Neurospora crassa glucose - repressible gene -1(Grg-1) promoter controls the expression of neurospora tyrosinase gene in a clock-controlled manner

International Nuclear Information System (INIS)

Tarawneh, A. K

1997-01-01

In this study sphareroplastes of white Neurospora crassa mutant auxotroph for aromatic am no acids a rom 9 q a-2 inv, was transformed by the pKF-Tyr7-wt DNA construct. This construct contains the promoter of neurospora crassa glucose-repressible gene-1 (G rg-1) usp stream of Neurospora tyrosinase gene. The co transformation of this mutant with pKF-Tyr-7-wt cincture's and the pKAL-1, a plasmid which contains the Neurospora q a-2+ gene transform it to photophor. The transform ant contains the tyrosinase gene which catalyzes the unique step in the synthesis of the black pigment melanin. The activity of the tyrosinase in this transform ant was followed by measuring the absorbance of the dark coloured pigment at 332 nm. The maximum of the tyrosinase activity was shown at 16.36 and 56 hours after the shift of the transformed mycelia from constant light (L L) to constant dark (Dd). The rate of the enzyme activity was changed according to ci radian cycle of 20 hours. This G rg 1/tyrosinase construct provides a good system to study to study the temporal control of gene expression and the interaction between the different environmental c uses that affects gene expression. (author). 20 refs., 4 figs

[Organization of mitochondria in the growing hyphae of Neurospora crassa].

Science.gov (United States)

Potapova, T V; Boĭtsova, L Iu; Golyshev, S A; Popinako, A V

2013-01-01

In vivo fluorescent labeling of mitochondria in Neurospora crassa showed the concentration of filamentous mitochondria within 30 μm of apex in growing hyphae. These mitochondrial assemblies propagated forward with the elongation of hyphae, split and segregated as the growing tip bifurcated and formed de novo when new branches formed farther away from the apex. The efficiency of the mitochondria concentration in the apical 30 μm zone is related to the growth rate and identical in hyphae cultivated in glucose- and sorbitol-containing media. The obtained data are discussed in connection with the behavior of microtubules in growing hyphae as well as with the electric heterogeneity of N. crassa hyphal apex described previously.

Neurospora crassa ncs-1, mid-1 and nca-2 double-mutant ...

Indian Academy of Sciences (India)

logue of Neuronal Calcium Sensor-1 has a role in growth, cal- cium stress tolerance, and ultraviolet survival. Genetica 139,. 885–894. Lew R. R., Abbas Z., Anderca M. I. and Free S. J. 2008 Phe- notype of a mechanosensitive channel mutant, mid-1, in a fil- amentous fungus, Neurospora crassa. Eukaryot. Cell. 7, 647–. 655.

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. Pt. 2

International Nuclear Information System (INIS)

Serres, F.J. de

1980-01-01

UV-induced inactivation and induction of mutations at the ad-3A and ad-3B loci of Neurospora crassa have been compared among 7 different UV-sensitive strains and a standard wild-type strain. The 7 strains show varying degrees of sensitivity to UV-induced inactivation, with the relative sensitivity being: uvs-2 > uvs-3 > uvs-4 > uvs-6 > upr-1 > uvs-5 > uvs-1. Studies on the induction of ad-3 mutants by UV show that the 2 excision-repair deficient mutants uvs-2 and upr-1 exhibit enhanced ad-3 mutant frequencies, while uvs-4 and uvs-5 exhibit reduced ad-3 mutant frequencies, and uvs-3 completely eliminates UV mutagenesis. The ad-3 mutation-induction curves obtained with uvs-1 or uvs-6 are not significantly different from that found with the wild-type strain. (orig.)

Conidiation of Neurospora crassa induced by treatment with natrium fluoride in submerged culture

Energy Technology Data Exchange (ETDEWEB)

Timberlake, W E; Turian, G

1975-01-01

A transient treatment of pregerminated conidia of Neurospora crassa with NaF induced young, submerged cultures to prematurely differentiate conidia. The inductive treatment decreased the rate of respiration (with lower RQ), reduced the relative concentration of nucleoside triphosphates, and inhibited leucine incorporation into protein and adenosine incorporation into RNA.

Functional Profiling of Transcription Factor Genes in Neurospora crassa

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Alexander J. Carrillo

2017-09-01

Full Text Available Regulation of gene expression by DNA-binding transcription factors is essential for proper control of growth and development in all organisms. In this study, we annotate and characterize growth and developmental phenotypes for transcription factor genes in the model filamentous fungus Neurospora crassa. We identified 312 transcription factor genes, corresponding to 3.2% of the protein coding genes in the genome. The largest class was the fungal-specific Zn2Cys6 (C6 binuclear cluster, with 135 members, followed by the highly conserved C2H2 zinc finger group, with 61 genes. Viable knockout mutants were produced for 273 genes, and complete growth and developmental phenotypic data are available for 242 strains, with 64% possessing at least one defect. The most prominent defect observed was in growth of basal hyphae (43% of mutants analyzed, followed by asexual sporulation (38%, and the various stages of sexual development (19%. Two growth or developmental defects were observed for 21% of the mutants, while 8% were defective in all three major phenotypes tested. Analysis of available mRNA expression data for a time course of sexual development revealed mutants with sexual phenotypes that correlate with transcription factor transcript abundance in wild type. Inspection of this data also implicated cryptic roles in sexual development for several cotranscribed transcription factor genes that do not produce a phenotype when mutated.

The Oxygenase CAO-1 of Neurospora crassa Is a Resveratrol Cleavage Enzyme

KAUST Repository

Diaz-Sanchez, V.; F. Estrada, A.; Limon, M. C.; Al-Babili, Salim; Avalos, J.

2013-01-01

The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving β-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cβ double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

The Oxygenase CAO-1 of Neurospora crassa Is a Resveratrol Cleavage Enzyme

KAUST Repository

Diaz-Sanchez, V.

2013-07-26

The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving β-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cβ double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

Epigenetic and Posttranslational Modifications in Light Signal Transduction and the Circadian Clock in Neurospora crassa

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Marco Proietto

2015-07-01

Full Text Available Blue light, a key abiotic signal, regulates a wide variety of physiological processes in many organisms. One of these phenomena is the circadian rhythm presents in organisms sensitive to the phase-setting effects of blue light and under control of the daily alternation of light and dark. Circadian clocks consist of autoregulatory alternating negative and positive feedback loops intimately connected with the cellular metabolism and biochemical processes. Neurospora crassa provides an excellent model for studying the molecular mechanisms involved in these phenomena. The White Collar Complex (WCC, a blue-light receptor and transcription factor of the circadian oscillator, and Frequency (FRQ, the circadian clock pacemaker, are at the core of the Neurospora circadian system. The eukaryotic circadian clock relies on transcriptional/translational feedback loops: some proteins rhythmically repress their own synthesis by inhibiting the activity of their transcriptional factors, generating self-sustained oscillations over a period of about 24 h. One of the basic mechanisms that perpetuate self-sustained oscillations is post translation modification (PTM. The acronym PTM generically indicates the addition of acetyl, methyl, sumoyl, or phosphoric groups to various types of proteins. The protein can be regulatory or enzymatic or a component of the chromatin. PTMs influence protein stability, interaction, localization, activity, and chromatin packaging. Chromatin modification and PTMs have been implicated in regulating circadian clock function in Neurospora. Research into the epigenetic control of transcription factors such as WCC has yielded new insights into the temporal modulation of light-dependent gene transcription. Here we report on epigenetic and protein PTMs in the regulation of the Neurospora crassa circadian clock. We also present a model that illustrates the molecular mechanisms at the basis of the blue light control of the circadian clock.

Biotechnological production of ethanol from renewable resources by Neurospora crassa: an alternative to conventional yeast fermentations?

Science.gov (United States)

Dogaris, Ioannis; Mamma, Diomi; Kekos, Dimitris

2013-02-01

Microbial production of ethanol might be a potential route to replace oil and chemical feedstocks. Bioethanol is by far the most common biofuel in use worldwide. Lignocellulosic biomass is the most promising renewable resource for fuel bioethanol production. Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/distillation. Conventional bioethanol processes for lignocellulosics apply commercial fungal cellulase enzymes for biomass hydrolysis, followed by yeast fermentation of resulting glucose to ethanol. The fungus Neurospora crassa has been used extensively for genetic, biochemical, and molecular studies as a model organism. However, the strain's potential in biotechnological applications has not been widely investigated and discussed. The fungus N. crassa has the ability to synthesize and secrete all three enzyme types involved in cellulose hydrolysis as well as various enzymes for hemicellulose degradation. In addition, N. crassa has been reported to convert to ethanol hexose and pentose sugars, cellulose polymers, and agro-industrial residues. The combination of these characteristics makes N. crassa a promising alternative candidate for biotechnological production of ethanol from renewable resources. This review consists of an overview of the ethanol process from lignocellulosic biomass, followed by cellulases and hemicellulases production, ethanol fermentations of sugars and lignocellulosics, and industrial application potential of N. crassa.

Neurospora crassa ASM-1 complements the conidiation defect in a stuA mutant of Aspergillus nidulans.

Science.gov (United States)

Chung, Dawoon; Upadhyay, Srijana; Bomer, Brigitte; Wilkinson, Heather H; Ebbole, Daniel J; Shaw, Brian D

2015-01-01

Aspergillus nidulans StuA and Neurospora crassa ASM-1 are orthologous APSES (ASM-1, PHD1, SOK2, Efg1, StuA) transcription factors conserved across a diverse group of fungi. StuA and ASM-1 have roles in asexual (conidiation) and sexual (ascospore formation) development in both organisms. To address the hypothesis that the last common ancestor of these diverse fungi regulated conidiation with similar genes, asm-1 was introduced into the stuA1 mutant of A. nidulans. Expression of asm-1 complemented defective conidiophore morphology and restored conidia production to wild type levels in stuA1. Expression of asm-1 in the stuA1 strain did not rescue the defect in sexual development. When the conidiation regulator AbaA was tagged at its C-terminus with GFP in A. nidulans, it localized to nuclei in phialides. When expressed in the stuA1 mutant, AbaA::GFP localized to nuclei in conidiophores but no longer was confined to phialides, suggesting that expression of AbaA in specific cell types of the conidiophore was conditioned by StuA. Our data suggest that the function in conidiation of StuA and ASM-1 is conserved and support the view that, despite the great morphological and ontogenic diversity of their condiphores, the last common ancestor of A. nidulans and N. crassa produced an ortholog of StuA that was involved in conidiophore development. © 2015 by The Mycological Society of America.

Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa.

Science.gov (United States)

Ao, Jie; Free, Stephen J

2017-04-01

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of the temperature-sensitive mutations un-7 and png-1 in Neurospora crassa.

Science.gov (United States)

Dieterle, Michael G; Wiest, Aric E; Plamann, Mike; McCluskey, Kevin

2010-05-18

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora.

Cell Wall Composition of Neurospora crassa Under Conditions of Copper Toxicity

OpenAIRE

Subramanyam, C.; Venkateswerlu, G.; Rao, S. L. N.

1983-01-01

The mycelia of Neurospora crassa grown in the presence of high concentrations of copper were blue in color, but only on a medium containing inorganic nitrate and phosphate as the nitrogen and phosphate sources, respectively. The cell wall isolate of the blue mycelia contained large amounts (12%) of copper and higher amounts of chitosan, phosphate, and amino groups, with a 42% decrease in the chitin content. Although all the glucosamine of the cell wall of control cultures could be released wi...

A Eukaryote without Catalase-Containing Microbodies : Neurospora crassa Exhibits a Unique Cellular Distribution of Its Four Catalases

NARCIS (Netherlands)

Schliebs, Wolfgang; Würtz, Christian; Kunau, Wolf-Hubert; Veenhuis, Marten; Rottensteiner, Hanspeter; Wuertz, Christian

2006-01-01

Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by the action of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing microbodies) exist in Neurospora crassa. Three distinct catalase isoforms were identified by native

Growth responses of Neurospora crassa to increased partial pressures of the noble gases and nitrogen.

Science.gov (United States)

Buchheit, R G; Schreiner, H R; Doebbler, G F

1966-02-01

Buchheit, R. G. (Union Carbide Corp., Tonawanda, N.Y.), H. R. Schreiner, and G. F. Doebbler. Growth responses of Neurospora crassa to increased partial pressures of the noble gases and nitrogen. J. Bacteriol. 91:622-627. 1966.-Growth rate of the fungus Neurospora crassa depends in part on the nature of metabolically "inert gas" present in its environment. At high partial pressures, the noble gas elements (helium, neon, argon, krypton, and xenon) inhibit growth in the order: Xe > Kr> Ar > Ne > He. Nitrogen (N(2)) closely resembles He in inhibitory effectiveness. Partial pressures required for 50% inhibition of growth were: Xe (0.8 atm), Kr (1.6 atm), Ar (3.8 atm), Ne (35 atm), and He ( approximately 300 atm). With respect to inhibition of growth, the noble gases and N(2) differ qualitatively and quantitatively from the order of effectiveness found with other biological effects, i.e., narcosis, inhibition of insect development, depression of O(2)-dependent radiation sensitivity, and effects on tissue-slice glycolysis and respiration. Partial pressures giving 50% inhibition of N. crassa growth parallel various physical properties (i.e., solubilities, solubility ratios, etc.) of the noble gases. Linear correlation of 50% inhibition pressures to the polarizability and of the logarithm of pressure to the first and second ionization potentials suggests the involvement of weak intermolecular interactions or charge-transfer in the biological activity of the noble gases.

Production of cellobionate from cellulose using an engineered Neurospora crassa strain with laccase and redox mediator addition

Science.gov (United States)

We report a novel production process for cellobionic acid from cellulose using an engineered fungal strain with the exogenous addition of laccase and a redox mediator. A previously engineered strain of Neurospora crassa (F5'ace-1'cre-1'ndvB) was shown to produce cellobionate directly from cellulose ...

A circadian rhythm of conidiation in Neurospora crassa (L-12)

Science.gov (United States)

Miyoshi, Yashuhiro

1993-01-01

Two fungi growth chambers containing six growth tubes each are used in this experiment. One chamber is for the space experiment; the other is for the simultaneous ground control experiment. The hyphae of Neurospora crassa band A mutant are inoculated at one end of each tube. Both the chambers are kept at 3 C plus or minus 1.5 C to stop hyphae growth until the Spacelab is activated. After the activation, each chamber is transferred simultaneously to the Spacelab and a phytotron in KSC and kept in continuous light at the same temperature. After about 24 hours of light exposure, each chamber is inserted into a growth chamber bag to keep it in constant darkness. The circadian rhythm of conidiation is initiated by this light to dark transition. After the dark incubation for 5 days at room temperature, both the growth chambers are kept at 3 C plus or minus 1.5 C to stop growth of the hyphae. After the space shuttle lands, both conidiation patterns are compared and analyzed. It has been known that numerous physiological phenomena show circadian rhythms. They are characterized by the fact that the oscillation can persist under constant conditions of light and temperature. Therefore, it has been accepted by most investigators that the generation mechanism of the circadian rhythm is endogeneous. However, one cannot reject the possibility that these rhythms are caused by some geophysical exogeneous factor having a 24-hour period, such as atmospheric pressure, gravity, or electromagnetic radiation. We use Neurospora crassa band A mutual which shows an obvious circadian rhythm in its spore-forming (conidiation) on the ground, and we intend to attempt the conidation of this mutant in the Spacelab where 24-hour periodicity is severely attenuated and to elucidate the effect of the geophysical exogeneous factor in the generation mechanism of the circadian rhythm.

Identification of TTAGGG-binding proteins in Neurospora crassa, a fungus with vertebrate-like telomere repeats.

Science.gov (United States)

Casas-Vila, Núria; Scheibe, Marion; Freiwald, Anja; Kappei, Dennis; Butter, Falk

2015-11-17

To date, telomere research in fungi has mainly focused on Saccharomyces cerevisiae and Schizosaccharomyces pombe, despite the fact that both yeasts have degenerated telomeric repeats in contrast to the canonical TTAGGG motif found in vertebrates and also several other fungi. Using label-free quantitative proteomics, we here investigate the telosome of Neurospora crassa, a fungus with canonical telomeric repeats. We show that at least six of the candidates detected in our screen are direct TTAGGG-repeat binding proteins. While three of the direct interactors (NCU03416 [ncTbf1], NCU01991 [ncTbf2] and NCU02182 [ncTay1]) feature the known myb/homeobox DNA interaction domain also found in the vertebrate telomeric factors, we additionally show that a zinc-finger protein (NCU07846) and two proteins without any annotated DNA-binding domain (NCU02644 and NCU05718) are also direct double-strand TTAGGG binders. We further find two single-strand binders (NCU02404 [ncGbp2] and NCU07735 [ncTcg1]). By quantitative label-free interactomics we identify TTAGGG-binding proteins in Neurospora crassa, suggesting candidates for telomeric factors that are supported by phylogenomic comparison with yeast species. Intriguingly, homologs in yeast species with degenerated telomeric repeats are also TTAGGG-binding proteins, e.g. in S. cerevisiae Tbf1 recognizes the TTAGGG motif found in its subtelomeres. However, there is also a subset of proteins that is not conserved. While a rudimentary core TTAGGG-recognition machinery may be conserved across yeast species, our data suggests Neurospora as an emerging model organism with unique features.

Bioconversion of dilute-acid pretreated sorghum bagasse to ethanol by Neurospora crassa

Energy Technology Data Exchange (ETDEWEB)

Dogaris, Ioannis; Gkounta, Olga; Mamma, Diomi; Kekos, Dimitris [National Technical Univ. of Athens, Zografou (Greece). Biotechnology Lab.

2012-07-15

Bioethanol production from sweet sorghum bagasse (SB), the lignocellulosic solid residue obtained after extraction of sugars from sorghum stalks, can further improve the energy yield of the crop. The aim of the present work was to evaluate a cost-efficient bioconversion of SB to ethanol at high solids loadings (16 % at pretreatment and 8 % at fermentation), low cellulase activities (1-7 FPU/g SB) and co-fermentation of hexoses and pentoses. The fungus Neurospora crassa DSM 1129 was used, which exhibits both depolymerase and co-fermentative ability, as well as mixed cultures with Saccharomyces cerevisiae 2541. A dilute-acid pretreatment (sulfuric acid 2 g/100 g SB; 210 C; 10 min) was implemented, with high hemicellulose decomposition and low inhibitor formation. The bioconversion efficiency of N. crassa was superior to S. cerevisiae, while their mixed cultures had negative effect on ethanol production. Supplementing the in situ produced N. crassa cellulolytic system (1.0 FPU/g SB) with commercial cellulase and {beta}-glucosidase mixture at low activity (6.0 FPU/g SB) increased ethanol production to 27.6 g/l or 84.7 % of theoretical yield (based on SB cellulose and hemicellulose sugar content). The combined dilute-acid pretreatment and bioconversion led to maximum cellulose and hemicellulose hydrolysis 73.3 % and 89.6 %, respectively. (orig.)

Performa Ayam dan Kualitas Telur yang Menggunakan Ransum Mengandung Onggok Fermentasi dengan Neurospora crassa

Directory of Open Access Journals (Sweden)

Nuraini

2008-12-01

Full Text Available An experiment was conducted to determine the effect of feeding tapioca by-products fermented by Neurospora crassa on layer performances and egg quality. Two hundred layers were randomly allocated into 20 pens. This experiment was arranged in a completely randomized design with four dietary treatments: 0%, 10%, 20% and 30% tapioca by product fermented by N. crassa in the diets and five replications. Variable measured were feed comsumption, egg production, feed conversion, egg cholesterol and egg yolk colour. Results of the experiment indicated that feed comsumption, egg production, feed conversion, egg cholesterol and egg yolk colour were affected (P<0.05 by using fermented tapioca by-product in the diets of layers. Results of Duncan multiple range test indicated that feed consumption, egg production, and egg yolk colour in D treatment (used 30% tapioca by-products fermented were the highest compared to those other treatments, but the lowest on feed conversion and egg cholesterol. The conclusion of the experiment that tapioca by-products fermented by N. crassa can be used up to 30% in the diet of layer.

Elimination of active tad elements during the sexual phase of the Neurospora crassa life cycle.

Science.gov (United States)

Anderson, C; Tang, Q; Kinsey, J A

2001-06-01

Tad is an active LINE-like retrotransposon isolated from the Adiopodoumé strain of Neurospora crassa. Extensive analysis of other Neurospora strains has revealed no other strain with active Tad, but all strains tested have multiple copies of defective Tad elements. We have examined the ability of Tad to survive during the sexual cycle of Neurospora and find that active Tad is rapidly eliminated. The characteristics of this elimination suggest that the repeat-induced point mutation (RIP) mechanism was responsible. By the use of transformation to switch the mating type of the Adiopodoumé strain we concluded that this strain is not defective in the RIP process. Analysis of defective Tad elements isolated from a variety of strains indicates that the major difference between these elements and active Tad is due to the presence of a large number of G-C to A-T transition mutations. This would be expected if the changes were due primarily to the RIP process. Mapping of a selection of defective Tad elements reveals that they are present on all of the chromosomes; however, many of the elements are not widely shared among strains. This suggests that repeated introduction and elimination of Tad elements has occurred. Mechanisms that might be responsible for this repeated introduction are discussed. Copyright 2001 Academic Press.

Transcriptomic profiling-based mutant screen reveals three new transcription factors mediating menadione resistance in Neurospora crassa.

Science.gov (United States)

Zhu, Jufen; Yu, Xinxu; Xie, Baogui; Gu, Xiaokui; Zhang, Zhenying; Li, Shaojie

2013-06-01

To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner

Bioconversion of dilute-acid pretreated sorghum bagasse to ethanol by Neurospora crassa.

Science.gov (United States)

Dogaris, Ioannis; Gkounta, Olga; Mamma, Diomi; Kekos, Dimitris

2012-07-01

Bioethanol production from sweet sorghum bagasse (SB), the lignocellulosic solid residue obtained after extraction of sugars from sorghum stalks, can further improve the energy yield of the crop. The aim of the present work was to evaluate a cost-efficient bioconversion of SB to ethanol at high solids loadings (16 % at pretreatment and 8 % at fermentation), low cellulase activities (1-7 FPU/g SB) and co-fermentation of hexoses and pentoses. The fungus Neurospora crassa DSM 1129 was used, which exhibits both depolymerase and co-fermentative ability, as well as mixed cultures with Saccharomyces cerevisiae 2541. A dilute-acid pretreatment (sulfuric acid 2 g/100 g SB; 210 °C; 10 min) was implemented, with high hemicellulose decomposition and low inhibitor formation. The bioconversion efficiency of N. crassa was superior to S. cerevisiae, while their mixed cultures had negative effect on ethanol production. Supplementing the in situ produced N. crassa cellulolytic system (1.0 FPU/g SB) with commercial cellulase and β-glucosidase mixture at low activity (6.0 FPU/g SB) increased ethanol production to 27.6 g/l or 84.7 % of theoretical yield (based on SB cellulose and hemicellulose sugar content). The combined dilute-acid pretreatment and bioconversion led to maximum cellulose and hemicellulose hydrolysis 73.3 % and 89.6 %, respectively.

Comparative sequence analysis of Sordaria macrospora and Neurospora crassa as a means to improve genome annotation.

Science.gov (United States)

Nowrousian, Minou; Würtz, Christian; Pöggeler, Stefanie; Kück, Ulrich

2004-03-01

One of the most challenging parts of large scale sequencing projects is the identification of functional elements encoded in a genome. Recently, studies of genomes of up to six different Saccharomyces species have demonstrated that a comparative analysis of genome sequences from closely related species is a powerful approach to identify open reading frames and other functional regions within genomes [Science 301 (2003) 71, Nature 423 (2003) 241]. Here, we present a comparison of selected sequences from Sordaria macrospora to their corresponding Neurospora crassa orthologous regions. Our analysis indicates that due to the high degree of sequence similarity and conservation of overall genomic organization, S. macrospora sequence information can be used to simplify the annotation of the N. crassa genome.

