affected by the absence of IMCD, descending limb of Henle and descending vasa recta urea transporters. Studies in urea transporter knockout mouse models have highlighted the essential role of urea for producing maximally concentrated urine.

SPERM MOTILITY IN HSF1 KNOCKOUT MICE AFTER HEAT SHOCK IS ASSOCIATED WITH FERTILITY DEFICITS

EPA Science Inventory

SPERM MOTILITY IN HSF1 KNOCKOUT MICE AFTER HEAT SHOCK IS ASSOCIATED WITH FERTILITY DEFICITS. L.F. Strader*, S.D. Perreault, J.C. Luft*, and D.J. Dix*. US EPA/ORD, Reproductive Toxicology Div., Research Triangle Park, NC
Heat shock proteins (HSPs) protect cells from environm...

Evaluation of cimi-shield knock-out bed bug eliminator against house fly (Musca domestica) adults

USDA-ARS?s Scientific Manuscript database

Cimi-Shield Knock-Out (CSKO) Bed Bug Eliminator is a green treatment labeled for use against bed bugs, carpet beetles, ants, roaches, fleas, ticks, silverfish, millipedes and centipedes. The active ingredient is soybean oil. If CSKO is formulated according to label instructions and sprayed directly ...

Evaluation of cimi-shield knock-out bed bug eliminator against house fly (Musca domestica) adults.

USDA-ARS?s Scientific Manuscript database

Cimi-Shield Knock-Out (CSKO) Bed Bug Eliminator is a green treatment labeled for use against bed bugs, carpet beetles, ants, roaches, fleas, ticks, silverfish, millipedes and centipedes. The active ingredient is soybean oil. If CSKO is formulated according to label instructions and sprayed directly ...

RETINOIC ACID INDUCTION OF CLEFT PALATE IN EGF AND TGF-ALPHA KNOCKOUT MICE: STAGE SPECIFIC INFLUENCES OF GROWTH FACTOR EXPRESSION

EPA Science Inventory

ABBOTT, B. D., LEFFLER, K.E. AND BUCKALEW, A.R, Reproductive Toxicology Division, NHEERL, ORD, US EPA, Research Triangle Park, North Carolina. Retinoic acid induction of cleft palate (CP) in EGF and TGF knockout mice: Stage specific influences of growth factor expression.
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Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound-Propelled Nanomotors.

PubMed

Hansen-Bruhn, Malthe; de Ávila, Berta Esteban-Fernández; Beltrán-Gastélum, Mara; Zhao, Jing; Ramírez-Herrera, Doris E; Angsantikul, Pavimol; Vesterager Gothelf, Kurt; Zhang, Liangfang; Wang, Joseph

2018-03-01

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound-powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA-loaded nanomotors to directly penetrate through the plasma membrane of GFP-expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA-loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor-based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing short-range correlations in asymmetric nuclei with quasi-free pair knockout reactions

NASA Astrophysics Data System (ADS)

Stevens, Sam; Ryckebusch, Jan; Cosyn, Wim; Waets, Andreas

2018-02-01

Short-range correlations (SRC) in asymmetric nuclei with an unusual neutron-to-proton ratio can be studied with quasi-free two-nucleon knockout processes following the collision between accelerated ions and a proton target. We derive an approximate factorized cross section for those SRC-driven p (A ,p‧N1N2) reactions. Our reaction model hinges on the factorization properties of SRC-driven A (e ,e‧N1N2) reactions for which strong indications are found in theory-experiment comparisons. In order to put our model to the test we compare its predictions with results of 12C (p ,p‧ pn) measurements conducted at Brookhaven National Laboratory (BNL) and find a fair agreement. The model can also reproduce characteristic features of SRC-driven two-nucleon knockout reactions, like back-to-back emission of the correlated nucleons. We study the asymmetry dependence of nuclear SRC by providing predictions for the ratio of proton-proton to proton-neutron knockout cross sections for the carbon isotopes 9-15C thereby covering neutron excess values (N - Z) / Z between -0.5 and +0.5.

Preliminary Characterization of a Leptin Receptor Knockout Rat Created by CRISPR/Cas9 System.

PubMed

Bao, Dan; Ma, Yuanwu; Zhang, Xu; Guan, Feifei; Chen, Wei; Gao, Kai; Qin, Chuan; Zhang, Lianfeng

2015-11-05

Leptin receptor, which is encoded by the diabetes (db) gene and is highly expressed in the choroid plexus, regulatesenergy homeostasis, the balance between food intake and energy expenditure, fertility and bone mass. Here, using CRISPR/Cas9 technology, we created the leptin receptor knockout rat. Homozygous leptin receptor null rats are characterized by obesity, hyperphagia, hyperglycemia, glucose intolerance, hyperinsulinemia and dyslipidemia. Due to long-term poor glycemic control, the leptin receptor knockout rats also develop some diabetic complications such as pancreatic, hepatic and renal lesions. In addition, the leptin receptor knockout rats show a significant decrease in bone volume and bone mineral density of the femur compared with their wild-type littermates. Our model has rescued some deficiency of the existing rodent models, such as the transient hyperglycemia of db/db mice in the C57BL/6J genetic background and the delayed onset of glucose intolerance in the Zucker rats, and it is proven to be a useful animal model for biomedical and pharmacological research on obesity and diabetes.

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus.

PubMed

Kim, Ji Woo; Kim, Hye-Min; Lee, Sang Mi; Kang, Man-Jong

2012-10-01

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks.

PubMed

Sauvage, M; Brabet, P; Holsboer, F; Bockaert, J; Steckler, T

2000-12-08

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

GENETIC CATHEPSIN B DEFICIENCY REDUCES β-AMYLOID IN TRANSGENIC MICE EXPRESSING HUMAN WILD-TYPE AMYLOID PRECURSOR PROTEIN

PubMed Central

Hook, Vivian Y. H.; Kindy, Mark; Reinheckel, Thomas; Peters, Christoph; Hook, Gregory

2009-01-01

Neurotoxic β-amyloid (Aβ) peptides participate in Alzheimer’s disease (AD); therefore, reduction of Aβ generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Aβ may identify targets for reducing Aβ. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decrease of Aβ40 and Aβ42 by 67% in brain, and decreases levels of the C-terminal β-secretase fragment (CTFβ) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Aβ. The difference in reduction of Aβ in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Aβ in AD. PMID:19501042

CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.

PubMed

Jafari, Naser; Kim, Hyunju; Park, Rackhyun; Li, Liqing; Jang, Minsu; Morris, Andrew J; Park, Junsoo; Huang, Cai

2017-01-01

Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

Deficits in learning and memory in mice with a mutation of the candidate dyslexia susceptibility gene Dyx1c1.

PubMed

Rendall, Amanda R; Tarkar, Aarti; Contreras-Mora, Hector M; LoTurco, Joseph J; Fitch, R Holly

2017-09-01

Dyslexia is a learning disability characterized by difficulty learning to read and write. The underlying biological and genetic etiology remains poorly understood. One candidate gene, dyslexia susceptibility 1 candidate 1 (DYX1C1), has been shown to be associated with deficits in short-term memory in dyslexic populations. The purpose of the current study was to examine the behavioral phenotype of a mouse model with a homozygous conditional (forebrain) knockout of the rodent homolog Dyx1c1. Twelve Dyx1c1 conditional homozygous knockouts, 7 Dyx1c1 conditional heterozygous knockouts and 6 wild-type controls were behaviorally assessed. Mice with the homozygous Dyx1c1 knockout showed deficits on memory and learning, but not on auditory or motor tasks. These findings affirm existing evidence that DYX1C1 may play an underlying role in the development of neural systems important to learning and memory, and disruption of this function could contribute to the learning deficits seen in individuals with dyslexia. Copyright © 2015 Elsevier Inc. All rights reserved.

Tailoring the Immune Response via Customization of Pathogen Gene Expression.

PubMed

Runco, Lisa M; Stauft, Charles B; Coleman, J Robert

2014-01-01

The majority of studies focused on the construction and reengineering of bacterial pathogens have mainly relied on the knocking out of virulence factors or deletion/mutation of amino acid residues to then observe the microbe's phenotype and the resulting effect on the host immune response. These knockout bacterial strains have also been proposed as vaccines to combat bacterial disease. Theoretically, knockout strains would be unable to cause disease since their virulence factors have been removed, yet they could induce a protective memory response. While knockout strains have been valuable tools to discern the role of virulence factors in host immunity and bacterial pathogenesis, they have been unable to yield clinically relevant vaccines. The advent of synthetic biology and enhanced user-directed gene customization has altered this binary process of knockout, followed by observation. Recent studies have shown that a researcher can now tailor and customize a given microbe's gene expression to produce a desired immune response. In this commentary, we highlight these studies as a new avenue for controlling the inflammatory response as well as vaccine development.

Tailoring the Immune Response via Customization of Pathogen Gene Expression

PubMed Central

Runco, Lisa M.; Stauft, Charles B.

2014-01-01

The majority of studies focused on the construction and reengineering of bacterial pathogens have mainly relied on the knocking out of virulence factors or deletion/mutation of amino acid residues to then observe the microbe's phenotype and the resulting effect on the host immune response. These knockout bacterial strains have also been proposed as vaccines to combat bacterial disease. Theoretically, knockout strains would be unable to cause disease since their virulence factors have been removed, yet they could induce a protective memory response. While knockout strains have been valuable tools to discern the role of virulence factors in host immunity and bacterial pathogenesis, they have been unable to yield clinically relevant vaccines. The advent of synthetic biology and enhanced user-directed gene customization has altered this binary process of knockout, followed by observation. Recent studies have shown that a researcher can now tailor and customize a given microbe's gene expression to produce a desired immune response. In this commentary, we highlight these studies as a new avenue for controlling the inflammatory response as well as vaccine development. PMID:24719769

[Expression of matrix metalloproteinase-19 in the human cornea. Wound healing in the MMP-19 knock-out mouse model].

PubMed

Treumer, F; Flöhr, C; Klettner, A; Nölle, B; Roider, J

2010-07-01

At present there are no data in the literature on the expression of matrix metalloprotein-19 in the human cornea. The aim of this study was to analyze the expression of matrix metalloproteinase-19 in the human cornea and to investigate its potential role in corneal wound healing using a MMP-19 knock-out mouse model. A method with Western blotting and immunohistological staining for MMP-19 was performed using paraffin embedded human corneas. Excimer laser keratectomy was performed in wild type (wt) and MMP-19 knock-out (ko) mice and the rate of re-epithelialization was analyzed after 8 h and 18 h. MMP-19 was strongly expressed in the human corneal epithelium mainly in the basal cell layer. MMP-19 was not expressed in the corneal stroma. In the mouse model the size of the corneal lesion after 8 h was 83% (wt) and 89.9% (ko) of the initial area (p=0.09). After 18 h the lesion was 17% (wt) and 13.3% (ko) of the initial area (p=0.01). Laminin-5 was expressed in the migrating epithelial cells with no differences between wild type and knock-out mouse. MMP-19 showed a strong expression in the basal cells of the human corneal epithelium. Corneal re-epithelialization was slightly faster in the MMP-19 knock-out mouse. No differences in the expression of laminin-5 could be detected.

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish

PubMed Central

Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L.; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

2013-01-01

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra. PMID:23447699

In vivo imaging of the Mouse Model of X-Linked Juvenile Retinoschisis Using Fourier Domain Optical Coherence Tomography

PubMed Central

Xu, Jing; Molday, Laurie L.; Molday, Robert S.; Sarunic, Marinko V.

2009-01-01

Purpose The purpose of this study is to investigate Fourier Domain Optical Coherence Tomography (FD OCT) as a non-invasive tool for retinal imaging in the Rs1h knockout mouse (model for X-linked Juvenile Retinoschisis). Methods A prototype spectrometer based FD OCT system was used in combination with a custom optical beam-scanning platform. Images of the retinas from wild type and Rs1h knockout mice were acquired non-invasively using FD OCT with the specimen anesthetized. At the completion of the non-invasive FD OCT imaging, invasive retinal cross sectional images (histology) were acquired from a nearby region for comparison to the FD OCT images. Results The retinal layers could be identified in the FD OCT images, permitting delineation and thickness measurement of the outer nuclear layer (ONL). During FD OCT in vivo imaging of the Rs1h knockout mouse, holes were observed in the inner nuclear layer (INL) and retinal cell disorganization was observed as a change in the backscattering intensity profile. Comparison of the ONL measurements acquired non-invasively using FD OCT to measurements taken using histology at nearby locations showed a degeneration of roughly thirty percent of the ONL by the age of two months in Rs1h knockout mice relative to wild type. Conclusions FD OCT has been demonstrated for non-invasive imaging of retinal degeneration and observation of retinal holes in Rs1h knockout mice. PMID:19182246

ATF6α/β-mediated adjustment of ER chaperone levels is essential for development of the notochord in medaka fish.

PubMed

Ishikawa, Tokiro; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Kamei, Yasuhiro; Shigenobu, Shuji; Tanaka, Minoru; Saito, Taro L; Yoshimura, Jun; Morishita, Shinichi; Toyoda, Atsushi; Sakaki, Yoshiyuki; Taniguchi, Yoshihito; Takeda, Shunichi; Mori, Kazutoshi

2013-05-01

ATF6α and ATF6β are membrane-bound transcription factors activated by regulated intramembrane proteolysis in response to endoplasmic reticulum (ER) stress to induce various ER quality control proteins. ATF6α- and ATF6β single-knockout mice develop normally, but ATF6α/β double knockout causes embryonic lethality, the reason for which is unknown. Here we show in medaka fish that ATF6α is primarily responsible for transcriptional induction of the major ER chaperone BiP and that ATF6α/β double knockout, but not ATF6α- or ATF6β single knockout, causes embryonic lethality, as in mice. Analyses of ER stress reporters reveal that ER stress occurs physiologically during medaka early embryonic development, particularly in the brain, otic vesicle, and notochord, resulting in ATF6α- and ATF6β-mediated induction of BiP, and that knockdown of the α1 chain of type VIII collagen reduces such ER stress. The absence of transcriptional induction of several ER chaperones in ATF6α/β double knockout causes more profound ER stress and impaired notochord development, which is partially rescued by overexpression of BiP. Thus ATF6α/β-mediated adjustment of chaperone levels to increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis before formation of the vertebra.

The Transient Role for Calcium and Vitamin D during the Developmental Hair Follicle Cycle.

PubMed

Mady, Leila J; Ajibade, Dare V; Hsaio, Connie; Teichert, Arnaud; Fong, Chak; Wang, Yongmei; Christakos, Sylvia; Bikle, Daniel D

2016-07-01

The role for 1,25-dihydroxyvitamin D3 and/or calcium in hair follicle cycling is not clear despite their impact on keratinocyte differentiation. We found that calbindin-D9k null (knockout) pups generated from calbindin-D9k knockout females fed a vitamin D-deficient, low-calcium (0.47%) diet develop transient alopecia. The pups appear phenotypically normal until 13 days of age, after which the hair progressively sheds in a caudocephalic direction, resulting in truncal alopecia totalis by 20-23 days, with spontaneous recovery by 28 days. Histological studies showed markedly dystrophic hair follicles, loss of hair shafts with increased apoptosis, and hyperplastic epidermis during this time. Ha1 expression is lost during catagen in all mice but recovers more slowly in the knockout pups on the vitamin D-deficient, low-calcium diet. Keratin 1 expression is reduced throughout days 19-28. The expressions of involucrin, loricrin, and cathepsin L is initially increased by day 19 but subsequently falls below those of controls by day 23, as does that of desmoglein 3. Feeding the mothers a high-vitamin D/high-calcium (2%)/lactose (20%) diet lessens the phenotype, and knockout pups fostered to mothers fed a normal diet do not develop alopecia. Our results show that in calbindin-D9k knockout pups, a maternal vitamin D-deficient/low-calcium diet leads to transient noncicatricial alopecia. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans

PubMed Central

Sangaletti, Rachele; Grant, Jeff; Della-Morte, David

2017-01-01

Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions. PMID:28820880

Mesenchyme-specific knockout of ESET histone methyltransferase causes ectopic hypertrophy and terminal differentiation of articular chondrocytes.

PubMed

Lawson, Kevin A; Teteak, Colin J; Zou, Junhui; Hacquebord, Jacques; Ghatan, Andrew; Zielinska-Kwiatkowska, Anna; Fernandes, Russell J; Chansky, Howard A; Yang, Liu

2013-11-08

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.

Cholecystokinin levels in prohormone convertase 2 knock-out mouse brain regions reveal a complex phenotype of region-specific alterations.

PubMed

Beinfeld, Margery C; Blum, Alissa; Vishnuvardhan, Daesety; Fanous, Sanya; Marchand, James E

2005-11-18

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.

CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development.

PubMed

Huang, Bonnie; Gomez-Rodriguez, Julio; Preite, Silvia; Garrett, Lisa J; Harper, Ursula L; Schwartzberg, Pamela L

2016-01-01

The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors.

Phosphoproteomic analysis of the striatum from pleiotrophin knockout and midkine knockout mice treated with cocaine reveals regulation of oxidative stress-related proteins potentially underlying cocaine-induced neurotoxicity and neurodegeneration.

PubMed

Vicente-Rodríguez, Marta; Gramage, Esther; Herradón, Gonzalo; Pérez-García, Carmen

2013-12-06

The neurotrophic factors pleiotrophin (PTN) and midkine (MK) are highly upregulated in different brain areas relevant to drug addiction after administrations of different drugs of abuse, including psychostimulants. We have previously demonstrated that PTN and MK modulate amphetamine-induced neurotoxicity and that PTN prevents cocaine-induced cytotoxicity in NG108-15 and PC12 cells. In an effort to dissect the different mechanisms of action triggered by PTN and MK to exert their protective roles against psychostimulant neurotoxicity, we have now used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN knockout, MK knockout and wild type mice treated with a single dose of cocaine (15mg/kg, i.p.). We identified 7 differentially expressed phosphoproteins: 5'(3')-deoxyribonucleotidase, endoplasmic reticulum resident protein 60 (ERP60), peroxiredoxin-6 (PRDX6), glutamate dehydrogenase 1 (GLUD1), aconitase and two subunits of hemoglobin. Most of these proteins are related to neurodegeneration processes and oxidative stress and their variations specially affect the PTN knockout mice, suggesting a protective role of endogenous PTN against cocaine-induced neural alterations. Further studies are needed to validate these proteins as possible targets against neural alterations induced by cocaine. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Enhanced effects of amphetamine but reduced effects of the hallucinogen, 5-MeO-DMT, on locomotor activity in 5-HT(1A) receptor knockout mice: implications for schizophrenia.

PubMed

van den Buuse, Maarten; Ruimschotel, Emma; Martin, Sally; Risbrough, Victoria B; Halberstadt, Adam L

2011-01-01

Serotonin-1A (5-HT(1A)) receptors may play a role in schizophrenia and the effects of certain antipsychotic drugs. However, the mechanism of interaction of 5-HT(1A) receptors with brain systems involved in schizophrenia, remains unclear. Here we show that 5-HT(1A) receptor knockout mice display enhanced locomotor hyperactivity to acute treatment with amphetamine, a widely used animal model of hyperdopaminergic mechanisms in psychosis. In contrast, the effect of MK-801 on locomotor activity, modeling NMDA receptor hypoactivity, was unchanged in the knockouts. The effect of the hallucinogen 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) was markedly reduced in 5-HT(1A) receptor knockout mice. There were no changes in apomorphine-induced disruption of PPI, a model of sensory gating deficits seen in schizophrenia. Similarly, there were no major changes in density of dopamine transporters (DAT) or dopamine D(1) or D(2) receptors which could explain the behavioural changes observed in 5-HT(1A) receptor knockout mice. These results extend our insight into the possible role of these receptors in aspects of schizophrenia. As also suggested by previous studies using agonist and antagonist drugs, 5-HT(1A) receptors may play an important role in hallucinations and to modulate dopaminergic activity in the brain. Copyright © 2011 Elsevier Ltd. All rights reserved.

Macrophage migration inhibitory factor (MIF) knockout preserves cardiac homeostasis through alleviating Akt-mediated myocardial autophagy suppression in high-fat diet-induced obesity.

PubMed

Xu, X; Ren, J

2015-03-01

Macrophage migration inhibitory factor (MIF) has a role in the development of obesity and diabetes. However, whether MIF has a role in fat diet-induced obesity and associated cardiac anomalies still remains unknown. The aim of this study was to examine the impact of MIF knockout on high-fat diet-induced obesity, obesity-associated cardiac anomalies and the underlying mechanisms involved with a focus on Akt-mediated autophagy. Adult male wild-type (WT) and MIF knockout (MIF(-/-)) mice were placed on 45% high-fat diet for 5 months. Oxygen consumption, CO2 production, respiratory exchange ratio, locomotor activity and heat generation were measured using energy calorimeter. Echocardiographic, cardiomyocyte mechanical and intracellular Ca2+ properties were assessed. Apoptosis was examined using terminal dUTP nick end labeling staining and western blot analysis. Akt signaling pathway and autophagy markers were evaluated. Cardiomyocytes isolated from WT and MIF(-/-) mice were treated with recombinant mouse MIF (rmMIF). High-fat diet feeding elicited increased body weight gain, insulin resistance and caloric disturbance in WT and MIF(-/-) mice. High-fat diet induced unfavorable geometric, contractile and histological changes in the heart, the effects of which were alleviated by MIF knockout. In addition, fat diet-induced cardiac anomalies were associated with Akt activation and autophagy suppression, which were nullified by MIF deficiency. In cardiomyocytes from WT mice, autophagy was inhibited by exogenous rmMIF through Akt activation. In addition, MIF knockout rescued palmitic acid-induced suppression of cardiomyocyte autophagy, the effect of which was nullified by rmMIF. These results indicate that MIF knockout preserved obesity-associated cardiac anomalies without affecting fat diet-induced obesity, probably through restoring myocardial autophagy in an Akt-dependent manner. Our findings provide new insights for the role of MIF in obesity and associated cardiac anomalies.

Age-dependent Changes of Cerebral Copper Metabolism in Atp7b−/− Knockout Mouse Model of Wilson’s Disease by [64Cu]CuCl2-PET/CT

PubMed Central

Xie, Fang; Xi, Yin; Pascual, Juan M.; Muzik, Otto; Peng, Fangyu

2017-01-01

Copper is a nutritional metal required for brain development and function. Wilson’s disease (WD), or hepatolenticular degeneration, is an inherited human copper metabolism disorder caused by mutation of ATP7B gene. Many WD patients present with variable neurological and psychiatric symptoms, which may be related to neurodegeneration secondary to copper metabolism imbalance. The objective of this study is to explore feasibility and use of copper-64 chloride ([64C]CuCl2) as a tracer for noninvasive assessment of age-dependence changes of cerebral copper metabolism in WD using an Atp7b−/− knockout mouse model of WD and a positron emission tomography/computed tomography (PET/CT) scanner. Continuing from recent study of biodistribution and radiation dosimetry of [64C]CuCl2 in Atp7b−/− knockout mice, PET quantitative analysis revealed low 64Cu radioactivity in the brains of Atp7b−/− knockout mice at 7th week of age, compared with the 64Cu radioactivity in the brains of age and gender-matched wild type C57BL/6 mice, at 24 hour (h) post intravenous injection of [64C]CuCl2 as a tracer. Furthermore, age-dependent increase of 64Cu radioactivity was detected in the brains of Atp7b−/− knockout mice from 13th to 21th week of age, using the data derived from a longitudinal [64C]CuCl2-PET/CT study of Atp7b−/− knockout mice with orally administered [64Cu]CuCl2 as a tracer. The findings of this study support the use of [64Cu]CuCl2-PET/CT as a tool for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD patients presenting with variable neurological and psychiatric symptoms. PMID:28130615

Thymosin Beta-4 Induces Mouse Hair Growth

PubMed Central

Hou, Fang; Zhang, Zhipeng; Nuo, Mingtu; Guo, Xudong; Liu, Dongjun

2015-01-01

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts. PMID:26083021

Thymosin Beta-4 Induces Mouse Hair Growth.

PubMed

Gao, Xiaoyu; Liang, Hao; Hou, Fang; Zhang, Zhipeng; Nuo, Mingtu; Guo, Xudong; Liu, Dongjun

2015-01-01

Thymosin beta-4 (Tβ4) is known to induce hair growth and hair follicle (HF) development; however, its mechanism of action is unknown. We generated mice that overexpressed Tβ4 in the epidermis, as well as Tβ4 global knockout mice, to study the role of Tβ4 in HF development and explore the mechanism of Tβ4 on hair growth. To study Tβ4 function, we depilated control and experimental mice and made tissue sections stained with hematoxylin and eosin (H&E). To explore the effect of Tβ4 on hair growth and HF development, the mRNA and protein levels of Tβ4 and VEGF were detected by real-time PCR and western blotting in control and experimental mice. Protein expression levels and the phosphorylation of P38, ERK and AKT were also examined by western blotting. The results of depilation indicated that hair re-growth was faster in Tβ4-overexpressing mice, but slower in knockout mice. Histological examination revealed that Tβ4-overexpressing mice had a higher number of hair shafts and HFs clustered together to form groups, while the HFs of control mice and knockout mice were separate. Hair shafts in knockout mice were significantly reduced in number compared with control mice. Increased Tβ4 expression at the mRNA and protein levels was confirmed in Tβ4-overexpressing mice, which also had increased VEGF expression. On the other hand, knockout mice had reduced levels of VEGF expression. Mechanistically, Tβ4-overexpressing mice showed increased protein expression levels and phosphorylation of P38, ERK and AKT, whereas knockout mice had decreased levels of both expression and phosphorylation of these proteins. Tβ4 appears to regulate P38/ERK/AKT signaling via its effect on VEGF expression, with a resultant effect on the speed of hair growth, the pattern of HFs and the number of hair shafts.

Akt2 knockout alleviates prolonged caloric restriction-induced change in cardiac contractile function through regulation of autophagy.

PubMed

Zhang, Yingmei; Han, Xuefeng; Hu, Nan; Huff, Anna F; Gao, Feng; Ren, Jun

2014-06-01

Caloric restriction leads to changes in heart geometry and function although the underlying mechanism remains elusive. Autophagy, a conserved pathway for degradation of intracellular proteins and organelles, preserves energy and nutrient in the face of caloric insufficiency. This study was designed to examine the role of Akt2 in prolonged caloric restriction-induced change in cardiac homeostasis and the underlying mechanism(s) involved. Wild-type (WT) and Akt2 knockout mice were calorie restricted (by 40%) for 30weeks. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, autophagy and its regulatory proteins were evaluated. Caloric restriction compromised echocardiographic indices (decreased left ventricular mass, left ventricular diameters and cardiac output), cardiomyocyte contractile and intracellular Ca(2+) properties associated with dampened SERCA2a phosphorylation, upregulated phospholamban and autophagy (Beclin-1, Atg7, LC3BII-to-LC3BI ratio), increased autophagy adaptor protein p62, elevated phosphorylation of AMPK, Akt2 and the Akt downstream signal molecule TSC2, the effects of which with the exception of autophagy protein markers (Beclin-1, Atg7, LC3B) and AMPK were mitigated or significantly alleviated by Akt2 knockout. Lysosomal inhibition using bafilomycin A1 negated Akt2 knockout-induced protective effect on p62. Evaluation of downstream signaling molecules of Akt and AMPK including mTOR and ULK1 revealed that caloric restriction suppressed and promoted phosphorylation of mTOR and ULK1, respectively, without affecting total mTOR and ULK1 expression. Akt2 knockout significantly augmented caloric restriction-induced responses on mTOR and ULK1. Taken together, these data suggest a beneficial role of Akt2 knockout in preservation of cardiac homeostasis against prolonged caloric restriction-induced pathological changes possibly through facilitating autophagy. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy." Copyright © 2013 Elsevier Ltd. All rights reserved.

Elevated progranulin contributes to synaptic and learning deficit due to loss of fragile X mental retardation protein.

PubMed

Zhang, Kun; Li, Yu-Jiao; Guo, Yanyan; Zheng, Kai-Yin; Yang, Qi; Yang, Le; Wang, Xin-Shang; Song, Qian; Chen, Tao; Zhuo, Min; Zhao, Ming-Gao

2017-12-01

Fragile X syndrome is an inheritable form of intellectual disability caused by loss of fragile X mental retardation protein (FMRP, encoded by the FMR1 gene). Absence of FMRP caused overexpression of progranulin (PGRN, encoded by GRN), a putative tumour necrosis factor receptor ligand. In the present study, we found that progranulin mRNA and protein were upregulated in the medial prefrontal cortex of Fmr1 knock-out mice. In Fmr1 knock-out mice, elevated progranulin caused insufficient dendritic spine pruning and late-phase long-term potentiation in the medial prefrontal cortex of Fmr1 knock-out mice. Partial progranulin knock-down restored spine morphology and reversed behavioural deficits, including impaired fear memory, hyperactivity, and motor inflexibility in Fmr1 knock-out mice. Progranulin increased levels of phosphorylated glutamate ionotropic receptor GluA1 and nuclear factor kappa B in cultured wild-type neurons. Tumour necrosis factor receptor 2 antibody perfusion blocked the effects of progranulin on GluA1 phosphorylation; this result indicates that tumour necrosis factor receptor 2 is required for progranulin-mediated GluA1 phosphorylation and late-phase long-term potentiation expression. However, high basal level of progranulin in Fmr1 knock-out mice prevented further facilitation of synaptic plasticity by exogenous progranulin. Partial downregulation of progranulin or tumour necrosis factor receptor 2/nuclear factor kappa B signalling restored synaptic plasticity and memory deficits in Fmr1 knock-out mice. These findings suggest that elevated PGRN is linked to cognitive deficits of fragile X syndrome, and the progranulin/tumour necrosis factor receptor 2 signalling pathway may be a putative therapeutic target for improving cognitive deficits in fragile X syndrome. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Age-dependent changes of cerebral copper metabolism in Atp7b -/- knockout mouse model of Wilson's disease by [64Cu]CuCl2-PET/CT.