Inactivation of normal and mutant Neurospora crassa conidia by visible light and near-UV: role of 1O2, carotenoid composition and sensitizer location

International Nuclear Information System (INIS)

Thomas, S.A.; Sargent, M.L.; Tuveson, R.W.

1981-01-01

Inactivation of Neurospora crassa conidia from wild-type and mutant strains by visible and near-ultraviolet light was investigated in the presence and absence of photosensitizing dyes. Inactivation by near-UV was virtually unchanged by the presence of deuterium oxide or azide suggesting that, contrary to the situation with visible light and photosensitizing dyes, 1 O 2 is not involved in any substantial way in the formation of lethal lesions. Carotenoid deficient strains were similar to wild-type strains in sensitivity to near-UV inactivation which is consistent with 1 O 2 not being involved. Photodynamic inactivation of conidia by visible light occurred in the presence of methylene blue (MB), toluidine blue O (TB), or acridine orange (AO). Carotenoid deficient strains were more sensitive to such inactivation only when MB and TB were used. This suggests that MB and TB mediated damage involves the cell membrane where carotenoids are available for quenching, whereas AO mediated damage occurs in the nucleus sequestered from the protective influence of carotenoids. A newly isolated, lemon-yellow mutant exhibited sensitivities to photodynamic inactivation similar to other pure-white mutants. The sensitivity of this pigmented mutant is apparently related to insufficient unsaturation of the two coloured carotenoids produced by the mutant. (author)

High-resolution autoradiography of nuclear modifications during and after heat treatment of neurospora crassa

Energy Technology Data Exchange (ETDEWEB)

Ton-That, C.; Turian, G. (Geneva Univ. (Switzerland))

1984-01-01

The appearance of perinucleolar electron-dense spots in the nuclei of macroconidia of Neurospora crassa incubated at 46/sup 0/C and their disaggregation after shift-down to 25/sup 0/C have been investigated by high-resolution autoradiography after (5-/sup 3/H) uridine pulses with or without chase periods. The RNA of these ribonucleoprotein-rich dense spots has been found to originate mainly from the heat-sensitive nucleolus; after return to 25/sup 0/C, the nucleolar activity was recovered and the RNA material stored either in an unprocessed or a mature rRNA form in the dense spots was found to be progressively extruded into the cytoplasm.

High-resolution autoradiography of nuclear modifications during and after heat treatment of neurospora crassa

International Nuclear Information System (INIS)

Ton-That, C.; Turian, G.

1984-01-01

The appearance of perinucleolar electron-dense spots in the nuclei of macroconidia of Neurospora crassa incubated at 46 0 C and their disaggregation after shift-down to 25 0 C have been investigated by high-resolution autoradiography after (5- 3 H) uridine pulses with or without chase periods. The RNA of these ribonucleoprotein-rich dense spots has been found to originate mainly from the heat-sensitive nucleolus; after return to 25 0 C, the nucleolar activity was recovered and the RNA material stored either in an unprocessed or a mature rRNA form in the dense spots was found to be progressively extruded into the cytoplasm. (Author)

Live imaging of β-1,3-glucan synthase FKS-1 in Neurospora crassa hyphae.

Science.gov (United States)

Sánchez-León, Eddy; Riquelme, Meritxell

2015-09-01

The subcellular localization and dynamics of FKS-1, the putative catalytic subunit of the β-1,3-glucan synthase complex, was analyzed in growing hyphae of Neurospora crassa by live confocal microscopy. GFP-tagged FKS-1 accumulated at the outer layer of the Spitzenkörper (Spk), and at the apical plasma membrane (PM). Fluorescence recovery after photobleaching analysis revealed arrival of FKS-1-containing carriers first at the immediate surroundings of the core region of the Spk, and thereafter to the Spk most outer region. The results obtained here and previous data suggest that FKS-1 is transported to the Spk in macrovesicles. Copyright © 2015 Elsevier Inc. All rights reserved.

Direct electrochemistry of nitrate reductase from the fungus Neurospora crassa.

Science.gov (United States)

Kalimuthu, Palraj; Ringel, Phillip; Kruse, Tobias; Bernhardt, Paul V

2016-09-01

We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39μM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained. Copyright © 2016. Published by Elsevier B.V.

A novel polyketide biosynthesis gene cluster is involved in fruiting body morphogenesis in the filamentous fungi Sordaria macrospora and Neurospora crassa.

Science.gov (United States)

Nowrousian, Minou

2009-04-01

During fungal fruiting body development, hyphae aggregate to form multicellular structures that protect and disperse the sexual spores. Analysis of microarray data revealed a gene cluster strongly upregulated during fruiting body development in the ascomycete Sordaria macrospora. Real time PCR analysis showed that the genes from the orthologous cluster in Neurospora crassa are also upregulated during development. The cluster encodes putative polyketide biosynthesis enzymes, including a reducing polyketide synthase. Analysis of knockout strains of a predicted dehydrogenase gene from the cluster showed that mutants in N. crassa and S. macrospora are delayed in fruiting body formation. In addition to the upregulated cluster, the N. crassa genome comprises another cluster containing a polyketide synthase gene, and five additional reducing polyketide synthase (rpks) genes that are not part of clusters. To study the role of these genes in sexual development, expression of the predicted rpks genes in S. macrospora (five genes) and N. crassa (six genes) was analyzed; all but one are upregulated during sexual development. Analysis of knockout strains for the N. crassa rpks genes showed that one of them is essential for fruiting body formation. These data indicate that polyketides produced by RPKSs are involved in sexual development in filamentous ascomycetes.

The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.

Science.gov (United States)

Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

2015-01-09

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Putative Cellodextrin Transporter-like Protein CLP1 Is Involved in Cellulase Induction in Neurospora crassa*

Science.gov (United States)

Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

2015-01-01

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. PMID:25398875

The pmr gene, encoding a Ca2+-ATPase, is required for calcium and manganese homeostasis and normal development of hyphae and conidia in Neurospora crassa.

Science.gov (United States)

Bowman, Barry J; Abreu, Stephen; Johl, Jessica K; Bowman, Emma Jean

2012-11-01

The pmr gene is predicted to encode a Ca(2+)-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr(RIP), in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl(2) (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca(2+)-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca(2+)-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr(RIP) Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.

A Mouse Neurodegenerative Dynein Heavy Chain Mutation Alters Dynein Motility and Localization in Neurospora crassa

Science.gov (United States)

Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

2013-01-01

Cytoplasmic dynein is responsible for the transport and delivery of cargoes in organisms ranging from humans to fungi. Dysfunction of dynein motor machinery due to mutations in dynein or its activating complex dynactin can result in one of several neurological diseases in mammals. The mouse Legs at odd angles (Loa) mutation in the tail domain of the dynein heavy chain has been shown to lead to progressive neurodegeneration in mice. The mechanism by which the Loa mutation affects dynein function is just beginning to be understood. In this work, we generated the dynein tail mutation observed in Loa mice into the Neurospora crassa genome and utilized cell biological and complementing biochemical approaches to characterize how that tail mutation affected dynein function. We determined that the Loa mutation exhibits several subtle defects upon dynein function in N. crassa that were not seen in mice, including alterations in dynein localization, impaired velocity of vesicle transport, and in the biochemical properties of purified motors. Our work provides new information on the role of the tail domain on dynein function and points out areas of future research that will be of interest to pursue in mammalian systems. PMID:22991199

Identification of two products of mitochondrial protein synthesis associated with mitochondrial adenosine triphosphatase from Neurospora crassa

International Nuclear Information System (INIS)

Jackl, G.; Sebald, W.

1975-01-01

Soluble mitochondrial ATPase (F 1 ) isolated from Neurospora crassa is resolved by dodecyl-sulfate-gel electrophoresis into five polypeptide bands with apparent molecular weights of 59,000, 55,000, 36,000, 15,000 and 12,000. At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X-100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F 1 ATPase. Two of the associated polypeptides with apparent molecular weights of 19,000 and 11,000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leucine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial (cycloheximide) protein synthesis. The appearance of mitochondrial translation products in the immunoprecipitated ATPase complex is inhibited by cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the ATPase complex. (orig.) [de

Influence of point mutations near the active site on the catalytic properties of fungal arylacetonitrilases from Aspergillus niger and Neurospora crassa

Czech Academy of Sciences Publication Activity Database

Petříčková, Alena; Sosedov, O.; Baum, S.; Stolz, A.; Martínková, Ludmila

2012-01-01

Roč. 77, MAY 2012 (2012), s. 74-80 ISSN 1381-1177 R&D Projects: GA ČR(CZ) GAP504/11/0394; GA ČR GD305/09/H008; GA AV ČR IAA500200708; GA MŠk(CZ) LC06010; GA MŠk OC09046 Institutional research plan: CEZ:AV0Z50200510 Keywords : Arylacetonitrilase variants * Aspergillus niger * Neurospora crassa Subject RIV: CE - Biochemistry Impact factor: 2.823, year: 2012

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa

International Nuclear Information System (INIS)

Serres, F.J. de

1989-01-01

More extensive genetic tests have been preformed on a series of 832 X-ray-induced specific-locus mutations in the ad-3 region of a 2-component heterokaryon of Neurospora crassa, reported earlier. Using new tester strains and techniques for performing large-scale genetic tests to characterize ad-3 mutants induced in 2-component heterokaryons, new data have been obtained on this sample of X-ray-induced mutants. These new data show that unexpectedly high frequencies of both single-locus mutations and multilocus deletions in the ad-3 region have addition, but separate, sites of recessive lethal damage in the imeediately adjacent genetic regions. The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory and classical models of chromosome structure during interphase. Current models of interphase chromosome structure in higher eukaryotes as revealed by chromosome 'painting' offer a possible explanation of the Neurospora data. (author). 25 refs.; 5 figs

Effect of benzene compounds from plants on the growth and hyphal morphology in Neurospora crassa Efeito de compostos benzênicos de plantas sobre o crescimento e a morfologia das hifas em Neurospora crassa

Directory of Open Access Journals (Sweden)

Fabrícia Mendonça Neves

2005-06-01

Full Text Available The effects of the benzene compounds from plants, respectively cinnamic acid, coumaric acid, ferulic acid, caffeic acid, and cinnamic aldehyde on growth and hyphal morphology of Neurospora crassa, were investigated. Cinnamic acid, ferulic acid and cinnamic aldehyde inhibited colony growth, but produced no visible alterations on hyphae. Caffeic acid and coumaric acid did not inhibit growth, but changed hyphal morphology. The results suggest that caffeic and coumaric acids probably affect polarity maintenance (the continued deposition of wall material at the extending tip, while cinnamic aldehyde, ferulic and cinnamic acids decrease growth rate, but did not change hyphal polarity. The actin cytoskeleton and the Spitzenkörper appeared diffuse and not clearly visible when one of the benzene compounds was present in the culture.Os efeitos de compostos benzênicos de plantas, respectivamente ácido cinâmico, ácido coumárico, ácido ferúlico, ácido cafeico e aldeído cinâmico, sobre o crescimento da colônia e a morfologia das hifas de Neurospora crassa foram investigados. Ácido cinâmico, ácido ferúlico e aldeído cinâmico inibiram o crescimento colonial, mas não produziram diferenças visíveis sobre as hifas. Ácido cafeico e ácido coumárico não inibiram o crescimento, mas alteraram a morfologia das hifas. Os resultados sugerem que os ácidos cafeico e coumárico afetam provavelmente a manutenção da polaridade (a contínua deposição de material da parede na ponta em extensão, enquanto aldeído cinâmico e os ácidos cinâmicos e ferúlico diminuem a velocidade de crescimento, mas não alteram a polaridade das hifas. Actina no citoesqueleto e no Spitzenkörper apareceu difuso e não estava claramente visível na presença de um dos compostos benzênicos na cultura.

SnoRNAs from the filamentous fungus Neurospora crassa: structural, functional and evolutionary insights

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Chen Chun-Long

2009-11-01

Full Text Available Abstract Background SnoRNAs represent an excellent model for studying the structural and functional evolution of small non-coding RNAs involved in the post-transcriptional modification machinery for rRNAs and snRNAs in eukaryotic cells. Identification of snoRNAs from Neurospora crassa, an important model organism playing key roles in the development of modern genetics, biochemistry and molecular biology will provide insights into the evolution of snoRNA genes in the fungus kingdom. Results Fifty five box C/D snoRNAs were identified and predicted to guide 71 2'-O-methylated sites including four sites on snRNAs and three sites on tRNAs. Additionally, twenty box H/ACA snoRNAs, which potentially guide 17 pseudouridylations on rRNAs, were also identified. Although not exhaustive, the study provides the first comprehensive list of two major families of snoRNAs from the filamentous fungus N. crassa. The independently transcribed strategy dominates in the expression of box H/ACA snoRNA genes, whereas most of the box C/D snoRNA genes are intron-encoded. This shows that different genomic organizations and expression modes have been adopted by the two major classes of snoRNA genes in N. crassa . Remarkably, five gene clusters represent an outstanding organization of box C/D snoRNA genes, which are well conserved among yeasts and multicellular fungi, implying their functional importance for the fungus cells. Interestingly, alternative splicing events were found in the expression of two polycistronic snoRNA gene hosts that resemble the UHG-like genes in mammals. Phylogenetic analysis further revealed that the extensive separation and recombination of two functional elements of snoRNA genes has occurred during fungus evolution. Conclusion This is the first genome-wide analysis of the filamentous fungus N. crassa snoRNAs that aids in understanding the differences between unicellular fungi and multicellular fungi. As compared with two yeasts, a more complex

Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa.

OpenAIRE

Hays, Shan M; Swanson, Johanna; Selker, Eric U

2002-01-01

We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged ...

The bZIP Transcription Factor HAC-1 Is Involved in the Unfolded Protein Response and Is Necessary for Growth on Cellulose in Neurospora crassa.

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Alejandro Montenegro-Montero

Full Text Available High protein secretion capacity in filamentous fungi requires an extremely efficient system for protein synthesis, folding and transport. When the folding capacity of the endoplasmic reticulum (ER is exceeded, a pathway known as the unfolded protein response (UPR is triggered, allowing cells to mitigate and cope with this stress. In yeast, this pathway relies on the transcription factor Hac1, which mediates the up-regulation of several genes required under these stressful conditions. In this work, we identified and characterized the ortholog of the yeast HAC1 gene in the filamentous fungus Neurospora crassa. We show that its mRNA undergoes an ER stress-dependent splicing reaction, which in N. crassa removes a 23 nt intron and leads to a change in the open reading frame. By disrupting the N. crassa hac-1 gene, we determined it to be crucial for activating UPR and for proper growth in the presence of ER stress-inducing chemical agents. Neurospora is naturally found growing on dead plant material, composed primarily by lignocellulose, and is a model organism for the study of plant cell wall deconstruction. Notably, we found that growth on cellulose, a substrate that requires secretion of numerous enzymes, imposes major demands on ER function and is dramatically impaired in the absence of hac-1, thus broadening the range of physiological functions of the UPR in filamentous fungi. Growth on hemicellulose however, another carbon source that necessitates the secretion of various enzymes for its deconstruction, is not impaired in the mutant nor is the amount of proteins secreted on this substrate, suggesting that secretion, as a whole, is unaltered in the absence of hac-1. The characterization of this signaling pathway in N. crassa will help in the study of plant cell wall deconstruction by fungi and its manipulation may result in important industrial biotechnological applications.

Rediscovery by Whole Genome Sequencing: Classical Mutations and Genome Polymorphisms in Neurospora crassa

Energy Technology Data Exchange (ETDEWEB)

McCluskey, Kevin; Wiest, Aric E.; Grigoriev, Igor V.; Lipzen, Anna; Martin, Joel; Schackwitz, Wendy; Baker, Scott E.

2011-06-02

Classical forward genetics has been foundational to modern biology, and has been the paradigm for characterizing the role of genes in shaping phenotypes for decades. In recent years, reverse genetics has been used to identify the functions of genes, via the intentional introduction of variation and subsequent evaluation in physiological, molecular, and even population contexts. These approaches are complementary and whole genome analysis serves as a bridge between the two. We report in this article the whole genome sequencing of eighteen classical mutant strains of Neurospora crassa and the putative identification of the mutations associated with corresponding mutant phenotypes. Although some strains carry multiple unique nonsynonymous, nonsense, or frameshift mutations, the combined power of limiting the scope of the search based on genetic markers and of using a comparative analysis among the eighteen genomes provides strong support for the association between mutation and phenotype. For ten of the mutants, the mutant phenotype is recapitulated in classical or gene deletion mutants in Neurospora or other filamentous fungi. From thirteen to 137 nonsense mutations are present in each strain and indel sizes are shown to be highly skewed in gene coding sequence. Significant additional genetic variation was found in the eighteen mutant strains, and this variability defines multiple alleles of many genes. These alleles may be useful in further genetic and molecular analysis of known and yet-to-be-discovered functions and they invite new interpretations of molecular and genetic interactions in classical mutant strains.

Neurospora tetrasperma crosses heterozygous for hybrid

Indian Academy of Sciences (India)

nucleus stage that follows meiosis and apost-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. We had previouslycreated novel TNt strains by introgressing four Neurospora crassa insertional ...

Neurospora tetrasperma crosses heterozygous for hybrid ...

Indian Academy of Sciences (India)

2017-02-10

nucleus stage that follows meiosis and a post-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. We had previously created novel TNt strains by introgressing four Neurospora crassa ...

Proteomic alterations induced by ionic liquids in Aspergillus nidulans and Neurospora crassa.

Science.gov (United States)

Martins, Isabel; Hartmann, Diego O; Alves, Paula C; Planchon, Sébastien; Renaut, Jenny; Leitão, M Cristina; Rebelo, Luís P N; Silva Pereira, Cristina

2013-12-06

This study constitutes the first attempt to understand at the proteomic level the fungal response to ionic liquid stress. Ascomycota are able to grow in media supplemented with high concentrations of an ionic liquid, which, in turn, lead to major alterations in the fungal metabolic footprint. Herein, we analysed the differential accumulation of mycelial proteins in Aspergillus nidulans and Neurospora crassa after their exposure to two of the most commonly used ionic liquids: 1-ethyl-3-methylimidazolium chloride or cholinium chloride. Data obtained showed that numerous stress-responsive proteins (e.g. anti-ROS defence proteins) as well as several critical biological processes and/or pathways were affected by either ionic liquid. Amongst other changes, these compounds altered developmental programmes in both fungi (e.g. promoting the development of Hülle cells or conidiation) and led to accumulation of osmolytes, some of which may play an important role in multiple stress responses. In particular, in N. crassa, both ionic liquids increased the levels of proteins which are likely involved in the biosynthesis of unusual metabolites. These data potentially open new perspectives on ionic liquid research, furthering their conscious design and their use to trigger production of targeted metabolites. The present study emphasises the importance of understanding ionic liquid's stress responses, crucial to further their safe large-scale usage. Knowledge of the alterations prompted at a cellular and biochemical level gives also fresh perspectives on how to employ these "novel" compounds to manipulate proteins or pathways of biotechnological value. The results presented here provide meaningful insights into the understanding of fungi stress and adaptation responses to anthropogenic chemicals used in industry. © 2013.

Unravelling the molecular basis for light modulated cellulase gene expression - the role of photoreceptors in Neurospora crassa

Science.gov (United States)

2012-01-01

Background Light represents an important environmental cue, which exerts considerable influence on the metabolism of fungi. Studies with the biotechnological fungal workhorse Trichoderma reesei (Hypocrea jecorina) have revealed an interconnection between transcriptional regulation of cellulolytic enzymes and the light response. Neurospora crassa has been used as a model organism to study light and circadian rhythm biology. We therefore investigated whether light also regulates transcriptional regulation of cellulolytic enzymes in N. crassa. Results We show that the N. crassa photoreceptor genes wc-1, wc-2 and vvd are involved in regulation of cellulase gene expression, indicating that this phenomenon is conserved among filamentous fungi. The negative effect of VVD on production of cellulolytic enzymes is thereby accomplished by its role in photoadaptation and hence its function in White collar complex (WCC) formation. In contrast, the induction of vvd expression by the WCC does not seem to be crucial in this process. Additionally, we found that WC-1 and WC-2 not only act as a complex, but also have individual functions upon growth on cellulose. Conclusions Genome wide transcriptome analysis of photoreceptor mutants and evaluation of results by analysis of mutant strains identified several candidate genes likely to play a role in light modulated cellulase gene expression. Genes with functions in amino acid metabolism, glycogen metabolism, energy supply and protein folding are enriched among genes with decreased expression levels in the wc-1 and wc-2 mutants. The ability to properly respond to amino acid starvation, i. e. up-regulation of the cross pathway control protein cpc-1, was found to be beneficial for cellulase gene expression. Our results further suggest a contribution of oxidative depolymerization of cellulose to plant cell wall degradation in N. crassa. PMID:22462823

Ascus dysgenesis in hybrid crosses of Neurospora and Sordaria (Sordariaceae).

Science.gov (United States)

Kasbekar, Durgadas P

2017-07-01

When two lineages derived from a common ancestor become reproductively isolated (e.g. Neurospora crassa and N. tetrasperma), genes that have undergone mutation and adaptive evolution in one lineage can potentially become dysfunctional when transferred into the other, since other genes have undergone mutation and evolution in the second lineage, and the derived alleles were never 'tested' together before hybrid formation. Bateson (1909), Dobzhansky (1936), and Muller (1942) recognized that incompatibility between the derived alleles could potentially make the hybrid lethal, sterile, or display some other detriment. Alternatively, the detrimental effects seen in crosses with the hybrids may result from the silencing of ascus-development genes by meiotic silencing by unpaired DNA (MSUD). Aberrant transcripts from genes improperly paired in meiosis are processed into single-stranded MSUD-associated small interfering RNA (masiRNA), which is used to degrade complementary mRNA. Recently, backcrosses of N. crassa / N. tetrasperma hybrid translocation strains with wild-type N. tetrasperma were found to elicit novel ascus dysgenesis phenotypes. One was a transmission ratio distortion that apparently disfavoured the homokaryotic ascospores formed following alternate segregation. Another was the production of heterokaryotic ascospores in eight-spored asci. Lewis (1969) also had reported sighting rare eight-spored asci with heterokaryotic ascospores in interspecific crosses in Sordaria, a related genus. Ordinarily, in both Neurospora and Sordaria, the ascospores are partitioned at the eight-nucleus stage, and ascospores in eight-spored asci are initially uninucleate. Evidently, in hybrid crosses of the family Sordariaceae, ascospore partitioning can be delayed until after one or more mitoses following the postmeiotic mitosis.

Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa.

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Amanda Cristina Campos Antoniêto

Full Text Available Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0, neutral (pH 7.0, and alkaline (pH 10.0 medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry.

Journal of Genetics | Indian Academy of Sciences

Indian Academy of Sciences (India)

Keywords. Spore killer; meiotic drive; Neurospora crassa; RIP. Abstract. A convenient assay to score repeat-induced point mutation (RIP) in Neurospora employs the erg-3 locus as a mutagenesis target. Using this assay we screened 132 wild-isolated Neurospora crassa strains for ability to dominantly suppress RIP.