PubMed

Xie, Fang; Xi, Yin; Pascual, Juan M; Muzik, Otto; Peng, Fangyu

2017-06-01

Copper is a nutritional metal required for brain development and function. Wilson's disease (WD), or hepatolenticular degeneration, is an inherited human copper metabolism disorder caused by a mutation of the ATP7B gene. Many WD patients present with variable neurological and psychiatric symptoms, which may be related to neurodegeneration secondary to copper metabolism imbalance. The objective of this study was to explore the feasibility and use of copper-64 chloride ([ 64 C]CuCl 2 ) as a tracer for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD using an Atp7b -/- knockout mouse model of WD and positron emission tomography/computed tomography (PET/CT) imaging. Continuing from our recent study of biodistribution and radiation dosimetry of [ 64 C]CuCl 2 in Atp7b -/- knockout mice, PET quantitative analysis revealed low 64 Cu radioactivity in the brains of Atp7b -/- knockout mice at 7th weeks of age, compared with 64 Cu radioactivity in the brains of age- and gender-matched wild type C57BL/6 mice, at 24 h (h) post intravenous injection of [ 64 C]CuCl 2 as a tracer. Furthermore, age-dependent increase of 64 Cu radioactivity was detected in the brains of Atp7b -/- knockout mice from the 13th to 21th weeks of age, based on the data derived from a longitudinal [ 64 C]CuCl 2 -PET/CT study of Atp7b -/- knockout mice with orally administered [ 64 Cu]CuCl 2 as a tracer. The findings of this study support clinical use of [ 64 Cu]CuCl 2 -PET/CT imaging as a tool for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD patients presenting with variable neurological and psychiatric symptoms.

GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus.

PubMed

Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte; Ostachowicz, Beata; Nowak, Gabriel

2014-10-31

Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail suspension test, and light/dark test. We also investigated whether lack of GPR39 would change levels of cAMP response element-binding protein (CREB),brain-derived neurotrophic factor (BDNF) and tropomyosin related kinase B (TrkB) protein in the hippocampus and frontal cortex of GPR39 knockout mice subjected to the forced swim test, as measured by Western-blot analysis. In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB and BDNF levels in the hippocampus, but not in the frontal cortex, which indicates region specificity for the impaired CREB/BDNF pathway (which is important in antidepressant response) in the absence of GPR39. There were no changes in TrkB protein in either structure. In the present study, we also investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. There were no changes in the GPR39 knockout mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn(2+)-sensing receptor in the pathophysiology of depression with component of anxiety. © The Author 2015. Published by Oxford University Press on behalf of CINP.

The Role of Beta-Adrenergic Receptors in the Regulation of Circadian Intraocular Pressure Rhythm in Mice.

PubMed

Tsuchiya, Shunsuke; Higashide, Tomomi; Toida, Kazunori; Sugiyama, Kazuhisa

2017-07-01

To investigate whether the elimination of β1- and β2-adrenergic receptors alters the diurnal intraocular pressure (IOP) rhythm in mice. β1-/β2-adrenergic receptor double-knockout and C57BL/6J mice were anesthetized intraperitoneally, with their IOPs measured via microneedle method. After entrainment to a 12-h light-dark (LD) cycle (light phase 6:00-18:00), IOPs were measured every 3 h from 9:00 to 24:00 (group 1, β1-/β2-adrenergic receptor double-knockout mice, n = 11; C57BL/6J, n = 15). The IOP measurements at 15:00 and 24:00 under a 12-h LD cycle and in the constant darkness (1 day and 8 days after exposure to darkness, respectively) were performed in another group of β1-/β2-adrenergic receptor double-knockout mice (group 2, n = 12). IOP variance throughout the day and mean IOP differences among time points were evaluated using a linear mixed model. β1-/β2-adrenergic receptor double-knockout and C57BL/6J mice showed biphasic IOP curves, low during the light phase and high during the dark phase; the fluctuation was significant (P < 0.001). The peak IOP (18.7 ± 1.4 mmHg) occurred at 24:00 and the trough IOP (13.5 ± 1.5 mmHg) occurred at 15:00 in β1-/β2-adrenergic receptor double-knockout mice group. IOP curves of β1-/β2-adrenergic receptor double-knockout and C57BL/6J were nearly parallel, and the IOPs of β1-/β2-adrenergic receptor double-knockout mice were significantly higher than those of C57BL/6J mice (P < 0.001). Under constant dark (DD) conditions, IOP at 24:00 (18.1 ± 1.5 mmHg) was significantly higher than that at 15:00 (13.3 ± 1.2 mmHg) (P < 0.001). The transition from the LD cycle to DD environment produced no significant change in IOP (P = 0.728). Elimination of both β1- and β2-adrenergic receptors did not disturb the biphasic diurnal IOP rhythm in mice.

β1/2 or M2/3 Receptors Are Required for Different Gastrointestinal Motility Responses Induced by Acupuncture at Heterotopic or Homotopic Acupoints

PubMed Central

Yu, Xiaochun; Cui, Changxiang; Yang, Zhaokun; Shi, Hong; Jing, Xianghong; Zhu, Bing

2016-01-01

Acupuncture at homotopic acupoints or heterotopic acupoints is known to either inhibit or facilitate gastrointestinal motility, depending on the acupoint location. However, little effort has been made to investigate the roles of specific receptors (such as adrenergic and muscarinic acetylcholine receptors) in mediating the effects of acupuncture at heterotopic and homotopic acupoints. Different adrenergic receptor subtypes or cholinergic receptor subtypes are predominantly expressed in various sections of the gut, resulting in variations between the effects of acupuncture at heterotopic or homotopic acupoints on gastrointestinal motility. Here, we investigated the role of β1/β2 receptors and M2/M3 receptors in gastrointestinal motility regulated by acupuncture at ST37, a heterotopic acupoint, and ST25, a homotopic acupoint, by simultaneously recording intraluminal pressures in the distal colon and stomach or jejunum and examining fecal phenol red excretion in β1/2 receptor-knockout mice and M2/3 receptor-knockout mice. We found that knockout of the M2/3 receptor significantly inhibited ST37 acupuncture-induced enhancement of gastric motility, jejunal motility, and colonic motility. Additionally, knocking out of the β1/2 receptor significantly diminished the ST25 acupuncture-induced inhibition of gastric motility and jejunal motility without significantly altering the enhancement of colonic motility induced by acupuncture at ST25. Acupuncture at ST37 significantly accelerated gastrointestinal transition in β1/2 receptor-knockout mice and their wild-type littermates. However, this acceleration of gastrointestinal transition was markedly diminished in M2/3 receptor-knockout mice relative to their wild-type littermates. Acupuncture at ST25 significantly increased gastrointestinal transition in β1/2 receptor-knockout mice and significantly decreased gastrointestinal transition in M2/3 receptor-knockout mice without altering gastrointestinal transition in wild-type littermates of either. Our study revealed that M2/3 receptors are required for the gastrointestinal motility associated with whole gastrointestinal transition enhanced by acupuncture at heterotopic acupoints, whereas β1/2 receptors are required for the same gastrointestinal motility processes inhibited by acupuncture at homotopic acupoints. Therefore, our findings reveal important biological mechanisms underlying acupuncture treatment of disorders involving gastrointestinal motility dysfunction. PMID:27978539

Development of a Genetic Map for Onion (Allium cepa L.) Using Reference-Free Genotyping-by-Sequencing and SNP Assays

PubMed Central

Jo, Jinkwan; Purushotham, Preethi M.; Han, Koeun; Lee, Heung-Ryul; Nah, Gyoungju; Kang, Byoung-Cheorl

2017-01-01

Single nucleotide polymorphisms (SNPs) play important roles as molecular markers in plant genomics and breeding studies. Although onion (Allium cepa L.) is an important crop globally, relatively few molecular marker resources have been reported due to its large genome and high heterozygosity. Genotyping-by-sequencing (GBS) offers a greater degree of complexity reduction followed by concurrent SNP discovery and genotyping for species with complex genomes. In this study, GBS was employed for SNP mining in onion, which currently lacks a reference genome. A segregating F2 population, derived from a cross between ‘NW-001’ and ‘NW-002,’ as well as multiple parental lines were used for GBS analysis. A total of 56.15 Gbp of raw sequence data were generated and 1,851,428 SNPs were identified from the de novo assembled contigs. Stringent filtering resulted in 10,091 high-fidelity SNP markers. Robust SNPs that satisfied the segregation ratio criteria and with even distribution in the mapping population were used to construct an onion genetic map. The final map contained eight linkage groups and spanned a genetic length of 1,383 centiMorgans (cM), with an average marker interval of 8.08 cM. These robust SNPs were further analyzed using the high-throughput Fluidigm platform for marker validation. This is the first study in onion to develop genome-wide SNPs using GBS. The resulting SNP markers and developed linkage map will be valuable tools for genetic mapping of important agronomic traits and marker-assisted selection in onion breeding programs. PMID:28959273

Regulation of miR394 in Response to Fusarium oxysporum f. sp. cepae (FOC) Infection in Garlic (Allium sativum L).

PubMed

Chand, Subodh K; Nanda, Satyabrata; Joshi, Raj K

2016-01-01

MicroRNAs (miRNAs) are a class of post-transcriptional regulators that negatively regulate gene expression through target mRNA cleavage or translational inhibition and play important roles in plant development and stress response. In the present study, six conserved miRNAs from garlic (Allium sativum L.) were analyzed to identify differentially expressed miRNAs in response to Fusarium oxysporum f. sp. cepae (FOC) infection. Stem-loop RT-PCR revealed that miR394 is significantly induced in garlic seedlings post-treatment with FOC for 72 h. The induction of miR394 expression during FOC infection was restricted to the basal stem plate tissue, the primary site of infection. Garlic miR394 was also upregulated by exogenous application of jasmonic acid. Two putative targets of miR394 encoding F-box domain and cytochrome P450 (CYP450) family proteins were predicted and verified using 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends) assay. Quantitative RT-PCR showed that the transcript levels of the predicted targets were significantly reduced in garlic plants exposed to FOC. When garlic cultivars with variable sensitivity to FOC were exposed to the pathogen, an upregulation of miR394 and down regulation of the targets were observed in both varieties. However, the expression pattern was delayed in the resistant genotypes. These results suggest that miR394 functions in negative modulation of FOC resistance and the difference in timing and levels of expression in variable genotypes could be examined as markers for selection of FOC resistant garlic cultivars.

Multiple garlic (Allium sativum L.) microRNAs regulate the immunity against the basal rot fungus Fusarium oxysporum f. sp. Cepae.

PubMed

Chand, Subodh Kumar; Nanda, Satyabrata; Mishra, Rukmini; Joshi, Raj Kumar

2017-04-01

The basal plate rot fungus, Fusarium oxysporum f. sp. cepae (FOC), is the most devastating pathogen posing a serious threat to garlic (Allium sativum L.) production worldwide. MicroRNAs (miRNAs) are key modulators of gene expression related to development and defense responses in eukaryotes. However, the miRNA species associated with garlic immunity against FOC are yet to be explored. In the present study, a small RNA library developed from FOC infected resistant garlic line was sequenced to identify immune responsive miRNAs. Forty-five miRNAs representing 39 conserved and six novel sequences responsive to FOC were detected. qRT-PCR analyses further classified them into three classes based on their expression patterns in susceptible line CBT-As11 and in the resistant line CBT-As153. North-blot analyses of six selective miRNAs confirmed the qRT-PCR results. Expression studies on a selective set of target genes revealed a negative correlation with the complementary miRNAs. Furthermore, transgenic garlic plant overexpresing miR164a, miR168a and miR393 showed enhanced resistance to FOC, as revealed by decreased fungal growth and up-regulated expression of defense-responsive genes. These results indicate that multiple miRNAs are involved in garlic immunity against FOC and that the overexpression of miR164a, miR168a and miR393 can augment garlic resistance to Fusarium basal rot infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Phytotoxicity of pesticides mancozeb and chlorpyrifos: correlation with the antioxidative defence system in Allium cepa.

PubMed

Fatma, Firdos; Verma, Sonam; Kamal, Aisha; Srivastava, Alka

2018-02-01

Pesticides are a group of chemical substances which are widely used to improve agricultural production. However, these substances could be persistent in soil and water, accumulative in sediment or bio-accumulative in biota depending on their solubility, leading to different types of environmental pollution. The present study was done to assess the impact of pesticides-mancozeb and chlorpyrifos, via morphological and physiological parameters using Allium cepa test system. Phytotoxic effects of pesticides were examined via germination percentage, survival percentage, root and shoot length, root shoot length ratio, seedling vigor index, percentage of phytotoxicity and tolerance index. Oxidative stress on Allium seedlings caused by pesticides was also assessed by investigating the activity of antioxidative enzymes viz. catalase, peroxidase and superoxide dismutase. Correlation was worked out between morphological parameters and antioxidative enzymes to bring out the alliance between them. Mancozeb and chlorpyrifos concentrations were significantly and positively correlated with the activity of antioxidative enzymes and negatively correlated with morphological parameters. Significant positive correlation between various morphological parameters showed their interdependency. However, negative correlation was obtained between activity of antioxidative enzymes and morphological parameters. The enzymes however, showed positive correlation with each other. Based on our result we can conclude that all morphological parameters were adversely affected by the two pesticides as reflected by phytotoxicity in Allium . Their negative correlation with activity of antioxidative enzymes indicates that upregulation of antioxidative enzymes is not sufficient to overcome the toxic effect, thereby signifying the threat being caused by the regular use of these pesticides.

The L locus, one of complementary genes required for anthocyanin production in onions (Allium cepa), encodes anthocyanidin synthase.

PubMed

Kim, Sunggil; Jones, Rick; Yoo, Kil-Sun; Pike, Leonard M

2005-06-01

Bulb color in onions (Allium cepa) is an important trait, but its complex, unclear mechanism of inheritance has been a limiting factor in onion cultivar improvement. The identity of the L locus, which is involved in the color difference between Brazilian yellow and red onions, is revealed in this study. A cross was made between a US-type yellow breeding line and a Brazilian yellow cultivar. The segregation ratio of nine red to seven yellow onions in the F(2) population supports the involvement of two complementary genes in anthocyanin production in the F(1) hybrids. The high-performance liquid chromatography (HPLC) and reverse-transcriptase (RT)-PCR analysis of the Brazilian yellow onions indicated that the genes are involved late in the anthocyanin synthesis pathway. The genomic sequence of the anthocyanidin synthase (ANS) gene in Brazilian yellow onions showed a point mutation, which results in an amino acid change of a glycine to an arginine at residue 229. Because this residue is located adjacent to a highly conserved iron-binding active site, this mutation is likely responsible for the inactivation of the ANS gene in Brazilian yellow onions. Following the isolation of the promoter sequence of the mutant allele, a PCR-based marker for allelic selection of the ANS gene was designed. This assay is based on an insertion (larger than 3 kb) mutation. The marker perfectly co-segregated with the color phenotypes in the F(2) populations, thereby indicating that the L locus encodes ANS.

Evaluation of genotoxicity of coal fly ash in Allium cepa root cells by combining comet assay with the Allium test.

PubMed

Chakraborty, Rajarshi; Mukherjee, Ashit Kumar; Mukherjee, Anita

2009-06-01

Fly ash is a by-product of coal-fired electricity generation plants. Its utilization and disposal is of utmost importance. Using onion (Allium cepa) root tip system, the present study was carried out to evaluate the potential toxic and genotoxic effects of fly ash, collected from a thermal power plant in West Bengal, India. Prior to testing, the collected fly ash sample was mixed with sand in different proportions. Allium bulbs were allowed to germinate directly in fly ash and after five days the germinating roots were processed for the Allium test. Additionally, the Allium test was adapted for detecting DNA damage through comet assay. The results from the Allium test indicate that fly ash at 100% concentration inhibits root growth and mitotic indices; induces binucleated cells as a function of the proportion, but is not toxic at very low concentration. In the comet assay, a statistical increase for DNA strand breaks was found only at higher concentrations. The sample was analyzed by flame atomic absorption spectrometer for Zn, Pb, Cu, Ni, Cd and As, whose presence could partly be responsible for the toxicity of fly ash. The study concludes that the classical Allium test can give a more comprehensive data when done in combination with the comet assay, which is faster, simpler and independent of mitosis. Also when fly ash is used for other purposes in combination with soils, it should be judiciously used at very low concentrations in order to protect the ecosystem health from any potential adverse effects.

Prostaglandin E2 Activates YAP and a Positive-Signaling Loop to Promote Colon Regeneration After Colitis but Also Carcinogenesis in Mice.

PubMed

Kim, Han-Byul; Kim, Minchul; Park, Young-Soo; Park, Intae; Kim, Tackhoon; Yang, Sung-Yeun; Cho, Charles J; Hwang, DaeHee; Jung, Jin-Hak; Markowitz, Sanford D; Hwang, Sung Wook; Yang, Suk-Kyun; Lim, Dae-Sik; Myung, Seung-Jae

2017-02-01

Prostaglandin E 2 (PGE 2 ) is mediator of inflammation that regulates tissue regeneration, but its continual activation has been associated with carcinogenesis. Little is known about factors in the PGE 2 signaling pathway that contribute to tumor formation. We investigated whether yes-associated protein 1 (YAP1), a transcriptional co-activator in the Hippo signaling pathway, mediates PGE 2 function. DLD-1 and SW480 colon cancer cell lines were transfected with vectors expressing transgenes or small hairpin RNAs and incubated with recombinant PGE 2 , with or without pharmacologic inhibitors of signaling proteins, and analyzed by immunoblot, immunofluorescence, quantitative reverse-transcription polymerase chain reaction, transcriptional reporter, and proliferation assays. Dextran sodium sulfate (DSS) was given to induce colitis in C57/BL6 (control) mice, as well as in mice with disruption of the hydroxyprostaglandin dehydrogenase 15 gene (15-PGDH-knockout mice), Yap1 gene (YAP-knockout mice), and double-knockout mice. Some mice also were given indomethacin to block PGE 2 synthesis. 15-PGDH knockout mice were crossed with mice with intestine-specific disruption of the salvador family WW domain containing 1 gene (Sav1), which encodes an activator of Hippo signaling. We performed immunohistochemical analyses of colon biopsy samples from 26 patients with colitis-associated cancer and 51 age-and sex-matched patients with colorectal cancer (without colitis). Incubation of colon cancer cell lines with PGE 2 led to phosphorylation of cyclic adenosine monophosphate-responsive element binding protein 1 and increased levels of YAP1 messenger RNA, protein, and YAP1 transcriptional activity. This led to increased transcription of the prostaglandin-endoperoxide synthase 2 gene (PTGS2 or cyclooxygenase 2) and prostaglandin E-receptor 4 gene (PTGER4 or EP4). Incubation with PGE 2 promoted proliferation of colon cancer cell lines, but not cells with knockdown of YAP1. Control mice developed colitis after administration of DSS, but injection of PGE 2 led to colon regeneration in these mice. However, YAP-knockout mice did not regenerate colon tissues and died soon after administration of DSS. 15-PGDH-knockout mice regenerated colon tissues more rapidly than control mice after withdrawal of DSS, and had faster recovery of body weight, colon length, and colitis histology scores. These effects were reversed by injection of indomethacin. SAV1-knockout or 15-PGDH-knockout mice did not develop spontaneous tumors after colitis induction, but SAV1/15-PGDH double-knockout mice developed polyps that eventually progressed to carcinoma in situ. Administration of indomethacin to these mice prevented spontaneous tumor formation. Levels of PGE 2 correlated with those of YAP levels in human sporadic colorectal tumors and colitis-associated tumors. PGE 2 signaling increases the expression and transcriptional activities of YAP1, leading to increased expression of cyclooxygenase 2 and EP4 to activate a positive signaling loop. This pathway promotes proliferation of colon cancer cell lines and colon tissue regeneration in mice with colitis. Constitutive activation of this pathway led to formation of polyps and colon tumors in mice. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

CD34 EXPRESSION BY HAIR FOLLICLE STEM CELLS IS REQUIRED FOR SKIN TUMOR DEVELOPMENT IN MICE

EPA Science Inventory

We used knockout mice to show that a cell surface protein called CD34 is required for skin tumor formation in mice. Wild type mice treated with 7-12-Dimethylbenz(a)anthracene (DMBA) and a tumor promoter developed papillomas. When we treated CD34 knockout (KO) mice the same way, n...

Linking cellular zinc status to body weight and fat mass: mapping quantitative trait loci in Znt7 knockout mice

USDA-ARS?s Scientific Manuscript database

Zinc transporter 7 (Znt7, Slc30a7) knockout (KO) mice display abnormalities in body weight gain and body adiposity. Regulation of body weight and fatness is complex, involving multiple genetic and environmental factors. To understand how zinc homeostasis influences body weight gain and fat deposit a...

TERATOGENIC RESPONSES OF TGFALPHA KNOCKOUT FETUSES TO 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

EPA Science Inventory

ABBOTT1, B.D., A.R. BUCKALEW1, and P.L. BRYANT2. 1Reproductive Toxicology Division, EPA, RAP, NC; 2Dept. Environ. Sciences & Engineering, UNC, Chapel Hill, NC. Teratogenic responses of TGF knockout fetuses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

TCDD induces cl...

Effects of blueberries in prevention of atherosclerosis in apoe knockout mice

USDA-ARS?s Scientific Manuscript database

ApoE knockout (ApoE-/-) mice were fed AIN-93G diet (CD) or CD formulated to contain 1% freeze-dried whole wild blueberries (CD1% BB). Mice were sacrificed after 20 weeks on the specified diet. Atherosclerotic lesions in aortic sinus were determined by staining cryosections (10 µm) with Oil Red O. Th...

Sarcocystis pantherophis, n. sp. from eastern rat snakes (Pantherophis alleghaniensis) definitive hosts and interferongamma gene knockout mice as experimental intermediate hosts

USDA-ARS?s Scientific Manuscript database

Here we report a new species, Sarcocystis pantherophisi with the Eastern rat snake (Pantherophis alleghaniensis) as natural definitive host and the interferon gamma gene knockout (KO) mouse as the experimental intermediate host. Sporocysts (n=15) from intestinal contents of the snake were 17.3 x 10....

INTERPRETATION OF THE CANCER RESPONSE TO POTENTIAL RENTAL CARCINOGENS IN THE TSC2 KNOCKOUT (EKER) RAT IS DEPENDENT ON LENGTH OF TREATMENT.

EPA Science Inventory

INTERPRETATION OF THE CANCER RESPONSE TO POTENTIAL RENAL CARCINOGENS IN THE TSC2 KNOCKOUT (EKER) RAT IS DEPENDENT ON LENGTH OF TREATMENT.

Genetically increasing the function of oncogenes or knocking out the function of a tumor supressor gene has dramatically increased the...

Shape and structure of N=Z 64Ge: electromagnetic transition rates from the application of the recoil distance method to a knockout reaction.

PubMed

Starosta, K; Dewald, A; Dunomes, A; Adrich, P; Amthor, A M; Baumann, T; Bazin, D; Bowen, M; Brown, B A; Chester, A; Gade, A; Galaviz, D; Glasmacher, T; Ginter, T; Hausmann, M; Horoi, M; Jolie, J; Melon, B; Miller, D; Moeller, V; Norris, R P; Pissulla, T; Portillo, M; Rother, W; Shimbara, Y; Stolz, A; Vaman, C; Voss, P; Weisshaar, D; Zelevinsky, V

2007-07-27

Transition rate measurements are reported for the 2(1)+ and 2(2)+ states in N=Z 64Ge. The experimental results are in excellent agreement with large-scale shell-model calculations applying the recently developed GXPF1A interactions. The measurement was done using the recoil distance method (RDM) and a unique combination of state-of-the-art instruments at the National Superconducting Cyclotron Laboratory (NSCL). States of interest were populated via an intermediate-energy single-neutron knockout reaction. RDM studies of knockout and fragmentation reaction products hold the promise of reaching far from stability and providing lifetime information for excited states in a wide range of nuclei.

Shape and Structure of N=Z Ge64: Electromagnetic Transition Rates from the Application of the Recoil Distance Method to a Knockout Reaction

NASA Astrophysics Data System (ADS)

Starosta, K.; Dewald, A.; Dunomes, A.; Adrich, P.; Amthor, A. M.; Baumann, T.; Bazin, D.; Bowen, M.; Brown, B. A.; Chester, A.; Gade, A.; Galaviz, D.; Glasmacher, T.; Ginter, T.; Hausmann, M.; Horoi, M.; Jolie, J.; Melon, B.; Miller, D.; Moeller, V.; Norris, R. P.; Pissulla, T.; Portillo, M.; Rother, W.; Shimbara, Y.; Stolz, A.; Vaman, C.; Voss, P.; Weisshaar, D.; Zelevinsky, V.

2007-07-01

Transition rate measurements are reported for the 21+ and 22+ states in N=Z Ge64. The experimental results are in excellent agreement with large-scale shell-model calculations applying the recently developed GXPF1A interactions. The measurement was done using the recoil distance method (RDM) and a unique combination of state-of-the-art instruments at the National Superconducting Cyclotron Laboratory (NSCL). States of interest were populated via an intermediate-energy single-neutron knockout reaction. RDM studies of knockout and fragmentation reaction products hold the promise of reaching far from stability and providing lifetime information for excited states in a wide range of nuclei.

Deconstructing mammalian reproduction: using knockouts to define fertility pathways.

PubMed

Roy, Angshumoy; Matzuk, Martin M

2006-02-01

Reproduction is the sine qua non for the propagation of species and continuation of life. It is a complex biological process that is regulated by multiple factors during the reproductive life of an organism. Over the past decade, the molecular mechanisms regulating reproduction in mammals have been rapidly unraveled by the study of a vast number of mouse gene knockouts with impaired fertility. The use of reverse genetics to generate null mutants in mice through targeted disruption of specific genes has enabled researchers to identify essential regulators of spermatogenesis and oogenesis in vivo and model human disorders affecting reproduction. This review focuses on the merits, utility, and the variations of the knockout technology in studies of reproduction in mammals.

Knockout and fragmentation reactions using a broad range of tin isotopes

NASA Astrophysics Data System (ADS)

Rodríguez-Sánchez, J. L.; Benlliure, J.; Bertulani, C. A.; Vargas, J.; Ayyad, Y.; Alvarez-Pol, H.; Atkinson, J.; Aumann, T.; Beceiro-Novo, S.; Boretzky, K.; Caamaño, M.; Casarejos, E.; Cortina-Gil, D.; Díaz-Cortes, J.; Fernández, P. Díaz; Estrade, A.; Geissel, H.; Kelić-Heil, A.; Litvinov, Yu. A.; Mostazo, M.; Paradela, C.; Pérez-Loureiro, D.; Pietri, S.; Prochazka, A.; Takechi, M.; Weick, H.; Winfield, J. S.

2017-09-01

Production cross sections of residual nuclei obtained by knockout and fragmentation reactions of different tin isotopes accelerated at 1 A GeV have been measured with the fragment separator (FRS) at GSI, Darmstadt. The new measurements are used to investigate the neutron-excess dependence of the neutron- and proton-knockout cross sections. These cross sections are compared to Glauber model calculations coupled to a nuclear de-excitation code in order to investigate the role of the remnant excitations. This bench marking shows an overestimation of the cross sections for the removal of deeply bound nucleons. A phenomenological increase in the excitation energy induced in the remnants produced in these cases allows us to reproduce the measured cross sections.

Optical–Mid-infrared Period–Luminosity Relations for W UMa-type Contact Binaries Based on Gaia DR 1: 8% Distance Accuracy

NASA Astrophysics Data System (ADS)

Chen, Xiaodian; Deng, Licai; de Grijs, Richard; Wang, Shu; Feng, Yuting

2018-06-01

W Ursa Majoris (W UMa)-type contact binary systems (CBs) are useful statistical distance indicators because of their large numbers. Here, we establish (orbital) period–luminosity relations (PLRs) in 12 optical to mid-infrared bands (GBVRIJHK s W1W2W3W4) based on 183 nearby W UMa-type CBs with accurate Tycho–Gaia parallaxes. The 1σ dispersion of the PLRs decreases from optical to near- and mid-infrared wavelengths. The minimum scatter, 0.16 mag, implies that W UMa-type CBs can be used to recover distances to 7% precision. Applying our newly determined PLRs to 19 open clusters containing W UMa-type CBs demonstrates that the PLR and open cluster CB distance scales are mutually consistent to within 1%. Adopting our PLRs as secondary distance indicators, we compiled a catalog of 55,603 CB candidates, of which 80% have distance estimates based on a combination of optical, near-infrared, and mid-infrared photometry. Using Fourier decomposition, 27,318 high-probability W UMa-type CBs were selected. The resulting 8% distance accuracy implies that our sample encompasses the largest number of objects with accurate distances within a local volume with a radius of 3 kpc available to date. The distribution of W UMa-type CBs in the Galaxy suggests that in different environments, the CB luminosity function may be different: larger numbers of brighter (longer-period) W UMa-type CBs are found in younger environments.