Maternal parentage influences spore production but not spore pigmentation in the anisogamous and hermaphroditic fungus Neurospora crassa

DEFF Research Database (Denmark)

Zimmerman, Kolea; Levitis, Daniel; Pringle, Anne

2014-01-01

. In this fungus, pigmented spores are viable and unpigmented spores are inviable. These results show that while both parents influence all these traits, maternal influence is strongest on both fertility and mortality traits until the spores are physiologically independent of the maternal cytoplasm.......In this study, we tested the hypothesis that maternal effects on offspring production and quality are greater than paternal effects in both offspring number (fertility) and offspring viability (mortality). We used the model filamentous fungus Neurospora crassa. This fungus is anisogamous......, and various ascospore characteristics. Mixed effects models of these data show that the female parent accounts for the majority of variation in perithecial production, number of spores produced, and spore germination. Surprisingly, both sexes equally influence the percentage of spores that are pigmented...

MTB-3, a microtubule plus-end tracking protein (+TIP of Neurospora crassa.

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Rosa R Mouriño-Pérez

Full Text Available The microtubule (MT "plus end" constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called "MT plus-end tracking proteins" (+TIPs. +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassaMicroTubule Binding protein-3 (MTB-3 is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.

Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

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Kathleen D Cusick

Full Text Available Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1 light/dark cycling: btl, asl, and vma1; (2 all-dark growth: btl, tbp, vma1, and vma2; (3 temperature flux: btl, vma1, act, and asl; (4 all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated, expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring

Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

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Cusick, Kathleen D; Fitzgerald, Lisa A; Pirlo, Russell K; Cockrell, Allison L; Petersen, Emily R; Biffinger, Justin C

2014-01-01

Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene

Neurospora crassa tox-1 Gene Encodes a pH- and Temperature-Tolerant Mini-Cellulase.

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Xiao, Yue; Zhang, Qiongsi; Luo, Yiquan; Zhang, Ying; Luo, Xi; Wang, Yuchuan; Cao, Weiguo; Pinto, Vito De; Liu, Qiuyun; Li, Gang

2016-06-15

Cellulases that endure extreme conditions are essential in various industrial sectors. This study reports a mini-cellulase gene tox-1 from Neurospora crassa. The gene tox-1 was cloned in Escherichia coli after chimerization with the YebF gene and substitutions of certain isoleucine and valine with leucine residues. The yeast transformants could grow on rice straw-agar medium. The 44-amino acid peptide and its two mutant variants displayed potent cellulase activities in Congo Red assay and enzymatic assays. Conservative replacements with leucine have substantially increased the stabilities and half-lives of the peptides at alkaline pH and low and high temperatures and also the tolerance to organic solvents and surfactants, on the basis of activities toward cellose. The small size of the mini-cellulase would allow for commercially viable automatic chemical peptide synthesis. This work suggests that conservative leucine replacements may serve as a general strategy in the engineering of more robust enzymes with special features with little loss of activities.

Mutations of the a mating-type gene in Neurospora crassa

International Nuclear Information System (INIS)

Griffiths, A.J.F.; DeLange, A.M.

1978-01-01

In Neurospora, the mating-type locus controls both mating (A + a is fertile) and heterokaryosis (A + a is incompatible). The two alleles appear stable: no novel fertility reactions have ever beeen reported, and attempts to separate fertility and heterokaryon incompatibility functions by recombination have been unsuccessful. In the present approach the locus was studied through a mutational analysis of heterokaryon incompatibility function. A selection system was used that detects vigorous (A + a) heterokaryotic colonies against a background of inhibited growth. Twenty-five mutants of an a strain were produced following mutagenic treatment with uv and NG: 15 were viable as homokaryons and 10 were not. All but one were infertile, but most showed an abortive mating reaction involving the production of barren, well-developed perithecia with A and (surprisingly) a testers. None of the mutants complement each other to restore fertility. Seven mutants have been mapped to the mating-type locus region of chromosome 1. Restoration of fertility was used to detect revertants, and these were found in five out of the eight mutants tested. (A dose response was observed). In four cases incompatibility was fully restored and in one case it was not. The results suggest two positive actions of the locus when in heterozygous (A/a) combination (the stimulation of some stage of ascus production and the inhibition of vegetative heterokaryosis), and one positive action in homozygous combinations

Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa.

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Bowman, Shaun M; Piwowar, Amy; Al Dabbous, Mash'el; Vierula, John; Free, Stephen J

2006-03-01

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.

Localization and role of MYO-1, an endocytic protein in hyphae of Neurospora crassa.

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Lara-Rojas, Fernando; Bartnicki-García, Salomón; Mouriño-Pérez, Rosa R

2016-03-01

The subapical endocytic collar is a prominent feature of hyphae of Neurospora crassa. It comprises a dynamic collection of actin patches associated with a number of proteins required for endocytosis, namely, ARP-2/3 complex, fimbrin, coronin, etc. We presently show that MYO-1 is another key component of this endocytic collar. A myo-1 sequence was identified in the genome of N. crassa and used it to generate a strain with a myo-1-sgfp allele under the ccg1 promoter. Examination of living hyphae by confocal microscopy, revealed MYO-1-GFP located mainly as a dynamic collection of small patches arranged in collar-like fashion in the hyphal subapex. Dual tagging showed MYO-1-GFP partially colocalized with two other endocytic proteins, fimbrin and coronin. MYO-1 was also present during septum formation. By recovering a viable strain, albeit severely inhibited, after deletion of myo-1, it was possible to investigate the phenotypic consequences of the elimination of MYO-1. Deletion of myo-1 caused a severe reduction in growth rate (95%), near absence of aerial mycelium and no conidiation. A reduced uptake of the lipophilic dye FM4-64 indicated a deficiency in endocytosis in the Δmyo-1 mutant. Hyphae were produced by the Δmyo-1 mutant but their morphogenesis was severely affected; hyphal morphology was distorted displaying irregular periods of isotropic and polarized growth. The morphological alterations were accompanied, and presumably caused, by a disruption in the organization and dynamics of a myosin-deprived actin cytoskeleton that, ultimately, compromised the stability and function of the Spitzenkörper as a vesicle supply center. Copyright © 2016 Elsevier Inc. All rights reserved.

The synthesis of Phosphate-repressible alkaline phosphatase do not appear to be regulated by ambient pH in the filamentous mould Neurospora crassa

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Nozawa Sérgio R.

2002-01-01

Full Text Available In order to investigate further the adaptive response of moulds to ambient pH, we have measured by ELISA the pho-2-encoded Pi-repressible alkaline phosphatase synthesised by Neurospora crassa. We showed that the 74A and pho-2A strains of this mould secrete similar amounts of the pho-2-encoded enzyme irrespective of ambient pH, when both the preg and pgov genes are not functional, i.e., in strains nuc-2+ growing under Pi-starvation. This suggests that pho-2, which is responsive to Pi starvation via the action of genes nuc-2, preg, pgov and nuc-1, is not a gene responsive to ambient pH and that the differential glycosylation observed for the Pi-repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted into the growth medium at pH 8.0 is the genetic response to ambient pH sensing in N. crassa.

Biochemical genetics of the circadian rhythm in Neurospora crassa: studies on the cel strain

International Nuclear Information System (INIS)

Lakin-Thomas, P.L.

1985-01-01

In Neurospora crassa, the cel mutation lengthens the period of the circadian rhythm when the medium is supplemented with linoleic acid (18:2). Double mutant strains were constructed between cel and the clock mutants prd-1 and four alleles at the frq locus. It was found that: (1) the effect of 18:2 on cel was blocked by prd-1, i.e., prd-1 is epistatic to cel. (2) cel and frq interact such that the percent increase in the period produced by 18:2 was inversely proportional to the period of the frq parent. (3) Data from the literature on period effects in double mutant strains support a multiplicative rather than an additive model. A biochemical interpretation of these interactions is discussed, based on the control of flux through metabolic pathways. Because the cel strain is known to be deficient in the pantothenate derivative normally attached to the fatty acid synthetase (FAS) complex, the possibility that cel may affect other pantothenate-modified proteins was investigated. It was found that in the cel + strain, five proteins of molecular weights (M/sub r/) 9000, 19,000, 22,000, 140,000, and 200,000 were labelled with [ 14 C]pantothenate. In the cel strain, only the 200 k (FAS) label was reduced in amount. Therefore, there is no evidence that cel affects circadian rhythmicity through any deficiency other than FAS. A biochemical model for circadian rhythmicity in Neurospora is presented. Oscillations in cytoplasmic and mitochondrial Ca 2+ are proposed; clock mutations are postulated to affect Ca 2+ transporters and the mitochondrial membrane; and phase-shifting effects are accounted for by changes in Ca 2+ or ATP levels

The Neurospora rca-1 gene complements an Aspergillus flbD sporulation mutant but has no identifiable role in Neurospora sporulation.

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Shen, W C; Wieser, J; Adams, T H; Ebbole, D J

1998-01-01

The Aspergillus nidulans flbD gene encodes a protein with a Myb-like DNA-binding domain that is proposed to act in concert with other developmental regulators to control initiation of conidiophore development. We have identified a Neurospora crassa gene called rca-1 (regulator of conidiation in Aspergillus) based on its sequence similarity to flbD. We found that N. crassa rca-1 can complement the conidiation defect of an A. nidulans flbD mutant and that induced expression of rca-1 caused coni...

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents Alteraciones de crecimiento y ramificación en Neurospora crassa provocadas por concentraciones subinhibitorias de agentes antimicóticos

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R. C. Pereira

2009-03-01

Full Text Available Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 μg/ml inhibited radial growth more than fluconazole at 5.0 μg/ml while amphotericin B at 0.05 μg/ml was more effective than nystatin at 0.05 μg/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9. All our results showed highly significant differences by ANOVA, (p Se investigó el efecto de seis agentes antimicóticos en concentraciones subinhibitorias sobre el crecimiento y la ramificación en Neurospora crassa. El agente azólico ketoconazol a la concentración de 0,5 μg/ml inhibió el crecimiento radial más que el fluconazol a 5,0 μg/ml, y la anfotericina B a 0,05 μg/ ml fue más eficiente que 0,05 μg/ml de nistatina, entre los agentes poliénicos usados. En presencia de griseofulvina, ketoconazol y terbinafina a las concentraciones establecidas se observaron alteraciones morfológicas en las hifas. Los agentes antimicóticos fueron más eficientes sobre el crecimiento vegetativo que sobre la germinación conidial. La terbinafina redujo marcadamente (54,89% la longitud de la unidad de crecimiento y provocó la hiperramificación del micelio. En todos los casos, existió gran correlación entre la longitud y el número de ápices de las hifas (r > 0,9. Todos los resultados mostraron diferencias altamente significativas de acuerdo con ANOVA (p < 0,001, α = 0,05. Considerando que el ápice de la hifa es la principal interfase entre

Bright to Dim Oscillatory Response of the Neurospora Circadian Oscillator

OpenAIRE

Gooch, Van D.; Johnson, Alicia E.; Larrondo, Luis F.; Loros, Jennifer J.; Dunlap, Jay C.

2014-01-01

The fungus Neurospora crassa constitutes an important model system extensively used in chronobiology. Several studies have addressed how environmental cues, such as light, can reset or synchronize a circadian system. By means of an optimized firefly luciferase reporter gene and a controllable lighting system, we show that Neurospora can display molecular circadian rhythms in dim light when cultures receive bright light prior to entering dim light conditions. We refer to this behavior as the “...

Screening of agricultural wastes as a medium production of catalase for enzymatic fuel cell by Neurospora crassa InaCC F226

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Santoso, Pugoh; Yopi

2017-12-01

Explorations of local microorganisms from Indonesia that can produce of catalase are still limited. Neurospora crassa is a fungus which resulting of two kinds of catalase, namely catalase-1 and catalase-3. We studied the production of catalase by Neurospora crassa (no. F226) from Indonesia Culture Collection (InaCC) in Solid State Fermentation (SSF). Among four screened agro wastes (corn cob, rice straw, oil palm empty fruit bunches, and bagasse), rice straw and oil palm empty fruit bunches (OPEFB) were remarked as the most promising substrate suited for the excellent growth and adequate production of catalase. Based on the result, the method of solid state fermentation was suitable to production of catalase. It is caused that the medium served to maintain microbial growth and metabolism. The filamentous filament is more suitable for living on solid media because it has a high tolerance to low water activity, and it has a high potential to excrete hydrolytic enzymes that caused of its morphology. The filamentous filament morphology allows the fungus to form colonies and penetrate the solid substrates in order to obtain nutrients. The results showed that the highest catalase activity was obtained on rice straw and oil palm empty fruit bunches medium with catalase activity of 39.1 U/mL and 37,7 U/mL in 50% moisture content medium, respectively. Optimization of humidity and pH medium in the rice straw were investigated which is the highest activity obtained in 30% moisture content and pH medium of 6. The catalase activity was reached in the value of 53.761 U/mL and 56.903 U/mL by incubated 48 hours and 96 hours, respectively.

The Neurospora crassa UVS-3 epistasis group encodes homologues of the ATR/ATRIP checkpoint control system.

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Kazama, Yusuke; Ishii, Chizu; Schroeder, Alice L; Shimada, Hisao; Wakabayashi, Michiyoshi; Inoue, Hirokazu

2008-02-01

The mutagen sensitive uvs-3 and mus-9 mutants of Neurospora show mutagen and hydroxyurea sensitivity, mutator effects and duplication instability typical of recombination repair and DNA damage checkpoint defective mutants. To determine the nature of these genes we used cosmids from a genomic library to clone the uvs-3 gene by complementation for MMS sensitivity. Mutation induction by transposon insertion and RIP defined the coding sequence. RFLP analysis confirmed that this sequence maps in the area of uvs-3 at the left telomere of LG IV. Analysis of the cDNA showed that the UVS-3 protein contains an ORF of 969 amino acids with one intron. It is homologous to UvsD of Aspergillus nidulans, a member of the ATRIP family of checkpoint proteins. It retains the N' terminal coiled-coil motif followed by four basic amino acids typical of these proteins and shows the highest homology in this region. The uvsD cDNA partially complements the defects of the uvs-3 mutation. The uvs-3 mutant shows a higher level of micronuclei in conidia and failure to halt germination and nuclear division in the presence of hydroxyurea than wild type, suggesting checkpoint defects. ATRIP proteins bind tightly to ATR PI-3 kinase (phosphatidylinositol 3-kinase) proteins. Therefore, we searched the Neurospora genome sequence for homologues of the Aspergillus nidulans ATR, UvsB. A uvsB homologous sequence was present in the right arm of chromosome I where the mus-9 gene maps. A cosmid containing this genomic DNA complemented the mus-9 mutation. The putative MUS-9 protein is 2484 amino acids long with eight introns. Homology is especially high in the C-terminal 350 amino acids that correspond to the PI-3 kinase domain. In wild type a low level of constitutive mRNA is present for both genes. It is transiently induced upon UV exposure.

Acetylglutamate synthase in Neurospora crassa: characterization, localization, and genetic behavior of a regulatory enzyme of arginine biosynthesis

International Nuclear Information System (INIS)

Jacobson, J.A.

1988-01-01

This study describes the characterization and localization of the first enzyme of arginine biosynthesis in Neurospora crassa. A radioactive assay was developed to detect this enzyme whereby radioactive substrate and product molecules could be separated by ion-exchange chromatography. The enzyme was found to have a pH optimum of 9.0 and K/sub m/ values for glutamate and acetyl-CoA of approximately 4.7 and 0.45 mM, respectively. The enzyme was shown to be feedback inhibited by arginine. Half-maximal inhibition was observed at 0.13 mM arginine, a concentration which is similar to be in vivo cytosolic concentration of 0.2 mM. Arginine was found to act as a competitive inhibitor with respect to acetyl-CoA. Acetylglutamate synthase was localized to the mitochondrion. However, in contrast to the mitochondrial matrix location of the other ornithine biosynthetic enzymes, this enzyme was found to reside on the mitochondrial inner membrane

Neurospora crassa female development requires the PACC and other signal transduction pathways, transcription factors, chromatin remodeling, cell-to-cell fusion, and autophagy.

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Jennifer L Chinnici

Full Text Available Using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus Neurospora crassa. We find that we can divide these genes into five general groups: 1 Genes encoding components of the PACC signal transduction pathway, 2 Other signal transduction pathway genes, including genes from the three N. crassa MAP kinase pathways, 3 Transcriptional factor genes, 4 Autophagy genes, and 5 Other miscellaneous genes. Complementation and RIP studies verified that these genes are needed for the formation of the female mating structure, the protoperithecium, and for the maturation of a fertilized protoperithecium into a perithecium. Perithecia grafting experiments demonstrate that the autophagy genes and the cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium. Deletion mutants for the PACC pathway genes palA, palB, palC, palF, palH, and pacC were found to be defective in two aspects of female development. First, they were unable to initiate female development on synthetic crossing medium. However, they could form protoperithecia when grown on cellophane, on corn meal agar, or in response to the presence of nearby perithecia. Second, fertilized perithecia from PACC pathway mutants were unable to produce asci and complete female development. Protein localization experiments with a GFP-tagged PALA construct showed that PALA was localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex. The N. crassa PACC signal transduction pathway appears to be similar to the PacC/Rim101 pathway previously characterized in Aspergillus nidulans and Saccharomyces cerevisiae. In N. crassa the pathway plays a key role in regulating female development.

Neurospora crassa female development requires the PACC and other signal transduction pathways, transcription factors, chromatin remodeling, cell-to-cell fusion, and autophagy.

Science.gov (United States)

Chinnici, Jennifer L; Fu, Ci; Caccamise, Lauren M; Arnold, Jason W; Free, Stephen J

2014-01-01

Using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus Neurospora crassa. We find that we can divide these genes into five general groups: 1) Genes encoding components of the PACC signal transduction pathway, 2) Other signal transduction pathway genes, including genes from the three N. crassa MAP kinase pathways, 3) Transcriptional factor genes, 4) Autophagy genes, and 5) Other miscellaneous genes. Complementation and RIP studies verified that these genes are needed for the formation of the female mating structure, the protoperithecium, and for the maturation of a fertilized protoperithecium into a perithecium. Perithecia grafting experiments demonstrate that the autophagy genes and the cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes) are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium. Deletion mutants for the PACC pathway genes palA, palB, palC, palF, palH, and pacC were found to be defective in two aspects of female development. First, they were unable to initiate female development on synthetic crossing medium. However, they could form protoperithecia when grown on cellophane, on corn meal agar, or in response to the presence of nearby perithecia. Second, fertilized perithecia from PACC pathway mutants were unable to produce asci and complete female development. Protein localization experiments with a GFP-tagged PALA construct showed that PALA was localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex. The N. crassa PACC signal transduction pathway appears to be similar to the PacC/Rim101 pathway previously characterized in Aspergillus nidulans and Saccharomyces cerevisiae. In N. crassa the pathway plays a key role in regulating female development.

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa.

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Gonçalves, A Pedro; Cordeiro, J Miguel; Monteiro, João; Lucchi, Chiara; Correia-de-Sá, Paulo; Videira, Arnaldo

2015-10-01

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics. Copyright © 2015 Elsevier B.V. All rights reserved.

Genome Defense Mechanisms in Neurospora and Associated Specialized Proteins

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Ranjan Tamuli

2010-06-01

Full Text Available Neurospora crassa, the filamentous fungus possesses widest array of genome defense mechanisms known to any eukaryotic organism, including a process called repeat-induced point mutation (RIP. RIP is a genome defense mechanism that hypermutates repetitive DNA sequences; analogous to genomic imprinting in mammals. As an impact of RIP, Neurospora possesses many fewer genes in multigene families than expected. A DNA methyltransferase homologue, RID was shown to be essential for RIP. Recently, a variant catalytic subunit of translesion DNA polymerase zeta (Pol zeta has been found to be essential for dominant RIP suppressor phenotype. Meiotic silencing and quelling are two other genome defense mechanisms in Neurospora, and proteins required for these two processes have been identified through genetic screens.

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.

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Wilfried Jonkers

2014-11-01

Full Text Available Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this

Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

International Nuclear Information System (INIS)

Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

1986-01-01

The H + translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ ≅ 70,000 and ≅ 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[ 14 C]ethylmaleimide and 7-chloro-4-nitro[ 14 C]benzo-2-oxa-1,3-diazole, labeled the M/sub r/ ≅ 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[ 14 C]dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ ≅ 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10 5 , 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ ≅ 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F 0 F 1 ATPases

Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle

International Nuclear Information System (INIS)

Bowman, B.J.; Berenski, C.J.; Jung, C.Y.

1985-01-01

Using radiation inactivation, the authors have measured the size of the H + -ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60 Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H + -ATPase in situ was found to be approximately 2.3 X 10(5) daltons. They also used radiation inactivation to measure the size of the Ca 2+ -ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, they directly compared the sizes of these two ATPases and found them to be essentially the same. The authors conclude that both H + -ATPase and Ca 2+ -ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides

Observing meiosis in filamentous fungi: Sordaria and Neurospora.

Science.gov (United States)

Zickler, Denise

2009-01-01

The filamentous fungi Neurospora crassa and Sordaria macrospora are materials of choice for recombination studies because each of the DNA strands involved in meiosis can be visually analyzed using spore-color mutants. Well-advanced molecular genetic methodologies have been developed for each of these fungi, and several mutants defective in recombination and/or pairing are available. Moreover, the complete genome sequence of N. crassa has made it possible to clone virtually any gene involved in their life cycle. Both fungi provide also a particularly attractive experimental system for cytological analysis of meiosis: stages can be determined independently of chromosomal morphology and their seven chromosomes are easily identified. The techniques for light, immunofluorescence and electron microscopy presented here have been used, with success, for monitoring of chromosome behavior during both meiotic and sporulation processes. They have also proved useful for the analysis of mitochondria and peroxisomes as well as cytoskeleton and spindle pole-body components. Moreover, all techniques of this chapter can be easily applied to other filamentous ascomycetes, including other Sordaria and Neurospora species as well as Podospora, Ascobolus, Ascophanus, Fusarium, Neotiella, and Aspergillus species.

Enabling a Community to Dissect an Organism: Overview of the Neurospora Functional Genomics Project

OpenAIRE

Dunlap, Jay C.; Borkovich, Katherine A.; Henn, Matthew R.; Turner, Gloria E.; Sachs, Matthew S.; Glass, N. Louise; McCluskey, Kevin; Plamann, Michael; Galagan, James E.; Birren, Bruce W.; Weiss, Richard L.; Townsend, Jeffrey P.; Loros, Jennifer J.; Nelson, Mary Anne; Lambreghts, Randy

2007-01-01

A consortium of investigators is engaged in a functional genomics project centered on the filamentous fungus Neurospora, with an eye to opening up the functional genomic analysis of all the filamentous fungi. The overall goal of the four interdependent projects in this effort is to acccomplish functional genomics, annotation, and expression analyses of Neurospora crassa, a filamentous fungus that is an established model for the assemblage of over 250,000 species of nonyeast fungi. Building fr...

Phylogenetics and Gene Structure Dynamics of Polygalacturonase Genes in Aspergillus and Neurospora crassa

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Jin-Sung Hong

2013-09-01

Full Text Available Polygalacturonase (PG gene is a typical gene family present in eukaryotes. Forty-nine PGs were mined from the genomes of Neurospora crassa and five Aspergillus species. The PGs were classified into 3 clades such as clade 1 for rhamno-PGs, clade 2 for exo-PGs and clade 3 for exo- and endo-PGs, which were further grouped into 13 sub-clades based on the polypeptide sequence similarity. In gene structure analysis, a total of 124 introns were present in 44 genes and five genes lacked introns to give an average of 2.5 introns per gene. Intron phase distribution was 64.5% for phase 0, 21.8% for phase 1, and 13.7% for phase 2, respectively. The introns varied in their sequences and their lengths ranged from 20 bp to 424 bp with an average of 65.9 bp, which is approximately half the size of introns in other fungal genes. There were 29 homologous intron blocks and 26 of those were sub-clade specific. Intron losses were counted in 18 introns in which no obvious phase preference for intron loss was observed. Eighteen introns were placed at novel positions, which is considerably higher than those of plant PGs. In an evolutionary sense both intron loss and gain must have taken place for shaping the current PGs in these fungi. Together with the small intron size, low conservation of homologous intron blocks and higher number of novel introns, PGs of fungal species seem to have recently undergone highly dynamic evolution.

Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa

International Nuclear Information System (INIS)

Heckman, J.E.; Hecker, L.I.; Schwartzbach, S.D.; Barnett, W.E.; Baumstark, B.; RajBhandary, U.L.

1978-01-01

Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5' terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from 3' end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence - T psiCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or psi)CG- as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56 to 60%) than to eucaryotic cytoplasmic initiator tRNAs

Alternative Oxidase Transcription Factors AOD2 and AOD5 of Neurospora crassa Control the Expression of Genes Involved in Energy Production and Metabolism.

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Qi, Zhigang; Smith, Kristina M; Bredeweg, Erin L; Bosnjak, Natasa; Freitag, Michael; Nargang, Frank E

2017-02-09

In Neurospora crassa , blocking the function of the standard mitochondrial electron transport chain results in the induction of an alternative oxidase (AOX). AOX transfers electrons directly from ubiquinol to molecular oxygen. AOX serves as a model of retrograde regulation since it is encoded by a nuclear gene that is regulated in response to signals from mitochondria. The N. crassa transcription factors AOD2 and AOD5 are necessary for the expression of the AOX gene. To gain insight into the mechanism by which these factors function, and to determine if they have roles in the expression of additional genes in N. crassa , we constructed strains expressing only tagged versions of the proteins. Cell fractionation experiments showed that both proteins are localized to the nucleus under both AOX inducing and noninducing conditions. Furthermore, chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) analysis revealed that the proteins are bound to the promoter region of the AOX gene under both conditions. ChIP-seq also showed that the transcription factors bind to the upstream regions of a number of genes that are involved in energy production and metabolism. Dependence on AOD2 and AOD5 for the expression of several of these genes was verified by quantitative PCR. The majority of ChIP-seq peaks observed were enriched for both AOD2 and AOD5. However, we also observed occasional sites where one factor appeared to bind preferentially. The most striking of these was a conserved sequence that bound large amounts of AOD2 but little AOD5. This sequence was found within a 310 bp repeat unit that occurs at several locations in the genome. Copyright © 2017 Qi et al.

Transnuclear retrotransposition of the Tad element of Neurospora.

OpenAIRE

Kinsey, J A

1993-01-01

Tad is a LINE-like DNA element found in Neutrospora crassa. A Neurospora artificial intron based on the first intron of the am (glutamate dehydrogenase) gene was constructed and introduced, in the correct orientation, into a unique Nru I site in open reading frame 1 of an active Tad element, Tad1-1. Transformants containing the Tad element with the artificial intron were placed in forced heterokaryons with strains lacking Tad elements. Tad was shown to transpose between nuclei in these hetero...

Alteration of light-dependent gene regulation by the absence of the RCO-1/RCM-1 repressor complex in the fungus Neurospora crassa.

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Carmen Ruger-Herreros

Full Text Available The activation of transcription by light in the fungus Neurospora crassa requires the White Collar Complex (WCC, a photoreceptor and transcription factor complex. After light reception two WCCs interact and bind the promoters of light-regulated genes to activate transcription. This process is regulated by VVD, a small photoreceptor that disrupts the interaction between WCCs and leads to a reduction in transcription after long exposures to light. The N. crassa RCO-1/RCM-1 repressor complex is the homolog of the Tup1-Ssn6 repressor complex in yeast, and its absence modifies photoadaptation. We show that the absence of the RCO-1/RCM-1 repressor complex leads to several alterations in transcription that are gene-specific: an increase in the accumulation of mRNAs in the dark, a repression of transcription, and a derepression of transcription after long exposures to light. The absence of the RCO-1/RCM-1 repressor complex leads to lower VVD levels that are available for the regulation of the activity of the WCC. The reduction in the amount of VVD results in increased WCC binding to the promoters of light-regulated genes in the dark and after long exposures to light, leading to the modification of photoadaptation that has been observed in rco-1 and rcm-1 mutants. Our results show that the photoadaptation phenotype of mutants in the RCO-1/RCM-1 repressor complex is, at least in part, an indirect consequence of the reduction of vvd transcription, and the resulting modification in the regulation of transcription by the WCC.

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator.

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Cupertino, Fernanda Barbosa; Virgilio, Stela; Freitas, Fernanda Zanolli; Candido, Thiago de Souza; Bertolini, Maria Célia

2015-04-01

The transcription factor CreA/Mig1/CRE-1 is a repressor protein that regulates the use of alternative carbon sources via a mechanism known as Carbon Catabolite Repression (CCR). In Saccharomyces cerevisiae, Mig1 recruits the complex Ssn6-Tup1, the Neurospora crassa RCM-1 and RCO-1 orthologous proteins, respectively, to bind to promoters of glucose-repressible genes. We have been studying the regulation of glycogen metabolism in N. crassa and the identification of the RCO-1 corepressor as a regulator led us to investigate the regulatory role of CRE-1 in this process. Glycogen content is misregulated in the rco-1(KO), rcm-1(RIP) and cre-1(KO) strains, and the glycogen synthase phosphorylation is decreased in all strains, showing that CRE-1, RCO-1 and RCM-1 proteins are involved in glycogen accumulation and in the regulation of GSN activity by phosphorylation. We also confirmed the regulatory role of CRE-1 in CCR and its nuclear localization under repressing condition in N. crassa. The expression of all glycogenic genes is misregulated in the cre-1(KO) strain, suggesting that CRE-1 also controls glycogen metabolism by regulating gene expression. The existence of a high number of the Aspergillus nidulans CreA motif (5'-SYGGRG-3') in the glycogenic gene promoters led us to analyze the binding of CRE-1 to some DNA motifs both in vitro by DNA gel shift and in vivo by ChIP-qPCR analysis. CRE-1 bound in vivo to all motifs analyzed demonstrating that it down-regulates glycogen metabolism by controlling gene expression and GSN phosphorylation. Copyright © 2015 Elsevier Inc. All rights reserved.

The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

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Carla J Eaton

Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

Regulation of Neurospora Catalase-3 by global heterochromatin formation and its proximal heterochromatin region.

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Wang, Yajun; Dong, Qing; Ding, Zhaolan; Gai, Kexin; Han, Xiaoyun; Kaleri, Farah Naz; He, Qun; Wang, Ying

2016-10-01

Catalase-3 (CAT-3) constitutes the main catalase activity in growing hyphae of Neurospora crassa, and its activity increases during exponential growth or is induced under different stress conditions. Although extensive progress has been made to identify catalase regulators, the regulation mechanism of CAT-3 at the chromatin level still remains unclear. Here, we aim at investigating the molecular regulation mechanisms of cat-3 at the chromatin level. We found that CAT-3 protein levels increased in mutants defective in proper global heterochromatin formation. Bioinformatics analysis identified a 5-kb AT-rich sequence adjacent to the cat-3 promoter as a heterochromatin region because of its enrichment of H3K9me3 and HP1. Expression of CAT-3 was induced by H 2 O 2 treatment in wild-type and such change occurred along with the accumulation of histone H3 acetylation at 5-kb heterochromatin boundaries and cat-3 locus, but without alteration of its H3K9me3 repressive modification. Moreover, disruption of 5-kb heterochromatin region results in elevated cat-3 expression, and higher levels of cat-3 expression were promoted by the combination with global heterochromatin defective mutants. Interestingly, the molecular weight and activity bands of CAT-3 protein are different in heterochromatin defective mutants compared with those in wild-type, suggesting that its N-terminal processing and modification may be altered. Our study indicates that the local chromatin structure creates a heterochromatin repressive environment to repress nearby gene expression. Copyright © 2016 Elsevier Inc. All rights reserved.

An F-actin-depleted zone is present at the hyphal tip of invasive hyphae of Neurospora crassa.

Science.gov (United States)

Suei, S; Garrill, A

2008-01-01

The distribution of filamentous actin (F-actin) in invasive and noninvasive hyphae of the ascomycete Neurospora crassa was investigated. Eighty six percent of noninvasive hyphae had F-actin in the tip region compared to only 9% of invasive hyphae. The remaining 91% of the invasive hyphae had no obvious tip high concentration of F-actin staining; instead they had an F-actin-depleted zone in this region, although some F-actin, possibly associated with the Spitzenkörper, remained at the tip. The size of the F-actin-depleted zone in invasive hyphae increased with an increase in agar concentration. The membrane stain FM 4-64 reveals a slightly larger accumulation of vesicles at the tips of invasive hyphae relative to noninvasive hyphae, although this difference is unlikely to be sufficient to account for the exclusion of F-actin from the depleted zone. Antibodies raised against the actin filament-severing protein cofilin from both yeast and human cells localize to the tips of invasive hyphae. The human cofilin antibody shows a more random distribution in noninvasive hyphae locating primarily at the hyphal periphery but with some diffuse cytoplasmic staining. This antibody also identifies a single band at 21 kDa in immunoblots of whole hyphal fractions. These data suggest that a protein with epitopic similarity to cofilin may function in F-actin dynamics that underlie invasive growth. The F-actin-depleted zone may play a role in the regulation of tip yielding to turgor pressure, thus increasing the protrusive force necessary for invasive growth.

Fulltext PDF

Indian Academy of Sciences (India)

Unknown

2001-08-07

Aug 7, 2001 ... (RIP) in crosses with the wild-isolated Neurospora crassa strains. Sugartown and ..... and complementation by proteins chimeric for human lamin B receptor sequences. ... α-tomatine to sensitivity to the pea phytoalexin pisatin.

Identification of a deoxyribonuclease controlled by the rad52 gene of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Chow, T.Y.K.; Resnick, M.A.

1983-01-01

We have examined deoxyribonuclease levels in extracts of wild-type and rad52 mutants and have observed no significant differences. However, major differences were observed when we employed anti-serum raised against a purified single strand DNA-binding endoexonuclease from Neurospora crassa. As much as sixty percent of the alkaline deoxyribonuclease in wild-type extracts exhibited immunocrossreactivity, whereas none was found in extracts from rad52 strains. This DNase activity was also followed through meiosis; maximum activity was observed in wild-type cells, at a time corresponding to an early stage of premeiotic DNA-synthesis and commitment to recombination. 14 references, 4 figures, 1 table

Comparative live-cell imaging analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa reveal novel features of the filamentous fungal polarisome.

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Alexander Lichius

Full Text Available A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk, whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture.

Partial characterization of GTP-binding proteins in Neurospora

International Nuclear Information System (INIS)

Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

1987-01-01

Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

Engineering Neurospora crassa for cellobionate production directly from cellulose without any enzyme addition

Science.gov (United States)

A previously engineered strain of N. crassa F5'ace-1'cre-1'ndvB) with six out of seven ß-glucosidase (bgl) genes, two transcription factors (cre1 and ace-1) and cellobionate phosphorylase (ndvB) deleted was able to produce cellobiose and cellobionate directly from cellulose without the addition of e...

The Aspergillus uvsH gene encodes a product homologous to yeast RAD18 and Neurospora UVS-2.

Science.gov (United States)

Yoon, J H; Lee, B J; Kang, H S

1995-07-28

The uvsH DNA repair gene of Aspergillus nidulans has been cloned by complementation of the uvsH77 mutation with a cosmid library containing genomic DNA inserts from a wild-type strain. Methylmethane sulfonate (MMS)-resistant transformants were obtained on medium containing 0.01% MMS, to which uvsH mutants exhibit high sensitivity. Retransformation of uvsH77 mutants with the rescued cosmids from the MMS-resistant transformants resulted in restoration of both UV and MMS resistance to wild-type levels. Nucleotide sequence analysis of the genomic DNA and cDNA of the uvsH gene shows that it has an open reading frame (ORF) of 1329 bp, interrupted by two introns of 51 and 61 bp. A 2.4 kb transcript of the uvsH gene was detected by Northern blot analysis. Primer extension analysis revealed that transcription starts at 31 bp upstream from the translation initiation codon. This gene encodes a predicted polypeptide of 443 amino acids, which has two unique zinc finger motifs. The proposed polypeptide displays 39% identity to the Neurospora crassa UVS-2 protein and 24% identity to the Saccharomyces cerevisiae RAD18 protein. The sequence similarity is particularly high in three domains. One zinc finger (RING finger) motif is located in the first domain close to the N-terminus. The other zinc finger motif is in the second domain. In the third domain, the mutation sites in both the uvsH77 and uvsH304 alleles were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

Science.gov (United States)

Honda, Shinji; Bicocca, Vincent T.; Gessaman, Jordan D.; Rountree, Michael R.; Yokoyama, Ayumi; Yu, Eun Y.; Selker, Jeanne M. L.; Selker, Eric U.

2016-01-01

DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation. PMID:27681634

Enabling a community to dissect an organism: overview of the Neurospora functional genomics project.

Science.gov (United States)

Dunlap, Jay C; Borkovich, Katherine A; Henn, Matthew R; Turner, Gloria E; Sachs, Matthew S; Glass, N Louise; McCluskey, Kevin; Plamann, Michael; Galagan, James E; Birren, Bruce W; Weiss, Richard L; Townsend, Jeffrey P; Loros, Jennifer J; Nelson, Mary Anne; Lambreghts, Randy; Colot, Hildur V; Park, Gyungsoon; Collopy, Patrick; Ringelberg, Carol; Crew, Christopher; Litvinkova, Liubov; DeCaprio, Dave; Hood, Heather M; Curilla, Susan; Shi, Mi; Crawford, Matthew; Koerhsen, Michael; Montgomery, Phil; Larson, Lisa; Pearson, Matthew; Kasuga, Takao; Tian, Chaoguang; Baştürkmen, Meray; Altamirano, Lorena; Xu, Junhuan

2007-01-01

A consortium of investigators is engaged in a functional genomics project centered on the filamentous fungus Neurospora, with an eye to opening up the functional genomic analysis of all the filamentous fungi. The overall goal of the four interdependent projects in this effort is to accomplish functional genomics, annotation, and expression analyses of Neurospora crassa, a filamentous fungus that is an established model for the assemblage of over 250,000 species of non yeast fungi. Building from the completely sequenced 43-Mb Neurospora genome, Project 1 is pursuing the systematic disruption of genes through targeted gene replacements, phenotypic analysis of mutant strains, and their distribution to the scientific community at large. Project 2, through a primary focus in Annotation and Bioinformatics, has developed a platform for electronically capturing community feedback and data about the existing annotation, while building and maintaining a database to capture and display information about phenotypes. Oligonucleotide-based microarrays created in Project 3 are being used to collect baseline expression data for the nearly 11,000 distinguishable transcripts in Neurospora under various conditions of growth and development, and eventually to begin to analyze the global effects of loss of novel genes in strains created by Project 1. cDNA libraries generated in Project 4 document the overall complexity of expressed sequences in Neurospora, including alternative splicing alternative promoters and antisense transcripts. In addition, these studies have driven the assembly of an SNP map presently populated by nearly 300 markers that will greatly accelerate the positional cloning of genes.

The NDR kinase scaffold HYM1/MO25 is essential for MAK2 map kinase signaling in Neurospora crassa.

Directory of Open Access Journals (Sweden)

Anne Dettmann

2012-09-01

Full Text Available Cell communication is essential for eukaryotic development, but our knowledge of molecules and mechanisms required for intercellular communication is fragmentary. In particular, the connection between signal sensing and regulation of cell polarity is poorly understood. In the filamentous ascomycete Neurospora crassa, germinating spores mutually attract each other and subsequently fuse. During these tropic interactions, the two communicating cells rapidly alternate between two different physiological states, probably associated with signal delivery and response. The MAK2 MAP kinase cascade mediates cell-cell signaling. Here, we show that the conserved scaffolding protein HYM1/MO25 controls the cell shape-regulating NDR kinase module as well as the signal-receiving MAP kinase cascade. HYM1 functions as an integral part of the COT1 NDR kinase complex to regulate the interaction with its upstream kinase POD6 and thereby COT1 activity. In addition, HYM1 interacts with NRC1, MEK2, and MAK2, the three kinases of the MAK2 MAP kinase cascade, and co-localizes with MAK2 at the apex of growing cells. During cell fusion, the three kinases of the MAP kinase module as well as HYM1 are recruited to the point of cell-cell contact. hym-1 mutants phenocopy all defects observed for MAK2 pathway mutants by abolishing MAK2 activity. An NRC1-MEK2 fusion protein reconstitutes MAK2 signaling in hym-1, while constitutive activation of NRC1 and MEK2 does not. These data identify HYM1 as a novel regulator of the NRC1-MEK2-MAK2 pathway, which may coordinate NDR and MAP kinase signaling during cell polarity and intercellular communication.

Journal of Genetics | Indian Academy of Sciences

Indian Academy of Sciences (India)

Evidence for dominant suppression of repeat-induced point mutation (RIP) in crosses with the wild-isolated Neurospora crassa strains Sugartown and Adiopodoume-7 · Felicite K. Noubissi K. Aparna Kevin McCluskey Durgadas P. Kasbekar · More Details Abstract Fulltext PDF. A convenient assay to score repeat-induced ...

Bright to dim oscillatory response of the Neurospora circadian oscillator.

Science.gov (United States)

Gooch, Van D; Johnson, Alicia E; Larrondo, Luis F; Loros, Jennifer J; Dunlap, Jay C

2014-02-01

The fungus Neurospora crassa constitutes an important model system extensively used in chronobiology. Several studies have addressed how environmental cues, such as light, can reset or synchronize a circadian system. By means of an optimized firefly luciferase reporter gene and a controllable lighting system, we show that Neurospora can display molecular circadian rhythms in dim light when cultures receive bright light prior to entering dim light conditions. We refer to this behavior as the "bright to dim oscillatory response" (BDOR). The bright light treatment can be applied up to 76 h prior to dim exposure, and it can be as short as 15 min in duration. We have characterized this response in respect to the duration of the light pulse, the time of the light pulse before dim, the intensity of dim light, and the oscillation dynamics in dim light. Although the molecular mechanism that drives the BDOR remains obscure, these findings suggest that a long-term memory of bright light exists as part of the circadian molecular components. It is important to consider the ecological significance of such dim light responses in respect to how organisms naturally maintain their timing mechanism in moonlight.

nuvA, an Aspergillus nidulans gene involved in DNA repair and recombination, is a homologue of Saccharomyces cerevisiae RAD18 and Neurospora crassa uvs-2.

Science.gov (United States)

Iwanejko, L; Cotton, C; Jones, G; Tomsett, B; Strike, P

1996-03-01

A 40 kb genomic clone and 2.3 kb EcoRI subclone that rescued the DNA repair and recombination defects of the Aspergillus nidulans nuvA11 mutant were isolated and the subclone sequenced. The subclone hybridized to a cosmid in a chromosome-specific library confirming the assignment of nuvA to linkage group IV and indicating its closeness to bimD. Amplification by PCR clarified the relative positions of nuvA and bimD. A region identified within the subclone, encoding a C3HC4 zinc finger motif, was used as a probe to retrieve a cDNA clone. Sequencing of this clone showed that the nuvA gene has an ORF of 1329 bp with two introns of 51 bp and 60 bp. Expression of nuvA appears to be extremely low. The putative NUVA polypeptide has two zinc finger motifs, a molecular mass of 48906 Da and has 39% identity with the Neurospora crassa uvs-2 and 25% identity with the Saccharomyces cerevisiae RAD18 translation products. Although mutations in nuvA, uvs-2 and RAD18 produce similar phenotypes, only the nuvA11 mutation affects meiotic recombination. A role for nuvA in both DNA repair and genetic recombination is proposed.

Neurospora circadian rhythms in space - A reexamination of the endogenous-exogenous question

Science.gov (United States)

Sulzman, F. M.; Ellman, D.; Wassmer, G.; Fuller, C. A.; Moore-Ede, M.

1984-01-01

To test the functioning of circadian rhythms removed from periodicities of the earth's 24-hour rotation, the conidiation rhythm of the fungus Neurospora crassa was monitored in constant darkness during spaceflight. The free-running period of the rhythm was the same in space as on the earth, but there was a marked reduction in the clarity of the rhythm, and apparent arrhythmicity in some tubes. At the current stage of analysis of the results there is insufficient evidence to determine whether the effect seen in space was related to removal from 24-hour periodicities and whether the circadian timekeeping mechanism, or merely its expression, was affected.

Mass flow and velocity profiles in Neurospora hyphae: partial plug flow dominates intra-hyphal transport.

Science.gov (United States)

Abadeh, Aryan; Lew, Roger R

2013-11-01

Movement of nuclei, mitochondria and vacuoles through hyphal trunks of Neurospora crassa were vector-mapped using fluorescent markers and green fluorescent protein tags. The vectorial movements of all three were strongly correlated, indicating the central role of mass (bulk) flow in cytoplasm movements in N. crassa. Profiles of velocity versus distance from the hyphal wall did not match the parabolic shape predicted by the ideal Hagen-Poiseuille model of flow at low Reynolds number. Instead, the profiles were flat, consistent with a model of partial plug flow due to the high concentration of organelles in the flowing cytosol. The intra-hyphal pressure gradients were manipulated by localized external osmotic treatments to demonstrate the dependence of velocity (and direction) on pressure gradients within the hyphae. The data support the concept that mass transport, driven by pressure gradients, dominates intra-hyphal transport. The transport occurs by partial plug flow due to the organelles in the cytosol.

DNA methylation inhibits expression and transposition of the Neurospora Tad retrotransposon.

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Zhou, Y; Cambareri, E B; Kinsey, J A

2001-06-01

Tad is a LINE-like retrotransposon of the filamentous fungus Neurospora crassa. We have analyzed both expression and transposition of this element using strains with a single copy of Tad located in the 5' noncoding sequences of the am (glutamate dehydrogenase) gene. Tad in this position has been shown to carry a de novo cytosine methylation signal which causes reversible methylation of both Tad and am upstream sequences. Here we find that methylation of the Tad sequences inhibits both Tad expression and transposition. This inhibition can be relieved by the use of 5-azacytidine, a drug which reduces cytosine methylation, or by placing the Tad/am sequences in a dim-2 genetic background.

Neurospora COP9 signalosome integrity plays major roles for hyphal growth, conidial development, and circadian function.

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Zhipeng Zhou

Full Text Available The COP9 signalosome (CSN is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EX(nHXHX(10D of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCF(FWD-1 complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa.

VIB1, a link between glucose signaling and carbon catabolite repression, is essential for plant cell wall degradation by Neurospora crassa.

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Yi Xiong

2014-08-01

Full Text Available Filamentous fungi that thrive on plant biomass are the major producers of hydrolytic enzymes used to decompose lignocellulose for biofuel production. Although induction of cellulases is regulated at the transcriptional level, how filamentous fungi sense and signal carbon-limited conditions to coordinate cell metabolism and regulate cellulolytic enzyme production is not well characterized. By screening a transcription factor deletion set in the filamentous fungus Neurospora crassa for mutants unable to grow on cellulosic materials, we identified a role for the transcription factor, VIB1, as essential for cellulose utilization. VIB1 does not directly regulate hydrolytic enzyme gene expression or function in cellulosic inducer signaling/processing, but affects the expression level of an essential regulator of hydrolytic enzyme genes, CLR2. Transcriptional profiling of a Δvib-1 mutant suggests that it has an improper expression of genes functioning in metabolism and energy and a deregulation of carbon catabolite repression (CCR. By characterizing new genes, we demonstrate that the transcription factor, COL26, is critical for intracellular glucose sensing/metabolism and plays a role in CCR by negatively regulating cre-1 expression. Deletion of the major player in CCR, cre-1, or a deletion of col-26, did not rescue the growth of Δvib-1 on cellulose. However, the synergistic effect of the Δcre-1; Δcol-26 mutations circumvented the requirement of VIB1 for cellulase gene expression, enzyme secretion and cellulose deconstruction. Our findings support a function of VIB1 in repressing both glucose signaling and CCR under carbon-limited conditions, thus enabling a proper cellular response for plant biomass deconstruction and utilization.

The mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma represents a model for early evolution of sex chromosomes.

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Audrius Menkis

2008-03-01

Full Text Available We combined gene divergence data, classical genetics, and phylogenetics to study the evolution of the mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma. In this species, a large non-recombining region of the mating-type chromosome is associated with a unique fungal life cycle where self-fertility is enforced by maintenance of a constant state of heterokaryosis. Sequence divergence between alleles of 35 genes from the two single mating-type component strains (i.e. the homokaryotic mat A or mat a-strains, derived from one N. tetrasperma heterokaryon (mat A+mat a, was analyzed. By this approach we were able to identify the boundaries and size of the non-recombining region, and reveal insight into the history of recombination cessation. The non-recombining region covers almost 7 Mbp, over 75% of the chromosome, and we hypothesize that the evolution of the mating-type chromosome in this lineage involved two successive events. The first event was contemporaneous with the split of N. tetrasperma from a common ancestor with its outcrossing relative N. crassa and suppressed recombination over at least 6.6 Mbp, and the second was confined to a smaller region in which recombination ceased more recently. In spite of the early origin of the first "evolutionary stratum", genealogies of five genes from strains belonging to an additional N. tetrasperma lineage indicate independent initiations of suppressed recombination in different phylogenetic lineages. This study highlights the shared features between the sex chromosomes found in the animal and plant kingdoms and the fungal mating-type chromosome, despite fungi having no separate sexes. As is often found in sex chromosomes of plants and animals, recombination suppression of the mating-type chromosome of N. tetrasperma involved more than one evolutionary event, covers the majority of the mating-type chromosome and is flanked by distal regions with obligate crossovers.

Neurospora ribosomal DNA sequences are indistinguishable within cell types but distinguishable among heterothallic species

International Nuclear Information System (INIS)

Chambers, C.; Dutta, S.K.

1983-01-01

High molecular nuclear DNAs were isolated from three developmental cell types of N. crassa: conidia, mycelia and germinated conidia, and from mycelial cells of two other heterothallic species, N. intermedia and N. sitophila. These nuclear DNAs were treated with several restriction enzymes: EcoR1, Bam H1, Hind III, Hinc II, Bgl II, Sma I and Pst 1. All seven restriction enzymes were tested on 0.7% agarose gels. EcoR1, Hind III, Pst 1, and Hinc II showed band differences among the species, but not among the cell types. Southern blot transfers of restricted DNA gels were then hybridized with 32 P-labelled pMF2 rDNAs (probe). This later DNA was prepared from N. crassa rDNA cloned into pBR322 plasmid, obtained from Dr. Robert Metzenberg of the University of Wisconsin. Autoradiograms of these hybrids between southern blots and probe DNA revealed similar rDNA band patterns confirming the observations on restriction gels. In the case of EcoR1 restriction analysis there were differences in fragments on 0.7% agarose gel, but after hybridization of southern blots no differences in band patterns were seen in autoradiograms. This raises the question whether the background bands were all of rDNA sequences. These studies are being continued using ITS (internal transcribed spacer) sequences of N. crassa rDNAs cloned in pBR322 plasmid

Traffic of chitin synthase 1 (CHS-1) to the Spitzenkörper and developing septa in hyphae of Neurospora crassa: actin dependence and evidence of distinct microvesicle populations.

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Sánchez-León, Eddy; Verdín, Jorge; Freitag, Michael; Roberson, Robert W; Bartnicki-Garcia, Salomon; Riquelme, Meritxell

2011-05-01

We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1-green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1-GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1-GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1-GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crass

International Nuclear Information System (INIS)

De Serres, F.J.

1990-01-01

More extensive complementation tests than those performed initially on a series of 832 X-ray-induced specific-locus mutations in the adenine-4 (ad-3) region of a two-component heterokaryon (H-12) of Neurospora crassa showed that unexpectedly high frequencies of specific-locus mutations in the ad-3 region have additional, but separate, sites of recessive lethal damage in the immediately adjacent genetic regions. In the present paper, X-ray-induced irreparable ad-3 mutants of the folowing genotypes and numbers (ad-3A ad-3B, ad-3A ad-3B nic-2, and ad-3B nic-2) have also subjected to the same genetic fine structure analysis. These experiments, in the previous and present papers, were designed to determine the extent of the functional inactivation in the ad-3 and immediately adjacent genetic regions in individual mutants classified as presumptive multilocus deletions or multiplelocus mutations. The data in the present paper have shown that in Neurospora crassa most X-ray-induced irreparable mutants of genotype ad-3A ad-3B, ad-3A ad-3B nic-2, and ad-3 nic-2 map as a series of overlapping multilocus deletions. In addition, genetic fine structure analysis has shown that some of the mutants classified, initially, as multilocus deletions, are actually multiple-locus mutations: multilocus deletions with closely linked, and separate, sites of recessive lethal damage with a wide variety of genotyes. Combining data from the present experiments with previously published date, the frequency of multiple-locus mutations among X-ray-induced gene/point mutations and multilocus deletions in the ad-3 region is 6.2%. (author). 27 refs.; 4 figs.; 7 tab

Mating-type genes from the homothallic fungus Sordaria macrospora are functionally expressed in a heterothallic ascomycete.

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Pöggeler, S; Risch, S; Kück, U; Osiewacz, H D

1997-10-01

Homokaryons from the homothallic ascomycte Sordaria macrospora are able to enter the sexual pathway and to form fertile fruiting bodies. To analyze the molecular basis of homothallism and to elucidate the role of mating-products during fruiting body development, we cloned and sequenced the entire S. macrospora mating-type locus. Comparison of the Sordaria mating-type locus with mating-type idiomorphs from the heterothallic ascomycetes Neurospora crassa and Podospora anserina revealed that sequences from both idiomorphs (A/a and mat-/mat+, respectively) are contiguous in S. macrospora. DNA sequencing of the S. macrospora mating-type region allowed the identification of four open reading frames (ORFs), which were termed Smt-a1, SmtA-1, SmtA-2 and SmtA-3. While Smt-a1, SmtA-1, and SmtA-2 show strong sequence similarities with the corresponding N. crassa mating-type ORFs, SmtA-3 has a chimeric character. It comprises sequences that are similar to the A and a mating-type idiomorph from N. crassa. To determine functionality of the S. macrospora mating-type genes, we show that all ORFs are transcriptionally expressed. Furthermore, we transformed the S. macrospora mating-type genes into mat- and mat+ strains of the closely related heterothallic fungus P. anserina. The transformation experiments show that mating-type genes from S. macrospora induce fruiting body formation in P. anserina.

Analysis of clock-regulated genes in Neurospora reveals widespread posttranscriptional control of metabolic potential

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Hurley, Jennifer M.; Dasgupta, Arko; Emerson, Jillian M.; Zhou, Xiaoying; Ringelberg, Carol S.; Knabe, Nicole; Lipzen, Anna M.; Lindquist, Erika A.; Daum, Christopher G.; Barry, Kerrie W.; Grigoriev, Igor V.; Smith, Kristina M.; Galagan, James E.; Bell-Pedersen, Deborah; Freitag, Michael; Cheng, Chao; Loros, Jennifer J.; Dunlap, Jay C.

2014-01-01

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation–based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter–luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level. PMID:25362047

Reorganization of plasma membrane lipid domains during conidial germination.

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Santos, Filipa C; Fernandes, Andreia S; Antunes, Catarina A C; Moreira, Filipe P; Videira, Arnaldo; Marinho, H Susana; de Almeida, Rodrigo F M

2017-02-01

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14.

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Jonkers, Wilfried; Fischer, Monika S; Do, Hung P; Starr, Trevor L; Glass, N Louise

2016-05-01

In filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). Anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. In Neurospora crassa, fusion of genetically identical germlings is a highly dynamic and regulated process that requires components of a MAP kinase signal transduction pathway. The kinase pathway components (NRC-1, MEK-2 and MAK-2) and the scaffold protein HAM-5 are recruited to hyphae and germling tips undergoing chemotropic interactions. The MAK-2/HAM-5 protein complex shows dynamic oscillation to hyphae/germling tips during chemotropic interactions, and which is out-of-phase to the dynamic localization of SOFT, which is a scaffold protein for components of the cell wall integrity MAP kinase pathway. In this study, we functionally characterize HAM-5 by generating ham-5 truncation constructs and show that the N-terminal half of HAM-5 was essential for function. This region is required for MAK-2 and MEK-2 interaction and for correct cellular localization of HAM-5 to "fusion puncta." The localization of HAM-5 to puncta was not perturbed in 21 different fusion mutants, nor did these puncta colocalize with components of the secretory pathway. We also identified HAM-14 as a novel member of the HAM-5/MAK-2 pathway by mining MAK-2 phosphoproteomics data. HAM-14 was essential for germling fusion, but not for hyphal fusion. Colocalization and coimmunoprecipitation data indicate that HAM-14 interacts with MAK-2 and MEK-2 and may be involved in recruiting MAK-2 (and MEK-2) to complexes containing HAM-5. Copyright © 2016 by the Genetics Society of America.

A UV-induced mutation in neurospora that affects translational regulation in response to arginine

International Nuclear Information System (INIS)

Freitag, M.; Dighde, N.; Sachs, M.S.

1996-01-01

The Neurospora crassa arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. The levels of arg-2 mRNA and mRNA translation are negatively regulated by arginine. An upstream open reading frame (uORF) in the transcript's 5' region has been implicated in arginine-specific control. An arg-2-hph fusion gene encoding hygromycin phosphotransferase conferred arginine-regulated resistance to hygromycin when introduced into N. crassa. We used an arg-2-hph strain to select for UV-induced mutants that grew in the presence of hygromycin and arginine, and we isolated 46 mutants that had either of two phenotypes. One phenotype indicated altered expression of both arg-2-hph and arg-2 genes; the other, altered expression of arg-2-hph but not arg-2. One of the latter mutations, which was genetically closely linked to arg-2-hph, was recovered from the 5' region of the arg-2-hph gene using PCR Sequence analyses and transformation experiments revealed a mutation at uORF codon 12 (Asp to Asn) that abrogated negative regulation. Examination of the distribution of ribosomes on arg-2-hph transcripts showed that loss of regulation had a translational component, indicating the uORF sequence was important for Arg-specific translational control. Comparisons with other uORFs suggest common elements in translational control mechanisms

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Indian Academy of Sciences (India)

PRAKASH KUMAR

natural populations of Neurospora are prevailingly sexual, not clonal (Perkins and Turner ... attesting to conidia being a highly palatable food for mites. .... Lauter F-R and Russo V E A 1989 Fast light regulated genes of Neurospora crassa;.

Potential production of carotenoids from Neurospora

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SRI PRIATNI

2014-05-01

Full Text Available Priatni S. 2014. Review: Potential production of carotenoids from Neurospora. Nusantara Bioscience 6: 63-68. Carotenoids are abundant and widely distributed in plants, animals and microorganisms. Commercial use of carotenoids competes between microorganisms and synthetic manufacture. Carotenoids production can be increased by improving the efficiency of carotenoid synthesis in microbes. Some of the cultural and environmental stimulants are positively affecting the carotenoid content of carotenogenic strains such as Neurospora. Neurospora is a fungus that exhibits the formation of spores and conidia, the part of the cell for carotenoids biosynthesis. The Indonesian traditional fermented food, red peanut cake or oncom, especially in West Java, is produced from legume residues of Neurospora sp. This fungus has been isolated and identified as Neurospora intermedia. In order to apply this pigment for food and cosmetic colorants, encapsulation techniques of carotenoids have been developed to improve its solubility and stability.

X-ray induced specific locus mutations in the ad-3 region of two-component heterokaryons of neurospora crassa

International Nuclear Information System (INIS)

Serres, F.J. de; Miller, I.R.

1988-01-01

The basis for the reduced growth rates of heterokaryons between strains carrying nonallelic combinations of gene/point mutations and multilocus deletion mutations has been investigated by a simple genetic test. The growth rates of forced 2-component heterokaryons (dikaryons) between multilocus deletion mutations were compared with forced 3-component heterokaryons (trikaryons) containing an ad-3A R ad-3B R double mutant as their third component. Since the third component has no genetic damage at other loci immediately adjacent to the ad-3A or ad-3B locus, the growth rate on minimal medium depends on the functional activity of the unaltered ad-3A and ad-3B loci in the first two components. Tests in the present experiments have shown the ad-3 IR mutations result not only in inactivation of the ad-3 loci by multilocus deletion byt also, in many cases, in partial gene inactivation by an unknown mechanisms at other loci in the immediately adacent regions. The heterozygous effects observed in our present experiments with multilocus deletions in Neurospora can be explained either by a spreading-type position effect of the type found by others in Drosophila, mice, Oenothera and Aspergillus or by undetected genetic damage in the immediately adjacent genetic regions. (author). 18 refs.; 8 figs.; 2 tabs

Discovering functions of unannotated genes from a transcriptome survey of wild fungal isolates.

Science.gov (United States)

Ellison, Christopher E; Kowbel, David; Glass, N Louise; Taylor, John W; Brem, Rachel B

2014-04-01

Most fungal genomes are poorly annotated, and many fungal traits of industrial and biomedical relevance are not well suited to classical genetic screens. Assigning genes to phenotypes on a genomic scale thus remains an urgent need in the field. We developed an approach to infer gene function from expression profiles of wild fungal isolates, and we applied our strategy to the filamentous fungus Neurospora crassa. Using transcriptome measurements in 70 strains from two well-defined clades of this microbe, we first identified 2,247 cases in which the expression of an unannotated gene rose and fell across N. crassa strains in parallel with the expression of well-characterized genes. We then used image analysis of hyphal morphologies, quantitative growth assays, and expression profiling to test the functions of four genes predicted from our population analyses. The results revealed two factors that influenced regulation of metabolism of nonpreferred carbon and nitrogen sources, a gene that governed hyphal architecture, and a gene that mediated amino acid starvation resistance. These findings validate the power of our population-transcriptomic approach for inference of novel gene function, and we suggest that this strategy will be of broad utility for genome-scale annotation in many fungal systems. IMPORTANCE Some fungal species cause deadly infections in humans or crop plants, and other fungi are workhorses of industrial chemistry, including the production of biofuels. Advances in medical and industrial mycology require an understanding of the genes that control fungal traits. We developed a method to infer functions of uncharacterized genes by observing correlated expression of their mRNAs with those of known genes across wild fungal isolates. We applied this strategy to a filamentous fungus and predicted functions for thousands of unknown genes. In four cases, we experimentally validated the predictions from our method, discovering novel genes involved in the

The transcriptional response to the inactivation of the PaMpk1 and PaMpk2 MAP kinase pathways in Podospora anserina.

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Bidard, Frédérique; Coppin, Evelyne; Silar, Philippe

2012-08-01

Transcription pattern during mycelium growth of Podospora anserina was assayed by microarray analysis in wild type and in mutants affected in the MAP kinase genes PaMpk1 and PaMpk2 and in the NADPH oxidase gene PaNox1. 15% of the genes have their expression modified by a factor two or more as growth proceeds in wild type. The genes whose expression is modified during growth in P. anserina are either not conserved or differently regulated in Neurospora crassa and Aspergillus niger, two fungi for which transcriptome data during growth are available. The P. anserina mutants display a similar alteration of their transcriptome profile, with nearly 1000 genes affected similarly in the three mutants, accounting for their similar growth phenotypes. Yet, each mutant has its specific set of modified transcripts, in line with particular phenotypes exhibited by each mutant. Again, there is limited conservation during evolution of the genes regulated at the transcription level by MAP kinases, as indicated by the comparison the P. anserina data, with those of Aspergillus fumigatus and N. crassa, two fungi for which gene expression data are available for mutants of the MAPK pathways. Among the genes regulated in wild type and affected in the mutants, those involved in carbohydrate and secondary metabolisms appear prominent. The vast majority of the genes differentially expressed are of unknown function. Availability of their transcription profile at various stages of development should help to decipher their role in fungal physiology and development. Copyright © 2012 Elsevier Inc. All rights reserved.

A factor in a wild isolated Neurospora crassa strain enables a ...

Indian Academy of Sciences (India)

in part, by a gene-silencing process called meiotic silencing by unpaired DNA, a presumed RNAi-mediated elimination of the transcripts of any gene not properly paired with a homolog in meiosis (Aramayo and Metzenberg 1996; Shiu et al. 2001, 2006). The semi-dominant Sad-1 suppressor of meiotic silencing was used to ...

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. Pt. 6

International Nuclear Information System (INIS)

De Serres, F.J.; Inoue, H.; Schuepbach, M.E.

1983-01-01

Genetic characterization of ad-3B mutants induced in wild-type and UV-sensitive strains has revealed qualitative differences between the spectra of genetic alterations at the molecular level. Ad-3B mutants induced in the two nucleotide excision-repair-deficient strains upr-1 and uvs-2 had significantly lower frequencies of nonpolarized complementation patterns and higher frequencies of noncomplementing mutants than ad-3B mutants induced in the wild-type strain in samples induced by either UV, #betta#-rays, 4NQO or MNNG. In these same samples ad-3B mutants induced in uvs-4, uvs-5 or uvs-6 did not differ significantly from those induced in the wild-type strain. After ICR-170 treatment, ad-3B mutants induced in the UV-sensitive strains did not differ significantly from those induced in wild-type. The comparisons in the present and previous studies demonstrate that the process of mutation-induction in the ad-3 region is under the control of other loci that not only alter mutant recovery quantitatively but also qualitatively. These data have important implications for comparative chemical mutagenesis, since the spectrum of genetic alterations produced by a given agent can be modified markedly as a result of defects in DNA repair. (orig./AJ)

A circadian rhythm regulating hyphal melanization in Cercospora kikuchii.

Science.gov (United States)

Bluhm, Burton H; Burnham, A Michele; Dunkle, Larry D

2010-01-01

Many metabolic and developmental processes in fungi are controlled by biological rhythms. Circadian rhythms approximate a daily (24 h) cycle and have been thoroughly studied in the model fungus, Neurospora crassa. However relatively few examples of true circadian rhythms have been documented among other filamentous fungi. In this study we describe a circadian rhythm underlying hyphal melanization in Cercospora kikuchii, an important pathogen of soybean. After growth in light or light : dark cycles, colonies transferred to darkness produced zonate bands of melanized hyphae interspersed with bands of hyaline hyphae. Rhythmic production of bands was remarkably persistent in the absence of external cues, lasting at least 7 d after transfer to darkness, and was compensated over a range of temperatures. As in N. crassa, blue light but not red light was sufficient to entrain the circadian rhythm in C. kikuchii, and a putative ortholog of white collar-1, one of the genes required for light responses in N. crassa, was identified in C. kikuchii. Circadian regulation of melanization is conserved in other members of the genus: Similar rhythms were identified in another field isolate of C. kikuchii as well as field isolates of C. beticola and C. sorghi, but not in wild-type strains of C. zeae-maydis or C. zeina. This report represents the first documented circadian rhythm among Dothideomycete fungi and provides a new opportunity to dissect the molecular basis of circadian rhythms among filamentous fungi.

Interaction between mating-type proteins from the homothallic fungus Sordaria macrospora.

Science.gov (United States)

Jacobsen, Sabine; Wittig, Michael; Pöggeler, Stefanie

2002-06-01

Mating-type genes control sexual development in ascomycetes. Little is known about their function in homothallic species, which are self-fertile and do not require a mating partner for sexual reproduction. The function of mating-type genes in the homothallic fungus Sordaria macrospora was assayed using a yeast system in order to find properties typical of eukaryotic transcription factors. We were able to demonstrate that the mating-type proteins SMTA-1 and SMTa-1 have domains capable of activating transcription of yeast reporter genes. Two-hybrid analysis for heterodimerization and homodimerization revealed the ability of SMTA-1 to interact with SMTa-1 and vice versa. These two proteins are encoded by different mating types in the related heterothallic species Neurospora crassa. The interaction between SMTA-1 and SMTa-1 was defined by experiments with truncated versions of SMTA-1 and in vitro by means of protein cross-linking. Moreover, we gained evidence for homodimerization of SMTA-1. Possible functions of mating-type proteins in the homothallic ascomycete S. macrospora are discussed.

Meiotic UV-sensitive mutant that causes deletion of duplications in neurospora

International Nuclear Information System (INIS)

Newmeyer, D.; Galeazzi, D.R.

1978-01-01

The meiotic-3 (mei-3) mutant of Neurospora crassa has several effects: (1) when homozygous, it almost completely blocks meiosis and ascospore formation, (2) it is sensitive to uv, (3) its growth is inhibited by histidine, and (4) it increases the instability of nontandem duplications. This was shown for duplications produced by five different rearrangements and was demonstrated by two different criteria. The effects on meiosis and duplication instability are expressed strongly at 25 0 ; the effects on sensitivity to uv and to histidine are expressed strongly at 38.5 0 but only slightly at 25 0 . Nevertheless, all four effects were shown to be due to a single gene. Mei-3 is not allelic with previously reported uv-sensitive mutants. Two other results were obtained that are not necessarily due to mei-3: (1) a cross involving mei-3 produced a new unlinked meiotic mutant, mei-4, which is not sensitive to uv or histidine, and (2) a burst of several new mutants occurred in a different mei-3 stock, including a partial revertant to mei-3. Mei-3 has previously been shown to cause frequent complete loss of a terminal duplicate segment, beginning exactly at the original rearrangement breakpoint. Possible mechanisms are discussed by which a uv-sensitive mutant could cause such precise deletions

Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora.

Science.gov (United States)

Nowrousian, Minou; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

2005-04-01

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.

Genetic control of anastomosis in Podospora anserina.

Science.gov (United States)

Tong, Laetitia Chan Ho; Silar, Philippe; Lalucque, Hervé

2014-09-01

We developed a new microscopy procedure to study anastomoses in the model ascomycete Podospora anserina and compared it with the previous method involving the formation of balanced heterokaryons. Both methods showed a good correlation. Heterokaryon formation was less quantifiable, but enabled to observe very rare events. Microscopic analysis evidenced that anastomoses were greatly influence by growth conditions and were severely impaired in the IDC mutants of the PaMpk1, PaMpk2, IDC1 and PaNox1 pathways. Yet some mutants readily formed heterokaryons, albeit with a delay when compared to the wild type. We also identified IDC(821), a new mutant presenting a phenotype similar to the other IDC mutants, including lack of anastomosis. Complete genome sequencing revealed that IDC(821) was affected in the orthologue of the Neurospora crassa So gene known to control anastomosis in several other ascomycetes. Copyright © 2014 Elsevier Inc. All rights reserved.

Journal of Genetics, Volume 92, 2013

Indian Academy of Sciences (India)

Molecular adaptation within the coat protein-encoding gene of Tunisian almond ... Neurospora crassa ncs-1, mid-1 and nca-2 double-mutant phe- notypes suggest .... based NCII designs: a simulation study (Research article) 529. Li, Shujuan.

The Effect of Chrysonilia crassa Additive on Duodenal & Caecal Morphology, Bacterial & Fungal Number, and Productivity of Ayam Kampung

OpenAIRE

Turrini Yudiarti; V. D. Yunianto B.I; R. Murwani; E. Kusdiyantini

2012-01-01

Fungi is a microorganism that can live in gastrointestinal tract of chicken. One type of fungi is multicellular or filamentous fungi. C.crassa is a species of filamentous fungi that has been isolated in the earlier study and it showed the best probiotic potency in vitro. The obyective of this research was to study the effect of addition of dried culture of  C.crassa in feed on intestinal & caecal morphology, bacterial & fungal number, and  productivity of indigenous chicken (ayam kamp...