Impaired Organization and Function of Myofilaments in Single Muscle Fibers from a Mouse Model of Pompe Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, S.; Galperin, M; Melvin, G

Pompe disease, a deficiency of lysosomal acid {alpha}-glucosidase, is a disorder of glycogen metabolism that can affect infants, children, or adults. In all forms of the disease, there is progressive muscle pathology leading to premature death. The pathology is characterized by accumulation of glycogen in lysosomes, autophagic buildup, and muscle atrophy. The purpose of the present investigation was to determine if myofibrillar dysfunction in Pompe disease contributes to muscle weakness beyond that attributed to atrophy. The study was performed on isolated myofibers dissected from severely affected fast glycolytic muscle in the {alpha}-glucosidase knockout mouse model. Psoas muscle fibers were firstmore » permeabilized, so that the contractile proteins could be directly relaxed or activated by control of the composition of the bathing solution. When normalized by cross-sectional area, single fibers from knockout mice produced 6.3 N/cm{sup 2} of maximum Ca{sup 2+}-activated tension compared with 12.0 N/cm{sup 2} produced by wild-type fibers. The total protein concentration was slightly higher in the knockout mice, but concentrations of the contractile proteins myosin and actin remained unchanged. Structurally, X-ray diffraction showed that the actin and myosin filaments, normally arranged in hexagonal arrays, were disordered in the knockout muscle, and a lower fraction of myosin cross bridges was near the actin filaments in the relaxed muscle. The results are consistent with a disruption of actin and myosin interactions in the knockout muscles, demonstrating that impaired myofibrillar function contributes to weakness in the diseased muscle fibers.« less

Reversal of disease-related pathologies in the fragile X mouse model by selective activation of GABAB receptors with arbaclofen.

PubMed

Henderson, Christina; Wijetunge, Lasani; Kinoshita, Mika Nakamoto; Shumway, Matthew; Hammond, Rebecca S; Postma, Friso R; Brynczka, Christopher; Rush, Roger; Thomas, Alexia; Paylor, Richard; Warren, Stephen T; Vanderklish, Peter W; Kind, Peter C; Carpenter, Randall L; Bear, Mark F; Healy, Aileen M

2012-09-19

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism, results from the transcriptional silencing of FMR1 and loss of the mRNA translational repressor protein fragile X mental retardation protein (FMRP). Patients with FXS exhibit changes in neuronal dendritic spine morphology, a pathology associated with altered synaptic function. Studies in the mouse model of fragile X have shown that loss of FMRP causes excessive synaptic protein synthesis, which results in synaptic dysfunction and altered spine morphology. We tested whether the pharmacologic activation of the γ-aminobutyric acid type B (GABA(B)) receptor could correct or reverse these phenotypes in Fmr1-knockout mice. Basal protein synthesis, which is elevated in the hippocampus of Fmr1-knockout mice, was corrected by the in vitro application of the selective GABA(B) receptor agonist STX209 (arbaclofen, R-baclofen). STX209 also reduced to wild-type values the elevated AMPA receptor internalization in Fmr1-knockout cultured neurons, a known functional consequence of increased protein synthesis. Acute administration of STX209 in vivo, at doses that modify behavior, decreased mRNA translation in the cortex of Fmr1-knockout mice. Finally, the chronic administration of STX209 in juvenile mice corrected the increased spine density in Fmr1-knockout mice without affecting spine density in wild-type mice. Thus, activation of the GABA(B) receptor with STX209 corrected synaptic abnormalities considered central to fragile X pathophysiology, a finding that suggests that STX209 may be a potentially effective therapy to treat the core symptoms of FXS.

Knockout of Epstein-Barr Virus BPLF1 Retards B-Cell Transformation and Lymphoma Formation in Humanized Mice

PubMed Central

Li, Guangming; Montgomery, Stephanie A.; Montgomery, Nathan D.; Su, Lishan; Pagano, Joseph S.

2015-01-01

ABSTRACT BPLF1 of Epstein-Barr virus (EBV) is classified as a late lytic cycle protein but is also found in the viral tegument, suggesting its potential involvement at both initial and late stages of viral infection. BPLF1 possesses both deubiquitinating and deneddylating activity located in its N-terminal domain and is involved in processes that affect viral infectivity, viral DNA replication, DNA repair, and immune evasion. A recently constructed EBV BPLF1-knockout (KO) virus was used in conjunction with a humanized mouse model that can be infected with EBV, enabling the first characterization of BPLF1 function in vivo. Results demonstrate that the BPLF1-knockout virus is approximately 90% less infectious than wild-type (WT) virus. Transformation of human B cells, a hallmark of EBV infection, was delayed and reduced with BPLF1-knockout virus. Humanized mice infected with EBV BPLF1-knockout virus showed less weight loss and survived longer than mice infected with equivalent infectious units of WT virus. Additionally, splenic tumors formed in 100% of mice infected with WT EBV but in only 25% of mice infected with BPLF1-KO virus. Morphological features of spleens containing tumors were similar to those in EBV-induced posttransplant lymphoproliferative disease (PTLD) and were almost identical to cases seen in human diffuse large B-cell lymphoma. The presence of EBV genomes was detected in all mice that developed tumors. The results implicate BPLF1 in human B-cell transformation and tumor formation in humanized mice. PMID:26489865

The role of KCNQ1/KCNE1 K(+) channels in intestine and pancreas: lessons from the KCNE1 knockout mouse.

PubMed

Warth, R; Garcia Alzamora, M; Kim, J K; Zdebik, A; Nitschke, R; Bleich, M; Gerlach, U; Barhanin, J; Kim, S J

2002-03-01

KCNE1 (IsK, minK) co-assembles with KCNQ1 (KvLQT1) to form voltage-dependent K(+) channels. Both KCNQ1 and KCNE1 are expressed in epithelial cells of gut and exocrine pancreas. We examined the role of KCNQ1/KCNE1 in Cl(-) secretion in small and large intestine and exocrine pancreas using the KCNE1 knockout mouse. Immunofluorescence revealed a similar basolateral localization of KCNQ1 in jejunum and colon of KCNE1 wild-type and knockout mice. Electrogenic Cl(-) secretion in the colon was not affected by gene disruption of KCNE1; in jejunum forskolin-induced short-circuit current was some 40% smaller but without being significantly different. Inhibition of KCNQ1 channels by 293B (IC(50) 1 micromol l(-1)) and by IKS224 (IC(50) 14 nmol l(-1)) strongly diminished intestinal Cl(-) secretion. In exocrine pancreas of wild-type mice, KCNQ1 was predominantly located at the basolateral membrane. In KCNE1 knockout mice, however, the basolateral staining was less pronounced and the distribution of secretory granules was irregular. A slowly activating and 293B-sensitive K(+) current was activated via cholinergic stimulation in pancreatic acinar cells of wild-type mice. In KCNE1 knockout mice this K(+) current was strongly reduced. In conclusion intestinal Cl(-) secretion is independent from KCNE1 but requires KCNQ1. In mouse pancreatic acini KCNQ1 probably co-assembled with KCNE1 leads to a voltage-dependent K(+) current that might be of importance for electrolyte and enzyme secretion.

Effects of vitamin D receptor knockout on cornea epithelium gap junctions.

PubMed

Lu, Xiaowen; Watsky, Mitchell A

2014-05-06

Gap junctions are present in all corneal cell types and have been shown to have a critical role in cell phenotype determination. Vitamin D has been shown to influence cell differentiation, and recent work demonstrates the presence of vitamin D in the ocular anterior segment. This study measured and compared gap junction diffusion coefficients among different cornea epithelium phenotypes and in keratocytes using a noninvasive technique, fluorescence recovery after photobleaching (FRAP), and examined the influence of vitamin D receptor (VDR) knockout on epithelial gap junction communication in intact corneas. Previous gap junction studies in cornea epithelium and keratocytes were performed using cultured cells or ex vivo invasive techniques. These invasive techniques were unable to measure diffusion coefficients and likely were disruptive to normal cell physiology. Corneas from VDR knockout and control mice were stained with 5(6)-carboxyfluorescein diacetate (CFDA). Gap junction diffusion coefficients of the corneal epithelium phenotypes and of keratocytes, residing in intact corneas, were detected using FRAP. Diffusion coefficients equaled 18.7, 9.8, 5.6, and 4.2 μm(2)/s for superficial squamous cells, middle wing cells, basal cells, and keratocytes, respectively. Corneal thickness, superficial cell size, and the superficial squamous cell diffusion coefficient of 10-week-old VDR knockout mice were significantly lower than those of control mice (P < 0.01). The superficial cell diffusion coefficient of heterozygous mice was significantly lower than control mice (P < 0.05). Our results demonstrate differences in gap junction dye spread among the epithelial cell phenotypes, mirroring the epithelial developmental axis. The VDR knockout influences previously unreported cell-to-cell communication in superficial epithelium.

Differential gene expression in Ndph-knockout mice in retinal development.

PubMed

Schäfer, Nikolaus F; Luhmann, Ulrich F O; Feil, Silke; Berger, Wolfgang

2009-02-01

Mutations in the NDP gene impair angiogenesis in the eyes of patients diagnosed with a type of blindness belonging to the group of exudative vitreoretinopathies. This study was conducted to investigate the differential gene expression caused by the absence of Norrin (the NDP protein) in the developing mouse retina and to elucidate early pathogenic events. A comparative gene expression analysis was performed on postnatal day (p)7 retinas from a knockout mouse model for Norrie disease using gene microarrays. Subsequently, results were verified by quantitative real-time PCR analyses. Immunohistochemistry was performed for the vascular permeability marker plasmalemma vesicle associated protein (Plvap). Our study identified expression differences in Ndph(y/-) versus wild-type mice retinas at p7. Gene transcription of the neutral amino acid transporter Slc38a5, apolipoprotein D (ApoD), and angiotensin II receptor-like 1 (Agtrl1) was decreased in the knockout mouse, whereas transcript levels of adrenomedullin (Adm) and of the plasmalemma vesicle associated protein (Plvap) were increased in comparison to the wild-type. In addition, ectopic expression of Plvap was found in the developing retinal vasculature of Norrin-knockout mice on the protein level. These data provide molecular evidence for a role of Norrin in the development of the retinal vasculature. Expression of two genes, Plvap and Slc38a5, is considerably altered in retinal development of Norrin-knockout mice and may reflect or contribute to the pathogenesis of the disease. In particular, ectopic expression of Plvap is consistent with hallmark disease symptoms in mice and humans.

Vascular changes in the cerebellum of Norrin /Ndph knockout mice correlate with high expression of Norrin and Frizzled-4.

PubMed

Luhmann, Ulrich F O; Neidhardt, John; Kloeckener-Gruissem, Barbara; Schäfer, Nikolaus F; Glaus, Esther; Feil, Silke; Berger, Wolfgang

2008-05-01

X-linked Norrie disease, familial exudative vitreoretinopathy (FEVR), Coat's disease and retinopathy of prematurity are severe human eye diseases and can all be caused by mutations in the Norrie disease pseudoglioma gene. They all show vascular defects and characteristic features of retinal hypoxia. Only Norrie disease displays additional neurological symptoms, which are sensorineural hearing loss and mental retardation. In the present study, we analysed transcript levels of the ligand Norrin (Ndph) and its two receptors Frizzled-4 (Fzd4) and LDL-related protein receptor 5 (Lrp5) in six different brain regions (cerebellum, cortex, hippocampus, olfactory bulb, pituitary and brain stem) of 6- to 8-month-old wild-type and Ndph knockout mice by quantitative real-time PCR. No effect of the Ndph knockout allele on Fzd4 or Lrp5 receptor expression was found. Furthermore, no alterations of the transcript levels of three hypoxia-regulated angiogenic factors (Vegfa, Itgrb3 and Tie1) were observed in the absence of Norrin. Interestingly, we identified significant differences in Ndph, Fzd4 and Lrp5 transcript levels in brain regions of wild-type mice and observed highest expression of Norrin and frizzled-4 in cerebellum. Transcript analyses were correlated with morphological data obtained from cerebellum and immunohistochemical studies of blood vessels in different brain regions. Vessel density was reduced in the cerebellum of Ndph knockout mice but the number of Purkinje and granular cells was not altered. This provides the first description of a brain phenotype in Ndph knockout mice, which will help to elucidate the role of Norrin in the brain.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yanyan; The Hamner Institutes for Health Sciences, Research Triangle Park, NC; Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-doublemore » knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.« less

EFFECTS OF HEAT AND BROMOCHLOROACETIC ACID ON MALE REPRODUCTION IN HEAT SHOCK FACTOR-1 GENE KNOCKOUT MICE

EPA Science Inventory

Effects of heat and bromochloroacetic acid on male reproduction in heat shock factor-1 gene knockout mice.
Luft JC1, IJ Benjamin2, JB Garges1 and DJ Dix1. 1Reproductive Toxicology Division, USEPA, RTP, NC, 27711 and 2Dept of Internal Medicine, Univ.of Texas Southwestern Med C...

THE EFFECT OF TARGETED KNOCKOUT MUTATION ON THE TRANSCRIPTIONAL PROFILE OF THE KIDNEY IN TSC2 MUTANT LONG-EVANS (EKER) RATS.

EPA Science Inventory

The effect of a targeted knockout mutation on the transcriptional profile of the kidney in
Tsc2 mutant Long-Evans (Eker) rats.

Renal cell carcinoma (RCC) is the most common tumor of the adult kidney, accounting
for up to 80% of malignant renal neoplasms. Hereditary...

Survival and Injury Outcome After TBI: Influence of Pre- and Post-Exposure to Caffeine

DTIC Science & Technology

2012-10-01

A1Rs. This notion is supported by findings that TBI in A1R knockout mice led to lethal status epilepticus (SE) (Kochanek et al., 2006). Likewise, A1R...G.E., Dixon, C.E., Schnermann, J., Jackson, E.K., 2006. Adenosine A1 receptor knockout mice develop lethal status epilepti- cus after experimental

Brief Report: Altered Social Behavior in Isolation-Reared "Fmr1" Knockout Mice

ERIC Educational Resources Information Center

Heitzer, Andrew M.; Roth, Alexandra K.; Nawrocki, Lauren; Wrenn, Craige C.; Valdovinos, Maria G.

2013-01-01

Social behavior abnormalities in Fragile X syndrome (FXS) are characterized by social withdrawal, anxiety, and deficits in social cognition. To assess these deficits, a model of FXS, the "Fmr1" knockout mouse ("Fmr1" KO), has been utilized. This mouse model has a null mutation in the fragile X mental retardation 1 gene ("Fmr1") and displays…

Kv4.2 Knockout Mice Have Hippocampal-Dependent Learning and Memory Deficits

ERIC Educational Resources Information Center

Lugo, Joaquin N.; Brewster, Amy L.; Spencer, Corinne M.; Anderson, Anne E.

2012-01-01

Kv4.2 channels contribute to the transient, outward K[superscript +] current (A-type current) in hippocampal dendrites, and modulation of this current substantially alters dendritic excitability. Using Kv4.2 knockout (KO) mice, we examined the role of Kv4.2 in hippocampal-dependent learning and memory. We found that Kv4.2 KO mice showed a deficit…

FOCuS: a metaheuristic algorithm for computing knockouts from genome-scale models for strain optimization.

PubMed

Mutturi, Sarma

2017-06-27

Although handful tools are available for constraint-based flux analysis to generate knockout strains, most of these are either based on bilevel-MIP or its modifications. However, metaheuristic approaches that are known for their flexibility and scalability have been less studied. Moreover, in the existing tools, sectioning of search space to find optimal knocks has not been considered. Herein, a novel computational procedure, termed as FOCuS (Flower-pOllination coupled Clonal Selection algorithm), was developed to find the optimal reaction knockouts from a metabolic network to maximize the production of specific metabolites. FOCuS derives its benefits from nature-inspired flower pollination algorithm and artificial immune system-inspired clonal selection algorithm to converge to an optimal solution. To evaluate the performance of FOCuS, reported results obtained from both MIP and other metaheuristic-based tools were compared in selected case studies. The results demonstrated the robustness of FOCuS irrespective of the size of metabolic network and number of knockouts. Moreover, sectioning of search space coupled with pooling of priority reactions based on their contribution to objective function for generating smaller search space significantly reduced the computational time.

Disrupting Hepatocyte Cyp51 from Cholesterol Synthesis Leads to Progressive Liver Injury in the Developing Mouse and Decreases RORC Signalling

NASA Astrophysics Data System (ADS)

Urlep, Žiga; Lorbek, Gregor; Perše, Martina; Jeruc, Jera; Juvan, Peter; Matz-Soja, Madlen; Gebhardt, Rolf; Björkhem, Ingemar; Hall, Jason A.; Bonneau, Richard; Littman, Dan R.; Rozman, Damjana

2017-01-01

Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51-/-) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51-/- and Rorc-/- expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51-/- females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity.

Genotype identification of Math1/LacZ knockout mice based on real-time PCR with SYBR Green I dye.

PubMed

Krizhanovsky, Valery; Golenser, Esther; Ben-Arie, Nissim

2004-07-30

Knockout mice are widely used in all fields of biomedical research. Determining the genotype of every newborn mouse is a tedious task, usually performed by Southern blot hybridization or Polymerase Chain Reaction (PCR). We describe here a quick and simple genotype identification assay based on real-time PCR and SYBR Green I dye, without using fluorescent primers. The discrimination between the wild type and targeted alleles is based on a PCR design that leads to a different melting temperature for each product. The identification of the genotype is obvious immediately after amplification, and no post-PCR manipulations are needed, reducing cost and time. Therefore, while the real-time PCR amplification increases the sensitivity, the fact that the reactions tubes are never opened after amplification, reduces the risk of contamination and eliminates errors, which are common during the repeated handling of dozens of samples from the same mouse line. The protocol we provide was tested on Math1 knockout mice, but is general, and may be utilized for any knockout line and real-time thermocycler, without any further modification, accessories or special reagents. Copyright 2004 Elsevier B.V.

Lithium ameliorates altered glycogen synthase kinase-3 and behavior in a mouse model of fragile X syndrome.

PubMed

Yuskaitis, Christopher J; Mines, Marjelo A; King, Margaret K; Sweatt, J David; Miller, Courtney A; Jope, Richard S

2010-02-15

Fragile X syndrome (FXS), the most common form of inherited mental retardation and a genetic cause of autism, results from mutated fragile X mental retardation-1 (Fmr1). This study examined the effects on glycogen synthase kinase-3 (GSK3) of treatment with a metabotropic glutamate receptor (mGluR) antagonist, MPEP, and the GSK3 inhibitor, lithium, in C57Bl/6 Fmr1 knockout mice. Increased mGluR signaling may contribute to the pathology of FXS, and the mGluR5 antagonist MPEP increased inhibitory serine-phosphorylation of brain GSK3 selectively in Fmr1 knockout mice but not in wild-type mice. Inhibitory serine-phosphorylation of GSK3 was lower in Fmr1 knockout, than wild-type, mouse brain regions and was increased by acute or chronic lithium treatment, which also increased hippocampal brain-derived neurotrophic factor levels. Fmr1 knockout mice displayed alterations in open-field activity, elevated plus-maze, and passive avoidance, and these differences were ameliorated by chronic lithium treatment. These findings support the hypothesis that impaired inhibition of GSK3 contributes to the pathogenesis of FXS and support GSK3 as a potential therapeutic target.

LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

PubMed Central

2012-01-01

Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation. We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis. Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes. PMID:22647713

Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line.

PubMed

Stephenson, Sarah E M; Aumann, Timothy D; Taylor, Juliet M; Riseley, Jessica R; Li, Ruili; Mann, Jeffrey R; Tomas, Doris; Lockhart, Paul J

2018-05-14

Mutations in PARK2 (parkin) can result in Parkinson's disease (PD). Parkin shares a bidirectional promoter with parkin coregulated gene (PACRG) and the transcriptional start sites are separated by only ~200 bp. Bidirectionally regulated genes have been shown to function in common biological pathways. Mice lacking parkin have largely failed to recapitulate the dopaminergic neuronal loss and movement impairments seen in individuals with parkin-mediated PD. We aimed to investigate the function of PACRG and test the hypothesis that parkin and PACRG function in a common pathway by generating and characterizing two novel knockout mouse lines harbouring loss of both parkin and Pacrg or Pacrg alone. Successful modification of the targeted allele was confirmed at the genomic, transcriptional and steady state protein levels for both genes. At 18-20 months of age, there were no significant differences in the behaviour of parental and mutant lines when assessed by openfield, rotarod and balance beam. Subsequent neuropathological examination suggested there was no gross abnormality of the dopaminergic system in the substantia nigra and no significant difference in the number of dopaminergic neurons in either knockout model compared to wildtype mice.

Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

PubMed

Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

2014-10-01

Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.

Genome Sequence Analysis of Vibrio cholerae clinical isolates from 2013 in Mexico reveals the presence of the strain responsible for the 2010 Haiti outbreak.

PubMed

Díaz-Quiñonez, José Alberto

2017-01-01

La primera semana de septiembre de 2013, el Sistema Nacional de Vigilancia Epidemiológica identificó dos casos de cólera en Ciudad de México. Los cultivos de ambas muestras se confirmaron como Vibrio cholerae serogrupo O1, serotipo Ogawa, biotipo El Tor. Los análisis iniciales por electroforesis por campos pulsados y por reacción en cadena de la polimerasa indicaron que ambas cepas eran similares, pero diferentes de las previamente reportadas en México. La semana siguiente se identificaron cuatro casos más en una comunidad del Estado de Hidalgo, ubicada a 121 kilómetros al noreste de Ciudad de México. Posteriormente se inició un brote de cólera en la región de La Huasteca. Los análisis genómicos de cuatro cepas obtenidas en este estudio confirmaron la presencia de las islas de patogenicidad VPI -1 y VPI-2, VSP-1 y VSP-2, y del elemento integrador SXT. La estructura genómica de los cuatro aislamientos fue similar a la de V. cholerae cepa 2010 EL-1786, identificada durante la epidemia en Haití en 2010. Este estudio pone de manifiesto que la epidemiología molecular es una herramienta muy poderosa para vigilar, prevenir y controlar enfermedades de importancia en salud pública en México. The first week of September 2013, the National Epidemiological Surveillance System identified two cases of cholera in Mexico City. The cultures of both samples were confirmed as Vibrio cholerae serogroup O1, serotype Ogawa, biotype El Tor. Initial analyses by pulsed-field gel electrophoresis and by polymerase chain reaction-amplification of the virulence genes, suggested that both strains were similar, but different from those previously reported in Mexico. The following week, four more cases were identified in a community in the state of Hidalgo, located 121 km northeast of Mexico City. Thereafter a cholera outbreak started in the region of La Huasteca. Genomic analyses of the strains obtained in this study confirmed the presence of pathogenicity islands VPI-1 and VPI-2, VSP-1 and VSP-2, and of the integrative element SXT. The genomic structure of the 4 isolates was similar to that of V. cholerae strain 2010 EL-1786, identified during the epidemic in Haiti in 2010. This study shows that molecular epidemiology is a very powerful tool to monitor, prevent and control diseases of public health importance in Mexico. Copyright: © 2017 SecretarÍa de Salud.

GeneKnockout by Targeted Mutagenesis in a Hemimetabolous Insect, the Two-Spotted Cricket Gryllus bimaculatus, using TALENs.

PubMed

Watanabe, Takahito; Noji, Sumihare; Mito, Taro

2016-01-01

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms.

A norm knockout method on indirect reciprocity to reveal indispensable norms

PubMed Central

Yamamoto, Hitoshi; Okada, Isamu; Uchida, Satoshi; Sasaki, Tatsuya

2017-01-01

Although various norms for reciprocity-based cooperation have been suggested that are evolutionarily stable against invasion from free riders, the process of alternation of norms and the role of diversified norms remain unclear in the evolution of cooperation. We clarify the co-evolutionary dynamics of norms and cooperation in indirect reciprocity and also identify the indispensable norms for the evolution of cooperation. Inspired by the gene knockout method, a genetic engineering technique, we developed the norm knockout method and clarified the norms necessary for the establishment of cooperation. The results of numerical investigations revealed that the majority of norms gradually transitioned to tolerant norms after defectors are eliminated by strict norms. Furthermore, no cooperation emerges when specific norms that are intolerant to defectors are knocked out. PMID:28276485

A norm knockout method on indirect reciprocity to reveal indispensable norms

NASA Astrophysics Data System (ADS)

Yamamoto, Hitoshi; Okada, Isamu; Uchida, Satoshi; Sasaki, Tatsuya

2017-03-01

Although various norms for reciprocity-based cooperation have been suggested that are evolutionarily stable against invasion from free riders, the process of alternation of norms and the role of diversified norms remain unclear in the evolution of cooperation. We clarify the co-evolutionary dynamics of norms and cooperation in indirect reciprocity and also identify the indispensable norms for the evolution of cooperation. Inspired by the gene knockout method, a genetic engineering technique, we developed the norm knockout method and clarified the norms necessary for the establishment of cooperation. The results of numerical investigations revealed that the majority of norms gradually transitioned to tolerant norms after defectors are eliminated by strict norms. Furthermore, no cooperation emerges when specific norms that are intolerant to defectors are knocked out.

Generation of knockout rabbits using transcription activator-like effector nucleases.

PubMed

Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

2014-01-01

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool.

PubMed

Wettstein, Rahel; Bodak, Maxime; Ciaudo, Constance

2016-01-01

CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.

Crimean-Congo Hemorrhagic Fever Virus Subunit Vaccines Induce High Levels of Neutralizing Antibodies But No Protection in STAT1 Knockout Mice.

PubMed

Kortekaas, Jeroen; Vloet, Rianka P M; McAuley, Alexander J; Shen, Xiaoli; Bosch, Berend Jan; de Vries, Laura; Moormann, Rob J M; Bente, Dennis A

2015-12-01

Crimean-Congo hemorrhagic fever virus is a tick-borne bunyavirus of the Nairovirus genus that causes hemorrhagic fever in humans with high case fatality. Here, we report the development of subunit vaccines and their efficacy in signal transducer and activator of transcription 1 (STAT1) knockout mice. Ectodomains of the structural glycoproteins Gn and Gc were produced using a Drosophila insect cell-based expression system. A single vaccination of STAT129 mice with adjuvanted Gn or Gc ectodomains induced neutralizing antibody responses, which were boosted by a second vaccination. Despite these antibody responses, mice were not protected from a CCHFV challenge infection. These results suggest that neutralizing antibodies against CCHFV do not correlate with protection of STAT1 knockout mice.

Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer.

PubMed

Shen, Youfeng; Xu, Kaixiang; Yuan, Zaimei; Guo, Jianxiong; Zhao, Heng; Zhang, Xuezeng; Zhao, Lu; Qing, Yubo; Li, Honghui; Pan, Weirong; Jia, Baoyu; Zhao, Hong-Ye; Wei, Hong-Jiang

2017-11-03

Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate. Transcription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting. The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping. Finally, six live piglets and one stillborn piglet were collected from two recipients by caesarean section. Sequencing analyses of the target site confirmed the P53 biallelic knockout in all fetuses and piglets, consistent with the genotype of the donor cells. The qPCR analysis showed that the expression of the P53 mRNA had significant reduction in various tissues of the knockout piglets. Furthermore, confocal microscopy and western blotting analyses demonstrated that the fibroblast cells of Diannan miniature piglets with a P53 biallelic knockout were defective in mediating DNA damage when incubated with doxorubicin. TALENs combined with SCNT was successfully used to generate P53 KO Diannan miniature pigs. Although these genetically engineered Diannan miniature pigs had no tumorigenic signs, the P53 gene was dysfunctional. We believe that these pigs will provide powerful new resources for preclinical oncology and basic cancer research.

Oscillations of Accretion Disks in Cataclysmic Variable Stars

NASA Astrophysics Data System (ADS)

Osaki, Y.

2013-12-01

The disk instability model for the outbursts of dwarf novae is reviewed, with particular attention given to the superoutburst of SU UMa stars. Two intrinsic instabilities in accretion disks of dwarf novae are known; the thermal instability and the tidal instability. The thermal-tidal instability model (abbreviated the TTI model), which combines these two instabilities, was first proposed in 1989 by Osaki (1989) to explain the superoutburst phenomenon of SU UMa stars. Recent Kepler observations of one SU UMa star, V1504 Cyg, have dramatically demonstrated that the superoutburst phenomenon of the SU UMa stars is explained by the thermal-tidal instability model.

The evolution of the lithium abundances of solar-type stars. II - The Ursa Major Group

NASA Technical Reports Server (NTRS)

Soderblom, David R.; Pilachowski, Catherine A.; Fedele, Stephen B.; Jones, Burton F.

1993-01-01

We draw upon a recent study of the membership of the Ursa Major Group (UMaG) to examine lithium among 0.3 Gyr old solar-type stars. For most G and K dwarfs, Li confirms the conclusions about membership in UMaG reached on the basis of kinematics and chromospheric activity. G and K dwarfs in UMaG have less Li than comparable stars in the Pleiades. This indicates that G and K dwarfs undergo Li depletion while they are on the main sequence, in addition to any pre-main-sequence depletion they may have experienced. Moreover, the Li abundances of the Pleiades K dwarfs cannot be attributed to main-sequence depletion alone, demonstrating that pre-main-sequence depletion of Li also takes place. The sun's Li abundance implies that the main-sequence mechanism becomes less effective with age. The hottest stars in UMaG have Li abundances like those of hot stars in the Pleiades and Hyades and in T Tauris, and the two genuine UMaG members with temperatures near Boesgaard's Li chasm have Li abundances consistent with that chasm developing fully by 0.3 Gyr for stars with UMaG's metallicity. We see differences in the abundance of Li between UMaG members of the same spectral types, indicating that a real spread in the lithium abundance exists within this group.