Triterpenes and antitubercular activity of Byrsonima crassa

Energy Technology Data Exchange (ETDEWEB)

Higuchi, Celio Takashi; Pavan, Fernando Rogerio; Leite, Clarice Queico Fujimura [Universidade Estadual Paulista, Araraquara, SP (Brazil). Fac. de Ciencias Farmaceuticas. Dept. de Ciencias Biologicas]. E-mail: leitecqf@fcfar.unesp.br; Sannomiya, Miriam; Vilegas, Wagner; Leite, Sergio Roberto de Andrade [Universidade Estadual Paulista (UNESP), Araraquara, SP (Brazil). Inst. de Quimica. Dept. de Quimica Geral e Inorganica; Sacramento, Luis Vitor S. [Universidade Estadual Paulista (UNESP), Araraquara, SP (Brazil). Fac. de Ciencias Farmaceuticas. Dept. de Principios Ativos Naturais e Toxicologia; Sato, Daisy Nakamura [Instituto Adolfo Lutz de Ribeirao Preto, SP (Brazil)

2008-07-01

We evaluated the potential antitubercular activity of triterpenes obtained from leaves and bark of Byrsonima crassa. From chloroform extracts of the leaves, by bioassay-guided fractionation, we obtained mixtures of known triterpenes: alpha-amyrin, beta-amyrin and their acetates, lupeol, oleanolic acid, ursolic acid and alpha-amyrinone. Tested against Mycobacterium tuberculosis, the triterpenes exhibited minimum inhibitory concentrations (MICs) of 31.25 - 312.25 mug/mL. beta-amyrin and friedelin, isolated from the chloroform extract of bark, showed MICs of 312.25 and 125 mug/mL respectively. This is the first report of the identification and determination of the activity of B. crassa triterpenes against M. tuberculosis. (author)

Multi-targeted priming for genome-wide gene expression assays

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Adomas Aleksandra B

2010-08-01

Full Text Available Abstract Background Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. Results We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Conclusions Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and

DNA vaccines encoding proteins from wild-type and attenuated canine distemper virus protect equally well against wild-type virus challenge.

Science.gov (United States)

Nielsen, Line; Jensen, Trine Hammer; Kristensen, Birte; Jensen, Tove Dannemann; Karlskov-Mortensen, Peter; Lund, Morten; Aasted, Bent; Blixenkrone-Møller, Merete

2012-10-01

Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.

Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens.

NARCIS (Netherlands)

Tierens, K.F.; Thomma, B.P.; Brouwer, M.H.J.; Schmidt, J.; Kistner, K.; Porzel, A.; Mauch-Mani, B.; Cammue, B.P.; Broekaert, W.F.

2001-01-01

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same

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Indian Academy of Sciences (India)

Prakash

complexes in mouse, pollen killer in wheat, and female gamete eliminator in tomato ...... me to arrange our meeting in Hyderabad. He wrote – “I have learned ... fungus Neurospora crassa; Nature (London) 422 859–868. Gallegos A, Jacobson ...

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

NARCIS (Netherlands)

Trip, H; Evers, ME; Driessen, AJM

2004-01-01

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and

Polymorphism for pKALILO based senescence in Hawaiian populations of Neurospora intermedia and Neurospora tetrasperma.

NARCIS (Netherlands)

Maas, M.F.P.M.; Mourik, van A.; Hoekstra, R.F.; Debets, A.J.M.

2005-01-01

The natural population of Neurospora intermedia from Hawaii is polymorphic for the presence of the linear mitochondrial plasmid pKALILO that is associated with an infectious senescence syndrome. Although inter-specific horizontal transmission is experimentally possible, thus far pKALILO associated

Molecular control of copper homeostasis in filamentous fungi: increased expression of a metallothionein gene during aging of Podospora anserina.

Science.gov (United States)

Averbeck, N B; Borghouts, C; Hamann, A; Specke, V; Osiewacz, H D

2001-01-01

The lifespan of the ascomycete Podospora anserina was previously demonstrated to be significantly increased in a copper-uptake mutant, suggesting that copper is a potential stressor involved in degenerative processes. In order to determine whether changes in copper stress occur in the cells during normal aging of cultures, we cloned and characterized a gene coding for a component of the molecular machinery involved in the control of copper homeostasis. This gene, PaMt1, is a single-copy gene that encodes a metallothionein of 26 amino acids. The coding sequence of PaMt1 is interrupted by a single intron. The deduced amino acid sequence shows a high degree of sequence identity to metallothioneins of the filamentous ascomycete Neurospora crassa and the basidiomycete Agaricus bisporus, and to the N-terminal portion of mammalian metallothioneins. Levels of PaMt1 transcript increase in response to elevated amounts of copper in the growth medium and during aging of wild-type cultures. In contrast, in the long-lived mutant grisea, transcript levels first increase but then decrease again. The ability of wild-type cultures to respond to exogenous copper stress via the induction of PaMt1 transcription is not affected as they grow older.

A Circadian Rhythm Regulating Hyphal Melanization in Cercospora Kikuchii

Science.gov (United States)

Circadian rhythms, biochemical or developmental processes with a period length of approximately 24 hours, are thoroughly documented in plants and animals. However, virtually all of what is currently known about circadian rhythms in fungi is derived from the model fungus, Neurospora crassa, including...

Dicty_cDB: Contig-U14020-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 1( AB434423 |pid:none) Rhodotorula glutinis CMK gene for ... 145 1e-33 S60303( S60303 ;S24578) serine/threon...nkara... 146 8e-34 DQ534193_1( DQ534193 |pid:none) Neurospora crassa checkpoint kinas... 145 1e-33 AB434423_

Intergeneric complementation of a circadian rhythmicity defect : phylogenetic conservation of structure and function of the clock gene frequency

NARCIS (Netherlands)

Merrow, Martha W.; Dunlap, Jay C.; Dover, G.

1994-01-01

The Neurospora crassa frequency locus encodes a 989 amino acid protein that is a central component, a state variable, of the circadian biological clock. We have determined the sequence of all or part of this protein and surrounding regulatory regions from additional fungi representing three genera

Journal of Genetics | Indian Academy of Sciences

Indian Academy of Sciences (India)

Heterokaryons of Neurospora crassa were generated by transformation of multinucleate conidia of a histidine-3 auxotroph with his-3+ plasmid. In one of the transformants, propagated on a medium with histidine supplementation, a gradual but drastic reduction occurred in the proportion of prototrophic nuclei that contained ...

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora

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Cebula Patricia

2005-11-01

Full Text Available Abstract Background The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development, a gene encoding a putative lectin homolog. Results Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Δtap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. Conclusion The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora.

Science.gov (United States)

Nowrousian, Minou; Cebula, Patricia

2005-11-03

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Deltatap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.

The Effect of Chrysonilia crassa Additive on Duodenal & Caecal Morphology, Bacterial & Fungal Number, and Productivity of Ayam Kampung

Directory of Open Access Journals (Sweden)

Turrini Yudiarti

2012-10-01

Full Text Available Fungi is a microorganism that can live in gastrointestinal tract of chicken. One type of fungi is multicellular or filamentous fungi. C.crassa is a species of filamentous fungi that has been isolated in the earlier study and it showed the best probiotic potency in vitro. The obyective of this research was to study the effect of addition of dried culture of  C.crassa in feed on intestinal & caecal morphology, bacterial & fungal number, and  productivity of indigenous chicken (ayam kampung. Research used completely randomized design with four treatments. The treatments were the level of  dried culture in basal diet (0%, 0.25 %,  0.50 % and 0.75 %. Each treatment was replicated 5 times and each replicate consists of 10 chickens. The parameters observed were : villi morphology, number of bacteria and fungi in the duodenum and cecum of chickens aged 1, 21 and 35 days and productivity i.e. feed intake, final body weight and feed conversion. The results showed that 0.50% dried culture of C.crassa could increase the duodenal villi width, decreased the number of bacterial and fungal colonies in duodenum and caecum, but it did not increase productivity. The conclusion : C.crassa could stimulate the duodenal villi development and decreased the number of the bacteria and fungi in the gastrointestinal tract, yet it has no positive impact on the chicken productivity.

The Effect of Chrysonilia crassa Additive on Duodenal & Caecal Morphology, Bacterial & Fungal Number, and Productivity of Ayam Kampung

Directory of Open Access Journals (Sweden)

T. Yudiarti

2012-10-01

Full Text Available Fungi is a microorganism that can live in gastrointestinal tract of chicken. One type of fungi is multicellular or filamentous fungi. C.crassa is a species of filamentous fungi that has been isolated in the earlier study and it showed the best probiotic potency in vitro. The obyective of this research was to study the effect of addition of dried culture of C.crassa in feed on intestinal & caecal morphology, bacterial & fungal number, and productivity of indigenous chicken (ayam kampung. Research used completely randomized design with four treatments. The treatments were the level of dried culture in basal diet (0%, 0.25 %, 0.50 % and 0.75 %. Each treatment was replicated 5 times and each replicate consists of 10 chickens. The parameters observed were : villi morphology, number of bacteria and fungi in the duodenum and cecum of chickens aged 1, 21 and 35 days and productivity i.e. feed intake, final body weight and feed conversion. The results showed that 0.50% dried culture of C.crassa could increase the duodenal villi width, decreased the number of bacterial and fungal colonies in duodenum and caecum, but it did not increase productivity. The conclusion : C.crassa could stimulate the duodenal villi development and decreased the number of the bacteria and fungi in the gastrointestinal tract, yet it has no positive impact on the chicken productivity

Byrsonima crassa Niedenzu (IK: antimicrobial activity and chemical study

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W. Vilegas

2009-01-01

Full Text Available

The methanolic extract of leaves from Byrsonima crassa, a Brazilian medicinal plant, was analyzed by CC and HPLC. Four constituents were isolated and identified as quercetin, methyl gallate, (--epigallocatechin gallate and quercetin-3-O-(2”-galloyl-a-L-arabinopyranoside. The methanolic and hydromethanolic extract, as well as fractions, were evaluated regarding their possible antimicrobial activity using in vitro methods. Results showed that both extracts and fractions exhibited significant antimicrobial activity against all tested strains. Keywords: Byrsonima crassa, antimicrobial activity, Malpighiaceae.

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Indian Academy of Sciences (India)

Unknown

Chapter I (Introduction) is six pages packed with use- ful information. It describes the Neurospora crassa life cycle ... information that is available on the Internet, and also provides information on scientific meetings. ... uses the per (perithecial color) mutant to show barrage formation as a manifestation of heterokaryon incom-.

FIRST HOST RECORD FOR THE ROOT PARASITE CORYNAEA CRASSA (BALANOPHORACEAE

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Joel Tupac Otero

2009-09-01

Full Text Available Corynaea crassa es una planta hemiparásita de raiz poco común y de la cual sabemos muy poco acerca  de su historia natural y en particular sobre su rango de hospederos. En este estudio escabamos 32 tuberculos de dicha especie y seguimos las raíces que estaban parasitando para determinar su identidad. Encontramos Bocconia frutescens (Papaveraceae, Verbesina sp. (Asteraceae, Cayaponia sp. (Cucurbitaceae y Palicourea sp.(Rubiaceae. Este es el primer registro de hospederos disponible para la especies y es información de gran utilidad para las conservación de C. crassa en habitats naturales.

Global DNA Methylation in the Chestnut Blight Fungus Cryphonectria parasitica and Genome-Wide Changes in DNA Methylation Accompanied with Sectorization

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Kum-Kang So

2018-02-01

Full Text Available Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase, demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

A family of DNA repeats in Aspergillus nidulans has assimilated degenerated retrotransposons

DEFF Research Database (Denmark)

Nielsen, M.L.; Hermansen, T.D.; Aleksenko, Alexei Y.

2001-01-01

In the course of a chromosomal walk towards the centromere of chromosome IV of Aspergillus nidulans, several cross- hybridizing genomic cosmid clones were isolated. Restriction mapping of two such clones revealed that their restriction patterns were similar in a region of at least 15 kb, indicati......) phenomenon, first described in Neurospora crassa, may have operated in A. nidulans. The data indicate that this family of repeats has assimilated mobile elements that subsequently degenerated but then underwent further duplications as a part of the host repeats....... the presence of a large repeat. The nature of the repeat was further investigated by sequencing and Southern analysis. The study revealed a family of long dispersed repeats with a high degree of sequence similarity. The number and location of the repeats vary between wild isolates. Two copies of the repeat...

Antiparasitic bromotyrosine derivatives from the Caribbean marine Sponge Aiolochroia crassa

International Nuclear Information System (INIS)

Galeano, Elkin; Martinez, Alejandro; Thomas, Olivier P.; Robledo, Sara; Munoz, Diana

2012-01-01

Six bromotyrosine-derived compounds were isolated from the Caribbean marine sponge Aiolochroia crassa: 3-bromo-5-hydroxy Ο-methyltyrosine (1), 3-bromo-N,N,N-trimethyltyrosinium (2), 3-bromo-N,N,N,ο-tetramethyltyrosinium (3), 3,5-dibromo-N,N,Ntrimethyltyrosinium (4), 3,5-dibromo-N,N,N,O-tetramethyltyrosinium (5), and aeroplysinin-1 (6). Structural determination was performed using NMR, MS and comparison with literature data. All isolated compounds were screened for their in vitro activity against Leishmania panamensis, Plasmodium falciparum and Trypanosoma cruzi. Compound 4 showed selective antiparasitic activity against Leishmania and Plasmodium parasites. This is the first report of compounds 1, 4 and 5 in the sponge A. crassa and the first biological activity reports for compounds 2-4. This work shows that bromotyrosines are potential antiparasitic agents. (author)

Bioavailability and Methylation Potential of Mercury Sulfides in Sediments

Science.gov (United States)

2014-08-01

well-characterized for the fungus Neurospora crassa 72 , and likely occurs within most microorganisms 73 . Another possibility introduced by Larose...of molecular biology (e.g. genomics, proteomics , metabolomics) could provide assistance to this problem 67 . For example, scientists are now... proteomics tools to characterize the proteins involved in metabolic pathways and to determine the proteome of microorganisms exposed to contaminants

Anti-Helicobacter pylori activity and immunostimulatory effect of extracts from Byrsonima crassa Nied. (Malpighiaceae

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Vilegas Wagner

2009-01-01

Full Text Available Abstract Background Several in vitro studies have looked at the effect of medicinal plant extracts against Helicobacter pylori (H. pylori. Regardless of the popular use of Byrsonima crassa (B. crassa as antiemetic, diuretic, febrifuge, to treat diarrhea, gastritis and ulcers, there is no data on its effects against H. pylori. In this study, we evaluated the anti-H. pylori of B. crassa leaves extracts and its effects on reactive oxygen/nitrogen intermediates induction by murine peritoneal macrophages. Methods The minimal inhibitory concentration (MIC was determined by broth microdilution method and the production of hydrogen peroxide (H2O2 and nitric oxide (NO by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. Results The methanolic (MeOH and chloroformic (CHCl3 extracts inhibit, in vitro, the growth of H. pylori with MIC value of 1024 μg/ml. The MeOH extract induced the production H2O2 and NO, but CHCl3 extract only NO. Conclusion Based in our results, B. crassa can be considered a source of compounds with anti-H. pylori activity, but its use should be done with caution in treatment of the gastritis and peptic ulcers, since the reactive oxygen/nitrogen intermediates are involved in the pathogenesis of gastric mucosal injury induced by ulcerogenic agents and H. pylori infections.

Functional characterization of MAT1-1-specific mating-type genes in the homothallic ascomycete Sordaria macrospora provides new insights into essential and nonessential sexual regulators.

Science.gov (United States)

Klix, V; Nowrousian, M; Ringelberg, C; Loros, J J; Dunlap, J C; Pöggeler, S

2010-06-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the alpha domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes.

Functional Characterization of MAT1-1-Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria macrospora Provides New Insights into Essential and Nonessential Sexual Regulators▿†

Science.gov (United States)

Klix, V.; Nowrousian, M.; Ringelberg, C.; Loros, J. J.; Dunlap, J. C.; Pöggeler, S.

2010-01-01

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the α domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes. PMID:20435701

Heterologous expression of the Aspergillus nidulans regulatory gene nirA in Fusarium oxysporum.

Science.gov (United States)

Daboussi, M J; Langin, T; Deschamps, F; Brygoo, Y; Scazzocchio, C; Burger, G

1991-12-20

We have isolated strains of Fusarium oxysporum carrying mutations conferring a phenotype characteristic of a loss of function in the regulatory gene of nitrate assimilation (nirA in Aspergillus nidulans, nit-4 in Neurospora crassa). One of these nir- mutants was successfully transformed with a plasmid containing the nirA gene of A. nidulans. The nitrate reductase of the transformants is still inducible, although the maximum activity is lower than in the wild type. Single and multiple integration events were found, as well as a strict correlation between the presence of the nirA gene and the Nir+ phenotype of the F. oxysporum transformants. We also investigated how the A. nidulans structural gene (niaD) is regulated in F. oxysporum. Enzyme assays and Northern experiments show that the niaD gene is subject to nitrate induction and that it responds to nitrogen metabolite repression in a F. oxysporum genetic background. This indicates that both the mechanisms of specific induction, mediated by a gene product isofunctional to nirA, and nitrogen metabolite repression, presumably mediated by a gene product isofunctional to the homologous gene of A. nidulans, are operative in F. oxysporum.

Evaluation of MIC Strip Isavuconazole test for susceptibility testing of wild-type and non-wild-type Aspergillus fumigatus isolates

DEFF Research Database (Denmark)

Arendrup, Maiken Cavling; Verweij, Paul; Nielsen, Henrik Vedel

2017-01-01

We evaluated the MIC Strip Isavuconazole test against EUCAST E.Def 9.3 by using 40 wild-type and 39 CYP51A mutant Aspergillus fumigatus strains. The strip full inhibition endpoint (FIE) and 80% growth inhibition endpoint were determined by two independent readers, reader 1 (R1) and R2. The essent......We evaluated the MIC Strip Isavuconazole test against EUCAST E.Def 9.3 by using 40 wild-type and 39 CYP51A mutant Aspergillus fumigatus strains. The strip full inhibition endpoint (FIE) and 80% growth inhibition endpoint were determined by two independent readers, reader 1 (R1) and R2...

Magic with moulds: Meiotic and mitotic crossing over in Neurospora ...

Indian Academy of Sciences (India)

2006-02-16

Feb 16, 2006 ... Home; Journals; Journal of Biosciences; Volume 31; Issue 1. Commentary: Magic with moulds: Meiotic and mitotic crossing over in Neurospora inversions and duplications. Durgadas P Kasbekar. Volume 31 Issue 1 March 2006 pp 3-4 ...

UV-sensitivity and repair of UV-damage in Salmonella of wild type

International Nuclear Information System (INIS)

Kondratiev, Y.S.; Brukhansky, G.V.; Andreeva, I.V.; Skavronskaya, A.G.

1977-01-01

The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E.coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4-5 times more sensitive than wild type E.coli and their inactivation curve is similar to that for E.coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E.coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed. (orig.) [de

UV-sensitivity and repair of UV-damage in Salmonella of wild type

Energy Technology Data Exchange (ETDEWEB)

Kondratiev, Y S; Brukhansky, G V; Andreeva, I V; Skavronskaya, A G [Akademiya Meditsinskikh Nauk SSSR, Moscow. Inst. Ehpidemiologii i Mikrobiologii

1977-12-01

The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E.coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4-5 times more sensitive than wild type E.coli and their inactivation curve is similar to that for E.coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E.coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed.

Sabin and wild type polioviruses from children who presented with ...

African Journals Online (AJOL)

Conclusion: This study further confirms the presence of Sabin and wild-type poliovirus among children in Nigeria. The isolation of Sabin strain of poliovirus is advantageous to the polio eradication program as it is capable of inducing natural immunity in susceptible hosts. Transmission of wild-type poliovirus among children ...

Un poète grec francophone : Théo Crassas (entretien

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Daniel Aranjo

2012-10-01

Full Text Available Dans cet entretien, l’auteur interroge le poète grec Théo Crassas sur ses raisons d’écrire en français. Le contexte familial - un oncle poète et francophone, ainsi que ses parents, éclaire ce choix. S’il est vrai que le français n’est plus la langue impériale qu’il fut longtemps, il n’en demeure pas moins une école universelle, de par ses auteurs classiques qui ont consacré son universalité. À ce titre, le français demeurera aussi capital dans l’histoire des humanités que le grec ancien ou le latin. Crassas se définit comme un cas singulier, car il n’écrit ses textes qu’en français.

Light activation of the LOV protein Vivid generates a rapidly exchanging dimer†‡

Science.gov (United States)

Zoltowski, Brian D.; Crane, Brian R.

2009-01-01

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (Light, Oxygen, Voltage) protein, couples light-induced cysteinyl-adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-to-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of Neurospora crassa, and indicate that LOV:LOV homo or hetero dimerization may be a mechanism for regulating light-activated gene expression. PMID:18553928

Constituents of Corynaea crassa "Peruvian Viagra"

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Gonzalo R. Malca Garcia

Full Text Available Abstract A phytochemical investigation of methanol and n-hexane extracts of tuber/roots of Corynaea crassa Hook. f., Balanophoraceae, led to the isolation and characterization of β-sitosterol, lupenone, β-amyrone, lupeol, and β-amyrine. Unusual complex 1:1 mixtures of lupenone/β-amyrone and lupeol/β-amyrine obtained from the extracts were identified by NMR and HR-MS experiments. The structure of the 1:1 lupenone/β-amyrone mixture was confirmed by X-ray analysis. These triterpene ketone derivatives, only distinguished either by 5- or 6-membered E ring, co-crystallize in one common unit cell in the solid state.

Activation Of Wild-Type Hras Suppresses The Earliest Stages Of Pancreatic Cancer

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Jamie Weyandt

2015-08-01

Conclusions: Loss of wild-type Hras promotes the earliest stages of pancreatic tumorigenesis, and moreover results in more rapid progression of the disease. As such, mechanisms leading to activation of wild-type Ras proteins, including but not limited to redox-dependent reactions, may influence the development of pancreatic cancer.

The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides 1O2-resistance genes in resistance to the photoactivated toxin cercosporin.

Science.gov (United States)

Beseli, Aydin; Goulart da Silva, Marilia; Daub, Margaret E

2015-01-01

The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular Diversity of Lactic Acid Bacteria on Ileum and Coecum Broiler Chicken Fed by Chrysonilia crassa Fermentation

Science.gov (United States)

Nur Jannah, Siti; Khotimah, Husnul; Siti Ferniah, Rejeki; Sugiharto

2018-05-01

The Lactid Acid Bakteria (LAB) are microflora in the digestive tract which has positive roles in poultry’s health. One of the factors diversity of LAB in the gatrointestinal tract are influenced by feeding factor. The purpose of this study was to analyze the LAB diversity in ileum and coecum after being fed on fermented Chrysonilia crassa molecularly. LAB species diversity was analysed to provide a baseline profile of the microbial community database on the ileum and coecum digestive tract of broiler chicken of control (commercial feed) and treatment (feed with Chrysonilia crassa fermentation) by the method of Terminal Restriction Fragment Lenght Polymorphism The calculated values werethe number of phylotypes, relative abundace, Shannon-Wiener diversity index (H’), evennes index (E’), and similarity. Group of LAB detected in the control group were Lactobacillus delbrueckii (180 bp), Lactobacillus sp. (187 bp), Lactobacillus plantarum (572 bp), uncultured bacterium (87 bp) and unidentified (50 bp, 582bp). The result of this study showed that by feeding on the fermented Chrysonilia crassa feed had resulted in the decreasing of LAB diversity, i.e. ileum (0.66), coecum (0.48) compared with commercial feed (control) that was ileum (0.84), coecum (1.05).

Diversification of Cercopithifilaria species (Nematoda: Filarioidea in Japanese wild ruminants with description of two new species

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Uni S.

2002-12-01

Full Text Available Twelve of the 17 Cervus nippon nippon deer from Kyushu Island, Japan, that we examined were infected with one or two Cercopithifilaria species. C. longa n. sp. adults were in the subcutaneous tissues of limbs and the abdomen, and C. crassa n. sp. adults were in the skin, mainly in the anterior part of the back ; the distribution of the dermal microfilariae generally matched that of the adult worms. The two new species were assigned to the group of primitive Cercopithifilaria species that parasitize ruminants (bovids and cervids, but the new species could readily be distinguished from others morphologically. C. longa was more primitive and resembled C. bulboidea, one of the five species from the serow Capricornis crispus, a Japanese member of the Caprinae, and species from Bovidae in Africa. C. crassa had a thick body and large spicules like C. rugosicauda from Capreolus capreolus in Europe, the only previously known Cercopithifilaria species from cervids, but it also had one or two hypertrophied pairs of caudal papillae, an unusual character found so far only in Japanese parasites. Among the 12 species known from ruminants, four are African, one is European and more highly evolved, and seven are Japanese, with some being primitive and some more evolved. The great diversity of Cercopithifilaria species in the two wild ruminants that live in Japan seems to have resulted from local speciation, which occurred during the Pleistocene, from a primitive form of the C. longa type derived from Eurasiatic ancestors, which has disappeared or, more probably, not yet been discovered.

The functions of grainy head-like proteins in animals and fungi and the evolution of apical extracellular barriers.

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Adam Paré

Full Text Available The Grainy head (GRH family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi--organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl. We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2 mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins.

Comparative behaviour of lab.-cultured and wild-type Dacus oleae flies in the field

International Nuclear Information System (INIS)

Prokopy, R.J.; Haniotakis, G.E.; Economopoulos, A.P.

1975-01-01

Under field conditions, the authors compared the responses of lab.-type (ca. 85 generations under artificial conditions) and wild-type Dacus oleae flies to host plant colour and odour, host fruit colour and shape, small rectangles of different colours and shades, and McPhail-type traps of different colours baited with different odours. Except for the lab.-type flies being relatively more attracted toward red fruit models and small red rectangles and relatively less attracted toward yellow fruit models and small yellow rectangles than the wild type, the qualitative nature of the responses of the two fly types toward the various experimental treatments was essentially the same. Quantitatively, however, consistently smaller percentages of the released lab.-type than the released wild-type flies were recaptured, suggesting that the mobility, flight pattern, or vigour of the two types of flies may be different. (author)

Structure and Composition of Protein Bodies from Wild-Type and High-Lysine Barley Endosperm

DEFF Research Database (Denmark)

Ingversen, J.

1975-01-01

Protein bodies were isolated from 13 and 28 day old endosperms of barley mutant 1508 and its wild type, Bomi barley. The fine structure of the isolated protein bodies was determined by electron microscopy, and the proteins present in the preparations characterized by amino-acid analysis and SDS......-polyacrylamidegel electrophoresis. Sections through pellets of isolated protein bodies from both the mutant and the wild type revealed protein body structures corresponding with those observed in sections through the intact starchy endosperms. The majority of the wild-type protein bodies was homogeneous spheres accompanied...... that the wild-type protein bodies contained large amounts of prolamines (the storage protein group which is soluble in 55 % isopropanol) and some glutelins (the storage proteins soluble in dilute alkali), whereas the mutant protein bodies have glutelin as the major component and little prolamines...

Porphyrin Interactions with Wild Type and Mutant Mouse Ferrochelatase

Energy Technology Data Exchange (ETDEWEB)

Ferreira, Gloria C.; Franco, Ricardo; Lu, Yi; Ma, Jian-Guo; Shelnutt, John A.

1999-05-19

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe²⁺ chelation into protoporphyrin IX. Resonance Raman and W-visible absorbance spectroscopes of wild type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into protoporphyrin by ferrochelatase. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. Addition of free base or metalated porphyrins to wild type ferrochelatase and H207N variant yields a quasi 1:1 complex, possibly a monomeric protein-bound species. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is sub-stoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive resonance Raman lines and the vinyl vibrational mode. Resonance Raman spectra of free base and metalated porphyrins bound to the wild type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle, although the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decomposition indicates a homogeneous ruffled distortion for the nickel protoporphyrin bound to the wild type ferrochelatase, whereas both a planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance , 3 Raman spectrum of the endogenous E287Q-bound porphyrin, which has

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

Science.gov (United States)

Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

2011-01-01

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species. PMID:22384347

Comparison of Pretreatment Methods on Vetiver Leaves for Efficient Processes of Simultaneous Saccharification and Fermentation by Neurospora sp.

Science.gov (United States)

Restiawaty, E.; Dewi, A.

2017-07-01

Lignocellulosic biomass is a potential raw material for bioethanol production. Neurospora sp. can be used to convert lignocellulosic biomass into bioethanol because of its ability to perform simultaneous saccharification and fermentation. However, lignin content, degree of polymerization, and crystallinity of cellulose contained in lignocellulosic biomass can inhibit cellulosic-biomass digestion by Neurospora sp, so that a suitable pretreatment method of lignocellulosic biomass is needed. The focus of this research was to investigate the suitable pretreatment method for vetiver leaves (Vetiveria zizanioides L. Nash) used as a raw material producing bioethanol in the process of simultaneous saccharification and fermentation (SSF) by Neurospora sp.. Vetiver plants obtained from Garut are deliberately cultivated to produce essential oils extracted from the roots of this plant. Since the vetiver leaves do not contain oil, some of harvested leaves are usually used for crafts and cattle feed, and the rest are burned. This study intended to look at other potential of vetiver leaves as a source of renewable energy. Pretreatments of the vetiver leaves were conducted using hot water, dilute acid, alkaline & dilute acid, and alkaline peroxide, in which each method was accompanied by thermal treatment. The results showed that the alkaline peroxide treatment is a suitable for vetiver leaves as indicated by the increase of cellulose content up to 65.1%, while the contents of hot water soluble, hemicellulose, lignin, and ash are 8.7%, 18.3%, 6.8%, and 1.1%, respectively. Using this pretreatment method, the vetiver leaves can be converted into bioethanol by SSF process using Neurospora sp. with a concentration of bioethanol of 6.7 g/L operated at room temperature.

Suppression subtractive hybridization and comparative expression analysis to identify developmentally regulated genes in filamentous fungi.

Science.gov (United States)

Gesing, Stefan; Schindler, Daniel; Nowrousian, Minou

2013-09-01

Ascomycetes differentiate four major morphological types of fruiting bodies (apothecia, perithecia, pseudothecia and cleistothecia) that are derived from an ancestral fruiting body. Thus, fruiting body differentiation is most likely controlled by a set of common core genes. One way to identify such genes is to search for genes with evolutionary conserved expression patterns. Using suppression subtractive hybridization (SSH), we selected differentially expressed transcripts in Pyronema confluens (Pezizales) by comparing two cDNA libraries specific for sexual and for vegetative development, respectively. The expression patterns of selected genes from both libraries were verified by quantitative real time PCR. Expression of several corresponding homologous genes was found to be conserved in two members of the Sordariales (Sordaria macrospora and Neurospora crassa), a derived group of ascomycetes that is only distantly related to the Pezizales. Knockout studies with N. crassa orthologues of differentially regulated genes revealed a functional role during fruiting body development for the gene NCU05079, encoding a putative MFS peptide transporter. These data indicate conserved gene expression patterns and a functional role of the corresponding genes during fruiting body development; such genes are candidates of choice for further functional analysis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis

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Yoshiaki Furukawa

2012-01-01

Full Text Available Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1 gene cause a familial form of amyotrophic lateral sclerosis (ALS. While it remains controversial how SOD1 mutations lead to onset and progression of the disease, many in vitro and in vivo studies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations in sod1 gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.

A Blumeria graminis f.sp. hordei BAC library - contig building and microsynteny studies

DEFF Research Database (Denmark)

Pedersen, C.; Wu, B.; Giese, H.

2002-01-01

A bacterial artificial chromosome (BAC) library of Blumeria graminis f.sp. hordei, containing 12,000 clones with an average insert size of 41 kb, was constructed. The library represents about three genome equivalents and BAC-end sequencing showed a high content of repetitive sequences, making...... contigs, at or close to avirulence loci, were constructed. Single nucleotide polymorphism (SNP) markers were developed from BAC-end sequences to link the contigs to the genetic maps. Two other BAC contigs were used to study microsynteny between B. graminis and two other ascomycetes, Neurospora crassa...

A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase

Science.gov (United States)

Witherow, D. Scott

2016-01-01

This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…

Differential induction of Toll-like receptors & type 1 interferons by Sabin attenuated & wild type 1 polioviruses in human neuronal cells.

Science.gov (United States)

Mohanty, Madhu C; Deshpande, Jagadish M

2013-01-01

Polioviruses are the causative agent of paralytic poliomyelitis. Attenuated polioviruses (Sabin oral poliovirus vaccine strains) do not replicate efficiently in neurons as compared to the wild type polioviruses and therefore do not cause disease. This study was aimed to investigate the differential host immune response to wild type 1 poliovirus (wild PV) and Sabin attenuated type 1 poliovirus (Sabin PV) in cultured human neuronal cells. By using flow cytometry and real time PCR methods we examined host innate immune responses and compared the role of toll like receptors (TLRs) and cytoplasmic RNA helicases in cultured human neuronal cells (SK-N-SH) infected with Sabin PV and wild PV. Human neuronal cells expressed very low levels of TLRs constitutively. Sabin PV infection induced significantly higher expression of TLR3, TLR7 and melanoma differentiation-associated protein-5 (MDA-5) m-RNA in neuronal cells at the beginning of infection (up to 4 h) as compared to wild PV. Further, Sabin PV also induced the expression of interferon α/β at early time point of infection. The induced expression of IFN α/β gene by Sabin PV in neuronal cells could be suppressed by inhibiting TLR7. Neuronal cell innate immune response to Sabin and wild polioviruses differ significantly for TLR3, TLR7, MDA5 and type 1 interferons. Effects of TLR7 activation and interferon production and Sabin virus replication in neuronal cells need to be actively investigated in future studies.

Differential induction of Toll-like receptors & type 1 interferons by Sabin attenuated & wild type 1 polioviruses in human neuronal cells

Directory of Open Access Journals (Sweden)

Madhu C Mohanty

2013-01-01

Full Text Available Background & objectives: Polioviruses are the causative agent of paralytic poliomyelitis. Attenuated polioviruses (Sabin oral poliovirus vaccine strains do not replicate efficiently in neurons as compared to the wild type polioviruses and therefore do not cause disease. This study was aimed to investigate the differential host immune response to wild type 1 poliovirus (wild PV and Sabin attenuated type 1 poliovirus (Sabin PV in cultured human neuronal cells. Methods: By using flow cytometry and real time PCR methods we examined host innate immune responses and compared the role of toll like receptors (TLRs and cytoplasmic RNA helicases in cultured human neuronal cells (SK-N-SH infected with Sabin PV and wild PV. Results: Human neuronal cells expressed very low levels of TLRs constitutively. Sabin PV infection induced significantly higher expression of TLR3, TLR7 and melanoma differentiation-associated protein-5 (MDA-5 m-RNA in neuronal cells at the beginning of infection (up to 4 h as compared to wild PV. Further, Sabin PV also induced the expression of interferon α/β at early time point of infection. The induced expression of IFN α/β gene by Sabin PV in neuronal cells could be suppressed by inhibiting TLR7. Interpretation & conclusions: Neuronal cell innate immune response to Sabin and wild polioviruses differ significantly for TLR3, TLR7, MDA5 and type 1 interferons. Effects of TLR7 activation and interferon production and Sabin virus replication in neuronal cells need to be actively investigated in future studies.

Modification of the Neurospora crassa plasma membrane [H+]-ATPase with N,N'-dicyclohexycarbodiimide

International Nuclear Information System (INIS)

Sussman, M.R.; Slayman, C.W.

1983-01-01

The carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the ATPase with pseudo-first order kinetics, suggesting that one site on the enzyme is involved. The rate constant for inactivation at pH 7.5 and 30 0 C is approximately 1000 M -1 min -1 , similar to values reported for the DCCD-binding proteolipid of F 0 -F 1 -type [H + ]-ATPases and for the sarcoplasmic reticulum [Ca +2 ]-ATPase. Although hydrophobic carbodiimides are inhibitory at micromolar concentrations, a hydrophilic analogue, 1-ethyl-3-(dimethylaminopropyl)-carbodiimide, is completely inactive even at millimolar concentrations. This result implies that the DCCD-reactive site is located in a lipophilic environment. [ 14 C]DCCD is incorporated into the M/sub r/ = 104,000 polypeptide at a rate similar to the rate of inactivation. There is no evidence for a separate low molecular weight DCCD-binding proteolipid. Using quantitative amino acid analysis, we established that complete inhibition occurs at a stoichiometry of 0.4 mol of DCCD/mol of polypeptide. Overall, the results are consistent with the idea the DCCD reacts with a single amino acid residue of the Neuspora [H + ]-ATPase, thereby blcoking ATP hydrolysis and proton translocation. 21 references, 5 figures, 2 tables

Pharmacologic Treatment Assigned for Niemann Pick Type C1 Disease Partly Changes Behavioral Traits in Wild-Type Mice.

Science.gov (United States)

Schlegel, Victoria; Thieme, Markus; Holzmann, Carsten; Witt, Martin; Grittner, Ulrike; Rolfs, Arndt; Wree, Andreas

2016-11-09

Niemann-Pick Type C1 (NPC1) is an autosomal recessive inherited disorder characterized by accumulation of cholesterol and glycosphingolipids. Previously, we demonstrated that BALB/c-npc1 nih Npc1 -/- mice treated with miglustat, cyclodextrin and allopregnanolone generally performed better than untreated Npc1 -/- animals. Unexpectedly, they also seemed to accomplish motor tests better than their sham-treated wild-type littermates. However, combination-treated mutant mice displayed worse cognition performance compared to sham-treated ones. To evaluate effects of these drugs in healthy BALB/c mice, we here analyzed pharmacologic effects on motor and cognitive behavior of wild-type mice. For combination treatment mice were injected with allopregnanolone/cyclodextrin weekly, starting at P7. Miglustat injections were performed daily from P10 till P23. Starting at P23, miglustat was embedded in the chow. Other mice were treated with miglustat only, or sham-treated. The battery of behavioral tests consisted of accelerod, Morris water maze, elevated plus maze, open field and hot-plate tests. Motor capabilities and spontaneous motor behavior were unaltered in both drug-treated groups. Miglustat-treated wild-type mice displayed impaired spatial learning compared to sham- and combination-treated mice. Both combination- and miglustat-treated mice showed enhanced anxiety in the elevated plus maze compared to sham-treated mice. Additionally, combination treatment as well as miglustat alone significantly reduced brain weight, whereas only combination treatment reduced body weight significantly. Our results suggest that allopregnanolone/cyclodextrin ameliorate most side effects of miglustat in wild-type mice.

Pharmacologic Treatment Assigned for Niemann Pick Type C1 Disease Partly Changes Behavioral Traits in Wild-Type Mice

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Victoria Schlegel

2016-11-01

Full Text Available Niemann-Pick Type C1 (NPC1 is an autosomal recessive inherited disorder characterized by accumulation of cholesterol and glycosphingolipids. Previously, we demonstrated that BALB/c-npc1nihNpc1−/− mice treated with miglustat, cyclodextrin and allopregnanolone generally performed better than untreated Npc1−/− animals. Unexpectedly, they also seemed to accomplish motor tests better than their sham-treated wild-type littermates. However, combination-treated mutant mice displayed worse cognition performance compared to sham-treated ones. To evaluate effects of these drugs in healthy BALB/c mice, we here analyzed pharmacologic effects on motor and cognitive behavior of wild-type mice. For combination treatment mice were injected with allopregnanolone/cyclodextrin weekly, starting at P7. Miglustat injections were performed daily from P10 till P23. Starting at P23, miglustat was embedded in the chow. Other mice were treated with miglustat only, or sham-treated. The battery of behavioral tests consisted of accelerod, Morris water maze, elevated plus maze, open field and hot-plate tests. Motor capabilities and spontaneous motor behavior were unaltered in both drug-treated groups. Miglustat-treated wild-type mice displayed impaired spatial learning compared to sham- and combination-treated mice. Both combination- and miglustat-treated mice showed enhanced anxiety in the elevated plus maze compared to sham-treated mice. Additionally, combination treatment as well as miglustat alone significantly reduced brain weight, whereas only combination treatment reduced body weight significantly. Our results suggest that allopregnanolone/cyclodextrin ameliorate most side effects of miglustat in wild-type mice.

Repair-resistant mutation in Neurospora

International Nuclear Information System (INIS)

Stadler, D.; Macleod, H.; Loo, M.

1987-01-01

Chronic UV treatment produces severalfold fewer mutations in Neurospora conidia than does the same total dose of acute UV. Experiments were designed to determine the conditions required for chronic UV mutagenesis. Measurement of the coincidence frequency for two independent mutations revealed the existence of a subset of cells which are mutable by chronic UV. Analysis of forward mutation at the mtr locus showed that the genetic alterations produced by chronic UV were virtually all point mutants, even though the assay system could detect alterations or deletions extending into neighboring genes. A significant fraction of the mutants produced by acute UV were multigenic deletions. The size of the dose-rate effect (acute UV mutation frequency divided by chronic UV mutation frequency) was compared for several different mutation assay systems. Forward mutations (recessive lethals and mtr) gave values ranging from four to nine. For events which were restricted to specific molecular sites (specific reversions and nonsense suppressor mutations), there was a wider range of dose-rate ratios. This suggests that chronic UV mutation may be restricted to certain molecular sequences or configurations

Wild type measles virus attenuation independent of type I IFN

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Horvat Branka

2008-02-01

Full Text Available Abstract Background Measles virus attenuation has been historically performed by adaptation to cell culture. The current dogma is that attenuated virus strains induce more type I IFN and are more resistant to IFN-induced protection than wild type (wt. Results The adaptation of a measles virus isolate (G954-PBL by 13 passages in Vero cells induced a strong attenuation of this strain in vivo. The adapted virus (G954-V13 differs from its parental strain by only 5 amino acids (4 in P/V/C and 1 in the M gene. While a vaccine strain, Edmonston Zagreb, could replicate equally well in various primate cells, both G954 strains exhibited restriction to the specific cell type used initially for their propagation. Surprisingly, we observed that both G954 strains induced type I IFN, the wt strain inducing even more than the attenuated ones, particularly in human plasmacytoid Dendritic Cells. Type I IFN-induced protection from the infection of both G954 strains depended on the cell type analyzed, being less efficient in the cells used to grow the viral strain. Conclusion Thus, mutations in M and P/V/C proteins can critically affect MV pathogenicity, cellular tropism and lead to virus attenuation without interfering with the α/β IFN system.

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate

International Nuclear Information System (INIS)

Zeeland, A.A. van; Aaron, C.S.; Mohn, G.R.; Hung, C.Y.; Brockman, H.E.

1983-01-01

Extending previous work with E. coli and mammalian cells in culture, forward-mutation frequencies induced by ethyl methanesulfonate (EMS) were quantitatively compared in Neurospora crassa and Saccharomyces cerevisiae under standardized conditions. Concomitantly, the actual dose to DNA was measured by determining the amount of radioactivity bound to DNA after treatment with tritium-labeled EMS. After exposure to EMS (2.5-50 mM), alkylation levels in N. crassa and S. cerevisiae were similar to those previously determined in E. coli and cultured mammalian cells. Consistently, there was a slightly less than proportional increase of the DNA alkylation level with the exposure concentration of the mutagen. Forward mutagenesis induced in yeast and N. crassa showed exponential kinetics with exponents of 1.5 and 2.6, respectively. These results are similar to those previously reported with E. coli, which differed from the results with cultured mammalian cells, where a linear dose-effect relationship between exposure and genetic effect was observed. These differences may reflect differences in the fate of EMS-induced adducts by cellular DNA repair systems, but are not due to initial differences in DNA alkylation levels. The fate and persistence of specific DNA adducts potentially responsible for pre-mutagenic changes are under investigation. (orig.)

Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan.

Science.gov (United States)

Angez, Mehar; Shaukat, Shahzad; Alam, Muhammad M; Sharif, Salmaan; Khurshid, Adnan; Zaidi, Syed Sohail Zahoor

2012-02-22

Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP) patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Our results emphasize that there was a persistent transmission of wild type 1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas, associated with high risk population groups which include migrants

Genetic relationships and epidemiological links between wild type 1 poliovirus isolates in Pakistan and Afghanistan

Directory of Open Access Journals (Sweden)

Angez Mehar

2012-02-01

Full Text Available Abstract Background/Aim Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. Methods In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. Results One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Conclusion Our results emphasize that there was a persistent transmission of wild type1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas

Genes encoding chimeras of Neurospora crassa erg-3 and human ...

Indian Academy of Sciences (India)

Unknown

(FGSC), University of Kansas Medical Center, Kansas. City, KS 66103, USA. .... pCH1-Hph digested with the same enzymes to produce ..... Wheat coding sequences are characterized by a high GC content and by a related strong bias of codon ...

Molecular mechanisms involved in the production of chromosomal aberrations. I. Utilization of Neurospora endonuclease for the study of aberration production in G2 stage of the cell cycle

Energy Technology Data Exchange (ETDEWEB)

Natarajan, A T; Obe, G [Rijksuniversiteit Leiden (Netherlands). J.A. Cohen Inst. voor Radiopathologie en Stralingsbescherming

1978-10-01

Chinese hamster ovary cells (CHO) were X-irradiated in G2 stage of the cell cycle and immediately treated, in the presence of inactivated Sendai virus, with Neurospora endonuclease (E.C. 3.1.4.), an enzyme which is specific for cleaving single-stranded DNA. With this treatment, the frequencies of all types of chromosome aberrations increased when compared to X-irradiated controls. These results are interpreted as due to the conversion of some of the X-ray induced single-stranded DNA breaks into double-strand breaks by this enzyme. Similar enhancement due to this enzyme was found following treatment with methyl methanesulfonate (MMS) and bleomycin, but not following UV and mitomycin C. Addition of Micrococcus endonuclease and Neurospora endonuclease to the cells did not alter the frequencies of aberrations induced by UV. The introduction of enzymes with specific DNA-repair function offers possibilities to probe into the molecular events involved in the formation of structural chromosome aberrations induced by different classes of physical and chemical mutagens.

Rearing in seawater mesocosms improves the spawning performance of growth hormone transgenic and wild-type coho salmon.