Sequential ultrasound-microwave assisted acid extraction (UMAE) of pectin from pomelo peels.

PubMed

Liew, Shan Qin; Ngoh, Gek Cheng; Yusoff, Rozita; Teoh, Wen Hui

2016-12-01

This study aims to optimize sequential ultrasound-microwave assisted extraction (UMAE) on pomelo peel using citric acid. The effects of pH, sonication time, microwave power and irradiation time on the yield and the degree of esterification (DE) of pectin were investigated. Under optimized conditions of pH 1.80, 27.52min sonication followed by 6.40min microwave irradiation at 643.44W, the yield and the DE value of pectin obtained was respectively at 38.00% and 56.88%. Based upon optimized UMAE condition, the pectin from microwave-ultrasound assisted extraction (MUAE), ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE) were studied. The yield of pectin adopting the UMAE was higher than all other techniques in the order of UMAE>MUAE>MAE>UAE. The pectin's galacturonic acid content obtained from combined extraction technique is higher than that obtained from sole extraction technique and the pectin gel produced from various techniques exhibited a pseudoplastic behaviour. The morphological structures of pectin extracted from MUAE and MAE closely resemble each other. The extracted pectin from UMAE with smaller and more regular surface differs greatly from that of UAE. This has substantiated the highest pectin yield of 36.33% from UMAE and further signified their compatibility and potentiality in pectin extraction. Copyright © 2016 Elsevier B.V. All rights reserved.

Merging first principle structure studies and few-body reaction formalism

NASA Astrophysics Data System (ADS)

Crespo, R.; Cravo, E.; Arriaga, A.; Wiringa, R.; Deltuva, A.; Diego, R.

2018-02-01

Calculations for nucleon knockout from a 7Li beam due to the collision with a proton target at 400 MeV/u are shown based on ab initio Quantum Monte Carlo (QMC) and conventional shell-model nuclear structure approaches to describe the relative motion between the knockout particle and the heavy fragment of the projectile. Structure effects on the total cross section are shown.

Ascorbate synthesis pathway, dual role of ascorbate in bone homeostasis

USDA-ARS?s Scientific Manuscript database

Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of D-glucuronate to L-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) m...

Proteomic Analyses of NF1-Interacting Proteins in Keratinocytes

DTIC Science & Technology

2015-04-01

and knockout mice further confirmed the interactions suggested by the proteomic analyses. In relation to the development of psoriasis -like symptoms...in the NF1 null epidermis, we analyzed NF1 expression in a mouse model of psoriasis (imiquimod-induced psoriasis -like skin inflammation) and...knockout of epidermal NF1 to elucidate the molecular underpinnings of psoriasis . 15. SUBJECT TERMS neurofibromin-1 (NF1), psoriasis , inflammation

Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

ERIC Educational Resources Information Center

Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

2016-01-01

Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

Hypercholesterolemia is associated with a T helper (Th) 1/Th2 switch of the autoimmune response in atherosclerotic apo E-knockout mice.

PubMed Central

Zhou, X; Paulsson, G; Stemme, S; Hansson, G K

1998-01-01

Atherosclerosis is an inflammatory-fibrotic response to accumulation of cholesterol in the artery wall. In hypercholesterolemia, low density lipoproteins (LDL) accumulate and are oxidized to proinflammatory compounds in the arterial intima, leading to activation of endothelial cells, macrophages, and T lymphocytes. We have studied immune cell activation and the autoimmune response to oxidized LDL in atherosclerotic apo E-knockout mice. Autoantibodies to oxidized LDL exhibited subclass specificities indicative of T cell help, and the increase in antibody titers in peripheral blood was associated with increased numbers of cytokine-expressing T cells in the spleen. In addition to T cell-dependent antibodies, IgM antibodies to oxidized LDL were also increased in apo E-knockout mice. This suggests that both T cell-dependent and T cell-independent epitopes may be present on oxidized LDL. In moderate hypercholesterolemia, IgG antibodies were largely of the IgG2a isotype, suggesting that T cell help was provided by proinflammatory T helper (Th) 1 cells, which are prominent components of atherosclerotic lesions. In severe hypercholesterolemia induced by cholesterol feeding of apo E-knockout mice, a switch to Th2-dependent help was evident. It was associated with a loss of IFN-gamma-producing Th1 cells in the spleen, whereas IL-4-producing Th2 cells were more resistant to hypercholesterolemia. IFN-gamma but not IL-4 mRNA was detected in atherosclerotic lesions of moderately hypercholesterolemic apo E-knockout mice, but IL-4 mRNA appeared in the lesions when mice were made severely hypercholesterolemic by cholesterol feeding. These data show that IFN-gamma-producing Th1 cells infiltrate atherosclerotic lesions and provide T cell help for autoimmune responses to oxidized LDL in apo E-knockout mice. However, severe hypercholesterolemia is associated with a switch from Th1 to Th2, which results not only in the formation of IgG1 autoantibodies to oxidized LDL, but also in the appearance of Th2-type cytokines in the atherosclerotic lesions. Since the two subsets of T cells counteract each other, this switch may have important consequences for the inflammatory/immune process in atherosclerosis. PMID:9541503

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanarsdall, Adam L.; Mikhailov, Victor S.; N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808

2007-08-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbpmore » knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma.« less

High-sensitivity O-glycomic analysis of mice deficient in core 2 β1,6-N-acetylglucosaminyltransferases

PubMed Central

Ismail, Mohd Nazri; Stone, Erica L; Panico, Maria; Lee, Seung Ho; Luu, Ying; Ramirez, Kevin; Ho, Samuel B; Fukuda, Minoru; Marth, Jamey D; Haslam, Stuart M; Dell, Anne

2011-01-01

Core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT), which exists in three isoforms, C2GnT1, C2GnT2 and C2GnT3, is one of the key enzymes in the O-glycan biosynthetic pathway. These isoenzymes produce core 2 O-glycans and have been correlated with the biosynthesis of core 4 O-glycans and I-branches. Previously, we have reported mice with single and multiple deficiencies of C2GnT isoenzyme(s) and have evaluated the biological and structural consequences of the loss of core 2 function. We now present more comprehensive O-glycomic analyses of neutral and sialylated glycans expressed in the colon, small intestine, stomach, kidney, thyroid/trachea and thymus of wild-type, C2GnT2 and C2GnT3 single knockouts and the C2GnT1–3 triple knockout mice. Very high-quality data have emerged from our mass spectrometry techniques with the capability of detecting O-glycans up to at least 3500 Da. We were able to unambiguously elucidate the types of O-glycan core, branching location and residue linkages, which allowed us to exhaustively characterize structural changes in the knockout tissues. The C2GnT2 knockout mice suffered a major loss of core 2 O-glycans as well as glycans with I-branches on core 1 antennae especially in the stomach and the colon. In contrast, core 2 O-glycans still dominated the O-glycomic profile of most tissues in the C2GnT3 knockout mice. Analysis of the C2GnT triple knockout mice revealed a complete loss of both core 2 O-glycans and branched core 1 antennae, confirming that the three known isoenzymes are entirely responsible for producing these structures. Unexpectedly, O-linked mannosyl glycans are upregulated in the triple deficient stomach. In addition, our studies have revealed an interesting terminal structure detected on O-glycans of the colon tissues that is similar to the RM2 antigen from glycolipids. PMID:20855471

Toll-Like Receptor 3 Is Critical for Coxsackievirus B4-Induced Type 1 Diabetes in Female NOD Mice

PubMed Central

Thuma, Jean R.; Courreges, Maria C.; Benencia, Fabian; James, Calvin B.L.; Malgor, Ramiro; Kantake, Noriko; Mudd, William; Denlinger, Nathan; Nolan, Bret; Wen, Li; Schwartz, Frank L.

2015-01-01

Group B coxsackieviruses (CVBs) are involved in triggering some cases of type 1 diabetes mellitus (T1DM). However, the molecular mechanism(s) responsible for this remain elusive. Toll-like receptor 3 (TLR3), a receptor that recognizes viral double-stranded RNA, is hypothesized to play a role in virus-induced T1DM, although this hypothesis is yet to be substantiated. The objective of this study was to directly investigate the role of TLR3 in CVB-triggered T1DM in nonobese diabetic (NOD) mice, a mouse model of human T1DM that is widely used to study both spontaneous autoimmune and viral-induced T1DM. As such, we infected female wild-type (TLR3+/+) and TLR3 knockout (TLR3−/−) NOD mice with CVB4 and compared the incidence of diabetes in CVB4-infected mice with that of uninfected counterparts. We also evaluated the islets of uninfected and CVB4-infected wild-type and TLR3 knockout NOD mice by immunohistochemistry and insulitis scoring. TLR3 knockout mice were markedly protected from CVB4-induced diabetes compared with CVB4-infected wild-type mice. CVB4-induced T-lymphocyte-mediated insulitis was also significantly less severe in TLR3 knockout mice compared with wild-type mice. No differences in insulitis were observed between uninfected animals, either wild-type or TLR3 knockout mice. These data demonstrate for the first time that TLR3 is 1) critical for CVB4-induced T1DM, and 2) modulates CVB4-induced insulitis in genetically prone NOD mice. PMID:25422874

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

PubMed

Kuwahara, Go; Hashimoto, Takuya; Tsuneki, Masayuki; Yamamoto, Kota; Assi, Roland; Foster, Trenton R; Hanisch, Jesse J; Bai, Hualong; Hu, Haidi; Protack, Clinton D; Hall, Michael R; Schardt, John S; Jay, Steven M; Madri, Joseph A; Kodama, Shohta; Dardik, Alan

2017-06-01

Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 μm; P =0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P =0.0015; interleukin-10: 7.6-fold, P =0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 μm; P =0.0306), increased collagen density (2.4-fold; P =0.0432), and increased number of M2 macrophages (2.1-fold; P =0.0335). CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation. © 2017 American Heart Association, Inc.

Importance of GluA1 Subunit-Containing AMPA Glutamate Receptors for Morphine State-Dependency

PubMed Central

Aitta-aho, Teemu; Möykkynen, Tommi P.; Panhelainen, Anne E.; Vekovischeva, Olga Yu.; Bäckström, Pia; Korpi, Esa R.

2012-01-01

In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days) using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test); in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards), in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity. PMID:22675452

Genetic loss of diazepam binding inhibitor in mice impairs social interest.

PubMed

Ujjainwala, A L; Courtney, C D; Rhoads, S G; Rhodes, J S; Christian, C A

2018-06-01

Neuropsychiatric disorders in which reduced social interest is a common symptom, such as autism, depression, and anxiety, are frequently associated with genetic mutations affecting γ-aminobutyric acid (GABA)ergic transmission. Benzodiazepine treatment, acting via GABA type-A receptors, improves social interaction in male mouse models with autism-like features. The protein diazepam binding inhibitor (DBI) can act as an endogenous benzodiazepine, but a role for DBI in social behavior has not been described. Here, we investigated the role of DBI in the social interest and recognition behavior of mice. The responses of DBI wild-type and knockout male and female mice to ovariectomized female wild-type mice (a neutral social stimulus) were evaluated in a habituation/dishabituation task. Both male and female knockout mice exhibited reduced social interest, and DBI knockout mice lacked the sex difference in social interest levels observed in wild-type mice, in which males showed higher social interest levels than females. The ability to discriminate between familiar and novel stimulus mice (social recognition) was not impaired in DBI-deficient mice of either sex. DBI knockouts could learn a rotarod motor task, and could discriminate between social and nonsocial odors. Both sexes of DBI knockout mice showed increased repetitive grooming behavior, but not in a manner that would account for the decrease in social investigation time. Genetic loss of DBI did not alter seminal vesicle weight, indicating that the social interest phenotype of males lacking DBI is not due to reduced circulating testosterone. Together, these studies show a novel role of DBI in driving social interest and motivation. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

PubMed

Marvel, Miranda; Spicer, Olivia Smith; Wong, Ten-Tsao; Zmora, Nilli; Zohar, Yonathan

2018-04-04

Gonadotropin-releasing hormone (GnRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a non-cell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

Roles of α- and β-estrogen receptors in mouse social recognition memory: effects of gender and the estrous cycle.

PubMed

Sánchez-Andrade, G; Kendrick, K M

2011-01-01

Establishing clear effects of gender and natural hormonal changes during female ovarian cycles on cognitive function has often proved difficult. Here we have investigated such effects on the formation and long-term (24 h) maintenance of social recognition memory in mice together with the respective involvement of α- and β-estrogen receptors using α- and β-estrogen receptor knockout mice and wildtype controls. Results in wildtype animals showed that while females successfully formed a memory in the context of a habituation/dishabituation paradigm at all stages of their ovarian cycle, only when learning occurred during proestrus (when estrogen levels are highest) was it retained after 24 h. In α-receptor knockout mice (which showed no ovarian cycles) both formation and maintenance of this social recognition memory were impaired, whereas β-receptor knockouts showed no significant deficits and exhibited the same proestrus-dependent retention of memory at 24 h. To investigate possible sex differences, male α- and β-estrogen receptor knockout mice were also tested and showed similar effects to females excepting that α-receptor knockouts had normal memory formation and only exhibited a 24 h retention deficit. This indicates a greater dependence in females on α-receptor expression for memory formation in this task. Since non-specific motivational and attentional aspects of the task were unaffected, our findings suggest a general α-receptor dependent facilitation of memory formation by estrogen as well as an enhanced long-term retention during proestrus. Results are discussed in terms of the differential roles of the two estrogen receptors, the neural substrates involved and putative interactions with oxytocin. Copyright © 2010 Elsevier Inc. All rights reserved.

Slc25a12 disruption alters myelination and neurofilaments: a model for a hypomyelination syndrome and childhood neurodevelopmental disorders.

PubMed

Sakurai, Takeshi; Ramoz, Nicolas; Barreto, Marta; Gazdoiu, Mihaela; Takahashi, Nagahide; Gertner, Michael; Dorr, Nathan; Gama Sosa, Miguel A; De Gasperi, Rita; Perez, Gissel; Schmeidler, James; Mitropoulou, Vivian; Le, H Carl; Lupu, Mihaela; Hof, Patrick R; Elder, Gregory A; Buxbaum, Joseph D

2010-05-01

SLC25A12, a susceptibility gene for autism spectrum disorders that is mutated in a neurodevelopmental syndrome, encodes a mitochondrial aspartate-glutamate carrier (aspartate-glutamate carrier isoform 1 [AGC1]). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and adenosine triphosphate production. We characterized mice with a disruption of the Slc25a12 gene, followed by confirmatory in vitro studies. Slc25a12-knockout mice, which showed no AGC1 by immunoblotting, were born normally but displayed delayed development and died around 3 weeks after birth. In postnatal day 13 to 14 knockout brains, the brains were smaller with no obvious alteration in gross structure. However, we found a reduction in myelin basic protein (MBP)-positive fibers, consistent with a previous report. Furthermore, the neocortex of knockout mice contained abnormal neurofilamentous accumulations in neurons, suggesting defective axonal transport and/or neurodegeneration. Slice cultures prepared from knockout mice also showed a myelination defect, and reduction of Slc25a12 in rat primary oligodendrocytes led to a cell-autonomous reduction in MBP expression. Myelin deficits in slice cultures from knockout mice could be reversed by administration of pyruvate, indicating that reduction in AGC1 activity leads to reduced production of aspartate/N-acetylaspartate and/or alterations in the dihydronicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide(+) ratio, resulting in myelin defects. Our data implicate AGC1 activity in myelination and in neuronal structure and indicate that while loss of AGC1 leads to hypomyelination and neuronal changes, subtle alterations in AGC1 expression could affect brain development, contributing to increased autism susceptibility. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

A modulatory role of the Rax homeobox gene in mature pineal gland function: Investigating the photoneuroendocrine circadian system of a Rax conditional knockout mouse.

PubMed

Rohde, Kristian; Bering, Tenna; Furukawa, Takahisa; Rath, Martin Fredensborg

2017-10-01

The retinal and anterior neural fold homeobox gene (Rax) controls development of the eye and the forebrain. Postnatal expression of Rax in the brain is restricted to the pineal gland, a forebrain structure devoted to melatonin synthesis. The role of Rax in pineal function is unknown. In order to investigate the role of Rax in pineal function while circumventing forebrain abnormalities of the global Rax knockout, we generated an eye and pineal-specific Rax conditional knockout mouse. Deletion of Rax in the pineal gland did not affect morphology of the gland, suggesting that Rax is not essential for pineal gland development. In contrast, deletion of Rax in the eye generated an anophthalmic phenotype. In addition to the loss of central visual pathways, the suprachiasmatic nucleus of the hypothalamus housing the circadian clock was absent, indicating that the retinohypothalamic tract is required for the nucleus to develop. Telemetric analyses confirmed the lack of a functional circadian clock. Arylalkylamine N-acetyltransferase (Aanat) transcripts, encoding the melatonin rhythm-generating enzyme, were undetectable in the pineal gland of the Rax conditional knockout under normal conditions, whereas the paired box 6 homeobox gene, known to regulate pineal development, was up-regulated. By injecting isoproterenol, which mimics a nocturnal situation in the pineal gland, we were able to induce pineal expression of Aanat in the Rax conditional knockout mouse, but Aanat transcript levels were significantly lower than those of Rax-proficient mice. Our data suggest that Rax controls pineal gene expression and via Aanat may modulate melatonin synthesis. © 2017 International Society for Neurochemistry.

Ascl1 (Mash1) Knockout Perturbs Differentiation of Nonneuronal Cells in Olfactory Epithelium

PubMed Central

Jang, Woochan; Wildner, Hendrik; Schwob, James E.

2012-01-01

The embryonic olfactory epithelium (OE) generates only a very few olfactory sensory neurons when the basic helix-loop-helix transcription factor, ASCL1 (previously known as MASH1) is eliminated by gene mutation. We have closely examined the structure and composition of the OE of knockout mice and found that the absence of neurons dramatically affects the differentiation of multiple other epithelial cell types as well. The most prominent effect is observed within the two known populations of stem and progenitor cells of the epithelium. The emergence of horizontal basal cells, a multipotent progenitor population in the adult epithelium, is anomalous in the Ascl1 knockout mice. The differentiation of globose basal cells, another multipotent progenitor population in the adult OE, is also aberrant. All of the persisting globose basal cells are marked by SOX2 expression, suggesting a prominent role for SOX2 in progenitors upstream of Ascl1. However, NOTCH1-expressing basal cells are absent from the knockout; since NOTCH1 signaling normally acts to suppress Ascl1 via HES1 and drives sustentacular (Sus) cell differentiation during adult epithelial regeneration, its absence suggests reciprocity between neurogenesis and the differentiation of Sus cells. Indeed, the Sus cells of the mutant mice express a markedly lower level of HES1, strengthening that notion of reciprocity. Duct/gland development appears normal. Finally, the expression of cKIT by basal cells is also undetectable, except in those small patches where neurogenesis escapes the effects of Ascl1 knockout and neurons are born. Thus, persistent neurogenic failure distorts the differentiation of multiple other cell types in the olfactory epithelium. PMID:23284756

PKCδ knockout mice are protected from para-methoxymethamphetamine-induced mitochondrial stress and associated neurotoxicity in the striatum of mice.

PubMed

Shin, Eun-Joo; Dang, Duy-Khanh; Tran, Hai-Quyen; Nam, Yunsung; Jeong, Ji Hoon; Lee, Young Hun; Park, Kyung Tae; Lee, Yong Sup; Jang, Choon-Gon; Hong, Jau-Shyong; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2016-11-01

Para-methoxymethamphetamine (PMMA) is a para-ring-substituted amphetamine derivative sold worldwide as an illegal psychotropic drug. Although PMMA use has been reported to lead to severe intoxication and even death, little is known about the mechanism(s) by which PMMA exerts its neurotoxic effects. Here we found that PMMA treatment resulted in phosphorylation of protein kinase Cδ (PKCδ) and subsequent mitochondrial translocation of cleaved PKCδ. PMMA-induced oxidative stress was more pronounced in mitochondria than in the cytosol. Moreover, treatment with PMMA consistently resulted in significant reductions in mitochondrial membrane potential, mitochondrial complex I activity, and mitochondrial Mn superoxide dismutase-immunoreactivity. In contrast, PMMA treatment led to a significant increase in intramitochondrial Ca 2+ level. Treatment with PMMA also significantly increased ionized calcium binding adaptor molecule 1 (Iba-1)-labeled microglial activation and upregulated tumor necrosis factor alpha (TNF-α) gene expression. PKCδ knockout attenuated these mitochondrial effects and dampened the neurotoxic effects of PMMA. Importantly, TNF-α knockout mice were significantly protected from PMMA-induced increases in phospho-PKCδ expression, mitochondrial translocation of cleaved PKCδ, and Iba-1-labeled microgliosis. Both rottlerin, a pharmacological inhibitor of PKCδ, and etanercept, a pharmacological inhibitor of TNF-α, significantly protected against PMMA-mediated induction of apoptosis, as assessed by terminal deoxynucleotidyl transferase dUDP nick end labeling (TUNEL) assays. In addition, PKCδ knockout and TNF-α knockout both resulted in decreased PMMA-mediated induction of dopaminergic loss. Therefore, our results suggest that PKCδ mediates PMMA-induced neurotoxicity by facilitating oxidative stress (mitochondria > cytosol), mitochondrial dysfunction, microglial activation, and pro-apoptotic signaling. Our results also indicate that PMMA-induced PKCδ activation requires the proinflammatory cytokine TNF-α. Copyright © 2016. Published by Elsevier Ltd.

Role of light and the circadian clock in the rhythmic oscillation of intraocular pressure: Studies in VPAC2 receptor and PACAP deficient mice.

PubMed

Fahrenkrug, Jan; Georg, Birgitte; Hannibal, Jens; Jørgensen, Henrik Løvendahl

2018-04-01

The intraocular pressure of mice displays a daily rhythmicity being highest during the dark period. The present study was performed to elucidate the role of the circadian clock and light in the diurnal and the circadian variations in intraocular pressure in mice, by using animals with disrupted clock function (VPAC2 receptor knockout mice) or impaired light information to the clock (PACAP knockout mice). In wildtype mice, intraocular pressure measured under light/dark conditions showed a statistically significant 24 h sinusoidal rhythm with nadir during the light phase and peak during the dark phase. After transfer of the wildtype mice into constant darkness, the intraocular pressure increased, but the rhythmic changes in intraocular pressure continued with a pattern identical to that obtained during the light/dark cycle. The intraocular pressure in VPAC2 receptor deficient mice during light/dark conditions also showed a sinusoidal pattern with significant changes as a function of a 24 h cycle. However, transfer of the VPAC2 receptor knockout mice into constant darkness completely abolished the rhythmic changes in intraocular pressure. The intraocular pressure in PACAP deficient mice oscillated significantly during both 24 h light and darkness and during constant darkness. During LD conditions, the amplitude of PACAP deficient was significantly lower compared to wildtype mice, resulting in higher daytime and lower nighttime values. In conclusion, by studying the VPAC2 receptor knockout mouse which lacks circadian control and the PACAP knockout mouse which displays impaired light signaling, we provided evidence that the daily intraocular pressure rhythms are primarily generated by the circadian master clock and to a lesser extent by environmental light and darkness. Copyright © 2018 Elsevier Ltd. All rights reserved.

Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

PubMed

Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K; Morales, Cyndi R; Contreras-Ferrat, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J; Somlo, Stefan; Rothermel, Beverly A; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

2015-06-16

L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. © 2015 American Heart Association, Inc.

The Xanthomonas oryzae pv. oryzae PhoPQ Two-Component System Is Required for AvrXA21 Activity, hrpG Expression, and Virulence▿ †

PubMed Central

Lee, Sang-Won; Jeong, Kyu-Sik; Han, Sang-Wook; Lee, Seung-Eun; Phee, Bong-Kwan; Hahn, Tae-Ryong; Ronald, Pamela

2008-01-01

The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the type one system-secreted molecule, AvrXA21. X. oryzae pv. oryzae requires a regulatory two-component system (TCS) called RaxRH to regulate expression of eight rax (required for AvrXA21 activity) genes and to sense population cell density. To identify other key components in this critical regulatory circuit, we assayed proteins expressed in a raxR gene knockout strain. This survey led to the identification of the phoP gene encoding a response regulator that is up-regulated in the raxR knockout strain. Next we generated a phoP knockout strain and found it to be impaired in X. oryzae pv. oryzae virulence and no longer able to activate the response regulator HrpG (hypersensitive reaction and pathogenicity G) in response to low levels of Ca2+. The impaired virulence of the phoP knockout strain can be partially complemented by constitutive expression of hrpG, indicating that PhoP controls a key aspect of X. oryzae pv. oryzae virulence through regulation of hrpG. A gene encoding the cognate putative histidine protein kinase, phoQ, was also isolated. Growth curve analysis revealed that AvrXA21 activity is impaired in a phoQ knockout strain as reflected by enhanced growth of this strain in rice lines carrying XA21. These results suggest that the X. oryzae pv. oryzae PhoPQ TCS functions in virulence and in the production of AvrXA21 in partnership with RaxRH. PMID:18203830

Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

PubMed Central

Stadler, Krisztian; Bonini, Marcelo G.; Dallas, Shannon; Duma, Danielle; Mason, Ronald P.; Kadiiska, Maria B.

2008-01-01

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the α-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression. PMID:18559982

CARD9 knockout ameliorates myocardial dysfunction associated with high fat diet-induced obesity.

PubMed

Cao, Li; Qin, Xing; Peterson, Matthew R; Haller, Samantha E; Wilson, Kayla A; Hu, Nan; Lin, Xin; Nair, Sreejayan; Ren, Jun; He, Guanglong

2016-03-01

Obesity is associated with chronic inflammation which plays a critical role in the development of cardiovascular dysfunction. Because the adaptor protein caspase recruitment domain-containing protein 9 (CARD9) in macrophages regulates innate immune responses via activation of pro-inflammatory cytokines, we hypothesize that CARD9 mediates the pro-inflammatory signaling associated with obesity en route to myocardial dysfunction. C57BL/6 wild-type (WT) and CARD9(-/-) mice were fed normal diet (ND, 12% fat) or a high fat diet (HFD, 45% fat) for 5months. At the end of 5-month HFD feeding, cardiac function was evaluated using echocardiography. Cardiomyocytes were isolated and contractile properties were measured. Immunofluorescence was performed to detect macrophage infiltration in the heart. Heart tissue homogenates, plasma, and supernatants from isolated macrophages were collected to measure the concentrations of pro-inflammatory cytokines using ELISA kits. Western immunoblotting analyses were performed on heart tissue homogenates and isolated macrophages to explore the underlying signaling mechanism(s). CARD9 knockout alleviated HFD-induced insulin resistance and glucose intolerance, prevented myocardial dysfunction with preserved cardiac fractional shortening and cardiomyocyte contractile properties. CARD9 knockout also significantly decreased the number of infiltrated macrophages in the heart with reduced myocardium-, plasma-, and macrophage-derived cytokines including IL-6, IL-1β and TNFα. Finally, CARD9 knockout abrogated the increase of p38 MAPK phosphorylation, the decrease of LC3BII/LC3BI ratio and the up-regulation of p62 expression in the heart induced by HFD feeding and restored cardiac autophagy signaling. In conclusion, CARD9 knockout ameliorates myocardial dysfunction associated with HFD-induced obesity, potentially through reduction of macrophage infiltration, suppression of p38 MAPK phosphorylation, and preservation of autophagy in the heart. Copyright © 2016 Elsevier Ltd. All rights reserved.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

PubMed

Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

2011-04-01

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization

PubMed Central

Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

2011-01-01

Abstract The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges. PMID:21300746

Neutral endopeptidase (EC 3.4.24.11) terminates colitis by degrading substance P.

PubMed

Sturiale, S; Barbara, G; Qiu, B; Figini, M; Geppetti, P; Gerard, N; Gerard, C; Grady, E F; Bunnett, N W; Collins, S M

1999-09-28

Neurogenic inflammation is regulated by sensory nerves and characterized by extravasation of plasma proteins and infiltration of neutrophils from post-capillary venules and arteriolar vasodilatation. Although it is well established that substance P (SP) interacts with the neurokinin 1 receptor (NK1R) to initiate neurogenic inflammation, the mechanisms that terminate inflammation are unknown. We examined whether neutral endopeptidase (NEP), a cell-surface enzyme that degrades SP in the extracellular fluid, terminates neurogenic inflammation in the colon. In NEP knockout mice, the SP concentration in the colon was approximately 2.5-fold higher than in wild-type mice, suggesting increased bioavailability of SP. The extravasation of Evans blue-labeled plasma proteins in the colon of knockout mice under basal conditions was approximately 4-fold higher than in wild-type mice. This elevated plasma leak was attenuated by recombinant NEP or the NK1R antagonist SR140333, and is thus caused by diminished degradation of SP. To determine whether deletion of NEP predisposes mice to uncontrolled inflammation, we compared dinitrobenzene sulfonic acid-induced colitis in wild-type and knockout mice. The severity of colitis, determined by macroscopic and histologic scoring and by myeloperoxidase activity, was markedly worse in knockout than wild-type mice after 3 and 7 days. The exacerbated inflammation in knockout mice was prevented by recombinant NEP and SR140333. Thus, NEP maintains low levels of SP in the extracellular fluid under basal conditions and terminates its proinflammatory effects. Because we have previously shown that intestinal inflammation results in down-regulation of NEP and diminished degradation of SP, our present results suggest that defects in NEP expression contribute to uncontrolled inflammation.