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Rosalind A Leggatt

Full Text Available Growth hormone (GH transgenes can significantly accelerate growth rates in fish and cause associated alterations to their physiology and behaviour. Concern exists regarding potential environmental risks of GH transgenic fish, should they enter natural ecosystems. In particular, whether they can reproduce and generate viable offspring under natural conditions is poorly understood. In previous studies, GH transgenic salmon grown under contained culture conditions had lower spawning behaviour and reproductive success relative to wild-type fish reared in nature. However, wild-type salmon cultured in equal conditions also had limited reproductive success. As such, whether decreased reproductive success of GH transgenic salmon is due to the action of the transgene or to secondary effects of culture (or a combination has not been fully ascertained. Hence, salmon were reared in large (350,000 L, semi-natural, seawater tanks (termed mesocosms designed to minimize effects of standard laboratory culture conditions, and the reproductive success of wild-type and GH transgenic coho salmon from mesocosms were compared with that of wild-type fish from nature. Mesocosm rearing partially restored spawning behaviour and success of wild-type fish relative to culture rearing, but remained lower overall than those reared in nature. GH transgenic salmon reared in the mesocosm had similar spawning behaviour and success as wild-type fish reared in the mesocosm when in full competition and without competition, but had lower success in male-only competition experiments. There was evidence of genotype×environmental interactions on spawning success, so that spawning success of transgenic fish, should they escape to natural systems in early life, cannot be predicted with low uncertainty. Under the present conditions, we found no evidence to support enhanced mating capabilities of GH transgenic coho salmon compared to wild-type salmon. However, it is clear that GH transgenic

Fungal intelligence; Or on the behaviour of microorganisms in confined micro-environments

International Nuclear Information System (INIS)

Held, M; Nicolau, D V; Edwards, C

2009-01-01

Filamentous fungi are very successful in colonising various microconfined environments, but their behaviour is usually tested on flat surfaces. This contribution presents the design, the fabrication and the use of microstructures, made of a biocompatible polymer (poly(dimethylsiloxane), PDMS) for studying the dynamic micro-scale behaviour of the filamentous fungus Neurospora crassa. The proposed methodology is simple to implement and uses low cost fabrication methods. The observations of the fungus growing through a variety of fabricated micro-environments revealed distinct structure-dependent and structure-induced responses. Generalising the proposed methodology we propose a tool for high-throughput studies of numerous fungal species.

The wild type as concept and in experimental practice: A history of its role in classical genetics and evolutionary theory.

Science.gov (United States)

Holmes, Tarquin

2017-06-01

Wild types in genetics are specialised strains of laboratory experimental organism which principally serve as standards against which variation is measured. As selectively inbred lineages highly isolated from ancestral wild populations, there appears to be little wild or typical about them. I will nonetheless argue that they have historically been successfully used as stand-ins for nature, allowing knowledge produced in the laboratory to be extrapolated to the natural world. In this paper, I will explore the 19th century origins of the wild type concept, the theoretical and experimental innovations which allowed concepts and organisms to move from wild nature to laboratory domestication c. 1900 (resulting in the production of standardised lab strains), and the conflict among early geneticists between interactionist and atomist accounts of wild type, which would eventually lead to the conceptual disintegration of wild types and the triumph of genocentrism and population genetics. I conclude by discussing how the strategy of using wild type strains to represent nature in the lab has nonetheless survived the downfall of the wild type concept and continues to provide, significant limitations acknowledged, an epistemically productive means of investigating heredity and evolutionary variation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fusarium spp. Associated with Field-Grown Grain of Near-Isogenic Low Lignin and Wild-Type Sorghum

Science.gov (United States)

Fusarium spp. associated with field-grown grain of near-isogenic low lignin and wild-type sorghum. Deanna Funnell-Harris and Jeff Pedersen, USDA-ARS, Lincoln, NE Previous studies indicated that low lignin brown midrib (bmr) sorghum may be more resistant to Fusarium spp. than wild-type and that phen...

Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

NARCIS (Netherlands)

Backx, A.; Heutink, C.G.; Rooij, van E.M.A.; Rijn, van P.A.

2009-01-01

Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven

Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

International Nuclear Information System (INIS)

Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank

2013-01-01

Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

Energy Technology Data Exchange (ETDEWEB)

Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank, E-mail: fkempken@bot.uni-kiel.de

2013-08-30

Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

POTENSI DARI KAPANG Aspergilus niger, Rhizophus oryzae DAN Neurospora sitophila SEBAGAI PENGHASIL EZIM FITASE DAN AMILASE PADA SUBSTRATE AMPAS TAHU

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Atit - Kanti

2017-02-01

Full Text Available Penambahan enzim hidrolisis untuk pakan ternak dapat meningkatkan nilai nutrisi pakan. Penelitian bertujuan untuk mendapatkan kondisi optimal untuk produksi enzim amilase dan fitase pada media ampas tahu menggunakan Aspergilus niger, Rhizophus oryzae dan Neurospora sitophila. Uji kemampuan produksi enzim fitase dan amilase oleh Aspergilus niger, Rhizophus oryzae dan Neurospora sitophila dilakukan menggunakan media ampas tahu yang disterilisasi. Pemilihan ketiga isolat ini diawali dengan uji produksi enzim amilase pada kultur cair yang mengandung 2 % pati, dan uji fitase dilakukan pada media yang mengandung 0.5 % sodium fitat. Hasil uji pada medium cair selanjutnya digunakan untuk uji produksi enzim fitase dan fitase pada sistem fermentasi padat (SSF menggunakan ampas tahu sebagai media fermentasi. Untuk mendapatkan produksi enzim yang tinggi dilakukan melalui optimasi waktu inkubasi, suhu inkubasi dan pH media. Fitase dan amilase dapat diproduksi dengan media ampas tahu oleh R. oryzae, A. niger dan N. sitophila. Kondisi optimum untuk produksi fitase, yaitu waktu inkubasi pada hari keempat untuk ketiga kapang, suhu 25 °C untuk R. oryzae dan A. niger, suhu 30°C untuk N. sitophila, pH 8 untuk R. oryzae, pH 6 untuk Aspergillus niger dan N. Sitophila. Neurospora sitophila menghasilkan amilase optimum pada suhu 35°C, sedangkan Aspergillus niger dan Rhizopus oryzae optimum pada suhu 30°C. Penurunan aktivitas produksi amilase menurun oleh R. oryzae pada suhu 40°C. Amilase diproduksi optimal pada pH 6-7. Pakan ternak yang mengandung asam fitat mampu dihidrolisis oleh fitase pada kondisi optimum. Ketiga kapang juga menghasilkan enzim amilase pada media ampas tahu mengindikasikan bahwa ampas tahu merupakan susbtrat yang baik untuk produksi enzim hidrolisis yang berguna untuk meningkatkan nilai nutrisi pakan ternak. (Kata kunci: Amilase, Aspergilus niger, Neurospora sitophila, phytase, Rhizophus oryzae

DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

International Nuclear Information System (INIS)

Cuthill, S.; Poellinger, L.

1988-01-01

The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [ 3 H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin

The effect of UVB on flavonoid biosynthesis in wild type and mutant petunia and arabidopsis

International Nuclear Information System (INIS)

Ryan, K.G.; Swinny, E.E.; Markham, K.R.; Winefield, C.

2000-01-01

Full text: Flavonoids may protect plants against damage by UVB radiation. Flavonoid composition and mRNA expression were determined following growth of plants under natural light, and under natural light with low UVB and with enhanced UVB. In wild-type Arabidopsis and Petunia, UVB induced an increase in total levels of flavonols and this was due to an up-regulation of, several genes coding for key enzymes in the phenylpropanoid pathway. In addition, UVB induced a higher rate of production of the di-hydroxylated si flavonol, quercetin glycoside than of the mono-hydroxylated equivalent, of kaempferol glycoside. Thus the ratio of quercetin to kaempferol increased with UVB treatment in wild type plants, and this suggests that the flavonoid r 3'hydroxylase (F3'H) enzyme, which converts dihydrokaempferol to dihydroquercetin, may play a key role in plant protection from UVB. Mutant plants of both species lacking this F3'H gene were grown under similar UV conditions. Leaves of the mutant Arabidopsis plant (tt7) did not contain quercetin, even under the enhanced UVB treatment. Under the low UVB treatment the total amount of flavonol was similar to the wild-type (Ler), but with increasing UVB, total flavonol (i.e. kaempferol) levels were significantly higher than in similarly treated wild type plants. In the Petunia F3'H mutant, low levels of quercetin were found even in the low UVB treatment, which indicates this variety may be producing some quercetin via an alternative pathway. Under UVB radiation, total flavonoids increased to levels significantly higher than in similarly treated wild type plants, and most of this material was kaempferol. These observations suggest that quercetin is the preferred protective flavonol in wild type plants, due perhaps to enhanced antioxidant or free radical scavenging activity. In mutant plants lacking the F3'H enzyme, the response is to produce a larger amount of a less effective photoprotectant

Effects of regionally applied heating on the respiration of wild type ...

African Journals Online (AJOL)

Nocturnal dark respiration (Rn) in wild type and transgenic soybean plants ... Illinois, USA under ambient and elevated CO2 conditions was examined in this study. ... Experimental plants were transferred to a controlled growth chamber at V4 ...

Comparative analysis of the protein compositions between wild type and body color mutant of helicoverpa armigera adult

International Nuclear Information System (INIS)

He Lihua; Chen Jin'e; Liu Yan; Wang Yongqiang; Liu Peigang; Meng Zhiqi

2012-01-01

To gain an in-depth understanding of the fineness and regulation mechanism of body color mutant of Helicoverpa armigera Hbner, the protein composition differences between adult of dominant mutant, recessive mutant and wild type were studied using the SDS-PAGE combined with MALDI-TOF-TOF/MS and bioinformatics analysis. The results indicated that the protein composition of the dominant mutant and wild type had little difference. However, there were obvious differences between the recessive mutant and wild-type. Three specific stripe were chosen for mass spectrometry and bioinformatics analysis, and two types of proteins related to energy metabolism and cytoskeleton were identified. These findings suggested that the two types of proteins may be associated with occurrence and regulation of body color mutant traits of H. armigera. (authors)

Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

Science.gov (United States)

Juréen, P; Angeby, K; Sturegård, E; Chryssanthou, E; Giske, C G; Werngren, J; Nordvall, M; Johansson, A; Kahlmeter, G; Hoffner, S; Schön, T

2010-05-01

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

Sabin and wild type polioviruses from children who presented with acute flaccid paralysis in Nigeria.

Science.gov (United States)

Adedeji, A O; Okonko, I O; Adu, F D

2012-09-01

Sensitive poliovirus surveillance to detect vaccine-derived-polioviruses will continue to increase in importance. Isolating and identifying poliovirus strains from children of pediatrics age in Nigeria. A total of 120 fecal samples were randomly collected from children under the age of five who presented with acute flaccid paralysis. Samples were tested by tissue culture technique and further characterized by intratypic differentiation testing using ELISA and PCR methods. The study confirmed the presence of 22(18.3%) enteroviral isolates comprising 19(86.4%) polioviruses and 3(13.6%) non-polio enteroviruses. These 19 polioviruses include: Sabin-type poliovirus-1 (15.8%), poliovirus-2 (10.5%), poliovirus-3 (10.5%) and wild-type poliovirus-1 (63.2%) isolates. It showed that poliovirus infection was higher in children ages 6-11 months (18.9%), females (18.4%), northern states (91.0%) with no vaccination record (75.0%). Wild-type poliovirus-1 was isolated from the stool samples of 12(54.6%) children from northern states and in all age groups except 18-23 months. No significant differences (P >0.05) between poliovirus infection and age (18.9% vs. 17.7%; 81.9% vs. 18.2%) and sex (18.3% vs. 18.4%). There was significant differences (Pvaccination (75.0% vs. 0.0%). No wild-type poliovirus was found in those with complete vaccination. This study further confirms the presence of Sabin and wild-type poliovirus among children in Nigeria. The isolation of Sabin strain of poliovirus is advantageous to the polio eradication program as it is capable of inducing natural immunity in susceptible hosts. Transmission of wild-type poliovirus among children with incomplete vaccination poses a serious threat to polio eradication program in Nigeria. Environmental and serological surveillance with larger sample size are important for monitoring poliovirus circulation in Nigeria.

Repair of gamma radiation damage in wild type and a radiation sensitive mutant of Deinococcus radiodurans

International Nuclear Information System (INIS)

Mizuma, Nagayo

1989-01-01

In an effort to examine production and repair of radiation-induced single and double strand breaks in the DNA, a repair-deficient wild type and a repair-deficient mutant, UV17, of Deinococcus radiodurans were subjected to Co-60 gamma irradiation at a dose rate of 6.3 kGy/hr for wild type and 3.9 kGy/hr for UV17 mutant. The shoulder of the curve of UV17 mutant was narrow but existed with the intercept of 0.7 kGy and the corresponding value of the wild type was 4.2 kGy. Mutant cells exhibited about 6 fold increases in sensitivity for the shoulder relative to the wild type. The D 37 doses in the wild type and the mutant were 0.57 kGy and 0.25 kGy, respectively. From the survival curves, difference in the sensitivity between two strains was mainly due to difference of repair capacity than the number of radiation sensitive target. Sedimentation rate of the main component in the irradiated cells of UV17 mutant increased almost to the level of unirradiated control by the postincubation at 30deg C for 3 hrs. The results indicated that this sensitive mutant also exhibited an ability to restore single strand breaks after exposure to a sublethal dose of 0.6 kGy. When restitution of double strand breaks was analyzed by sedimentation in a neutral sucrose gradient, the wild type showed restitution to DNA-membrane complex from large part of the breaks. For UV17 mutant, the apparent increase in DNA-membrane complex formation was seen after 3 hours incubation. Large part of the decrease in the activities of peak 2 was recovered in the peak 1 for the wild type. For the mutant, there was little restitution to peak 1. Almost free DNA component in UV17 mutant, therefore, was merely degraded into shorter pieces. Restoration of DNA-membrane complex from free DNA derived from gamma-ray induced double strand scission involved closely in the repair of gamma-induced damage and survival. (N.K.)

Parasitic castration in Fissurella crassa (Archaeogastropoda) due to an adult Digenea, Proctoeces lintoni (Fellodistomidae)

OpenAIRE

Oliva,Marcelo E.

1992-01-01

Specimens of Fissurella crassa (Archaeogastropoda) from Ilo, southern Perú, are infected with the adult stage of the digenetic trematode Proctoeces lintoni (Fellodistomidae). The histopatological analysis of the male and female gonads show a strong effect of the parasite on the structure and function of these organs. P. lintoni live unencysted in the gonads, and the main mechanical damage is originated by the action of a well developed acetabulum. Chemical actions of parasitic secretions may ...

Genetic recombination of tick-borne flaviviruses among wild-type strains.

Science.gov (United States)

Norberg, Peter; Roth, Anette; Bergström, Tomas

2013-06-05

Genetic recombination has been suggested to occur in mosquito-borne flaviviruses. In contrast, tick-borne flaviviruses have been thought to evolve in a clonal manner, although recent studies suggest that recombination occurs also for these viruses. We re-analyzed the data and found that previous conclusions on wild type recombination were probably falsely drawn due to misalignments of nucleotide sequences, ambiguities in GenBank sequences, or different laboratory culture histories suggestive of recombination events in laboratory. To evaluate if reliable predictions of wild type recombination of tick-borne flaviviruses can be made, we analyzed viral strains sequenced exclusively for this study, and other flavivirus sequences retrieved from GenBank. We detected genetic signals supporting recombination between viruses within the three clades of TBEV-Eu, TBEV-Sib and TBEV-Fe, respectively. Our results suggest that the tick-borne encephalitis viruses may undergo recombination under natural conditions, but that geographic barriers restrict most recombination events to involve only closely genetically related viruses. Copyright © 2013 Elsevier Inc. All rights reserved.

Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections▿

Science.gov (United States)

Juréen, P.; Ängeby, K.; Sturegård, E.; Chryssanthou, E.; Giske, C. G.; Werngren, J.; Nordvall, M.; Johansson, A.; Kahlmeter, G.; Hoffner, S.; Schön, T.

2010-01-01

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis. PMID:20237102

Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

Energy Technology Data Exchange (ETDEWEB)

Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey, E-mail: carey.pope@okstate.edu

2011-11-15

Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and

Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

International Nuclear Information System (INIS)

Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey

2011-01-01

Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (−/−) mice. Mice of both genotypes (n = 5–6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82–95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 μM) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20–23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: ► C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. ► Wild type and cannabinoid CB1 receptor knockout littermates

Omics for Investigating Chitosan as an Antifungal and Gene Modulator

Directory of Open Access Journals (Sweden)

Federico Lopez-Moya

2016-03-01

Full Text Available Chitosan is a biopolymer with a wide range of applications. The use of chitosan in clinical medicine to control infections by fungal pathogens such as Candida spp. is one of its most promising applications in view of the reduced number of antifungals available. Chitosan increases intracellular oxidative stress, then permeabilizes the plasma membrane of sensitive filamentous fungus Neurospora crassa and yeast. Transcriptomics reveals plasma membrane homeostasis and oxidative metabolism genes as key players in the response of fungi to chitosan. A lipase and a monosaccharide transporter, both inner plasma membrane proteins, and a glutathione transferase are main chitosan targets in N. crassa. Biocontrol fungi such as Pochonia chlamydosporia have a low content of polyunsaturated free fatty acids in their plasma membranes and are resistant to chitosan. Genome sequencing of P. chlamydosporia reveals a wide gene machinery to degrade and assimilate chitosan. Chitosan increases P. chlamydosporia sporulation and enhances parasitism of plant parasitic nematodes by the fungus. Omics studies allow understanding the mode of action of chitosan and help its development as an antifungal and gene modulator.

Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

Science.gov (United States)

Ren, Zhao-Yu; Xu, Xiao-Ming; Wang, Shui-Cai; Xin, Yue-Yong; He, Jun-Fang; Hou, Xun

2003-10-01

A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wild-type rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wild-type. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

Structural Characterization of Lignin in Wild-Type versus COMT Down-Regulated Switchgrass

Energy Technology Data Exchange (ETDEWEB)

Samuel, Reichel [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States); Pu, Yunqiao, E-mail: yunqiao.pu@ipst.gatech.edu [BioEnergy Science Center, Oak Ridge, TN (United States); Institute of Paper Science and Technology, Georgia Institute of Technology, Atlanta, GA (United States); Jiang, Nan [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States); Fu, Chunxiang [Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK (United States); Wang, Zeng-Yu [BioEnergy Science Center, Oak Ridge, TN (United States); Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK (United States); Ragauskas, Arthur, E-mail: yunqiao.pu@ipst.gatech.edu [School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA (United States); BioEnergy Science Center, Oak Ridge, TN (United States)

2014-01-20

This study examined the chemical structural characteristics of cellulolytic enzyme lignin isolated from switchgrass focusing on comparisons between wild-type control and caffeic acid 3-O-methyltransferase (COMT) down-regulated transgenic line. Nuclear magnetic resonance techniques including {sup 13}C, {sup 31}P, and two-dimensional {sup 13}C-{sup 1}H heteronuclear single quantum coherence as well as gel permeation chromatography were employed. Compared to the wild-type, the COMT down-regulated transgenic switchgrass lignin demonstrated a decrease in syringyl (S):guaiacyl (G) ratio and p-coumarate:ferulate ratio, an increase in relative abundance of phenylcoumaran unit, and a comparable content of total free phenolic OH groups along with formation of benzodioxane unit. In addition, COMT down-regulation had no significant effects on the lignin molecular weights during its biosynthesis process.

An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis

Directory of Open Access Journals (Sweden)

Melissa S Rotunno

2013-12-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s of sporadic ALS, which accounts for approximately 90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1 protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a toxic conformation that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS.

Dicty_cDB: Contig-U01385-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available DRAF... 46 2.0 1 ( EJ296094 ) 1095388038537 Global-Ocean-Sampling_GS-27-01-01-1... 46 2.0 1 ( CB895163 ) EST647955 HOGA Med...067741 |pid:none) Homo sapiens proteasome (prosome, ... 35 2.7 AF323913_1( AF323913 |pid:none) Neurospora crassa interm...094293 ) 1092962025663 Global-Ocean-Sampling_GS-31-01-01-1... 36 1.4 2 ( CU570868 ) M.truncatula DNA sequenc...:none) Leishmania major strain Friedlin... 37 0.94 CP001213_467( CP001213 |pid:none) Bifidobacterium animali...icago truncatula clone mth2-165c4, complete se... 46 2.0 1 ( AC150776 ) Medicago truncatula c

No evidence for functional inactivation of wild-type p53 protein by MDM2 overexpression in gastric carcinogenesis

NARCIS (Netherlands)

Blok, P.; Craanen, M. E.; Dekker, W.; Offerhaus, G. J.; Tytgat, G. N.

1998-01-01

Inactivation of wild-type p53 during gastric carcinogenesis is usually caused by mutations within exons 5-8 of the p53 gene leading to mutated, usually immunohistochemically detectable p53 proteins. However, functional inactivation of wild-type p53, mimicking mutational inactivation, may also result

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines

Directory of Open Access Journals (Sweden)

ÁNGELA D ARMENDÁRIZ

2006-01-01

Full Text Available The role of metallothioneins (MT in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-. As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80% belong to two major categories: 1 metabolism; and 2 cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.

Panitumumab and pegylated liposomal doxorubicin in platinum-resistant epithelial ovarian cancer with KRAS wild-type

DEFF Research Database (Denmark)

Steffensen, Karina Dahl; Waldstrøm, Marianne; Pallisgård, Niels

2013-01-01

OBJECTIVE: The increasing number of negative trials for ovarian cancer treatment has prompted an evaluation of new biologic agents, which in combination with chemotherapy may improve survival. The aim of this study was to investigate the response rate in platinum-resistant, KRAS wild-type ovarian...... cancer patients treated with pegylated liposomal doxorubicin (PLD) supplemented with panitumumab. PATIENTS AND METHODS: Major eligibility criteria were relapsed ovarian/fallopian/peritoneal cancer patients with platinum-resistant disease, measurable disease by GCIG CA125 criteria and KRAS wild-type...

La formacion de anillos de crecimiento en Fissurella crassa en el norte de Chile

OpenAIRE

Bretos, Marta

1980-01-01

Growth discontinuity in molluscs result in the formation of rings on their shells. Two kind of rings may be formed: disturbance and growth rings. Growth rings can be used to determine the age of molluscs with long life span. For this, it is necessary to know how many growth rings are formed per year. It has been demonstrated experimentally, using marked specimens, that F. crassa forms two growth rings per year at Huayquique, Northern Chile. They are formed during winter and summer. Disturbanc...

Cloning of an E. coli RecA and yeast RAD51 homolog, radA, an allele of the uvsC in Aspergillus nidulans and its mutator effects.

Science.gov (United States)

Seong, K Y; Chae, S K; Kang, H S

1997-04-30

An E. coli RecA and yeast RAD51 homolog from Aspergillus nidulans, radA, has been cloned by screening genomic and cDNA libraries with a PCR-amplified probe. This probe was generated using primers carrying the conserved sequences of eukaryotic RecA homologs. The deduced amino acid sequence revealed two conserved Walker-A and -B type nucleotide-binding domains and exhibited 88%, 60%, and 53% identity with Mei-3 of Neurospora crassa, rhp51+ of Schizosaccharomyces pombe, and Rad51 of Saccharomyces cerevisiae, respectively. radA null mutants constructed by replacing the whole coding region with a selection marker showed high methyl methanesulfonate (MMS) sensitivity. Heterozygous diploids of radA disruptant with the uvsC114 mutant failed to complement with respect to MMS-sensitivity, indicating that radA is an allele of uvsC. In selecting spontaneous forward selenate resistant mutations, mutator effects were observed in radA null mutants similarly to those shown in uvsC114 mutant strains.

Evolution and diversity of a fungal self/nonself recognition locus.

Directory of Open Access Journals (Sweden)

Charles Hall

2010-11-01

Full Text Available Self/nonself discrimination is an essential feature for pathogen recognition and graft rejection and is a ubiquitous phenomenon in many organisms. Filamentous fungi, such as Neurospora crassa, provide a model for analyses of population genetics/evolution of self/nonself recognition loci due to their haploid nature, small genomes and excellent genetic/genomic resources. In N. crassa, nonself discrimination during vegetative growth is determined by 11 heterokaryon incompatibility (het loci. Cell fusion between strains that differ in allelic specificity at any of these het loci triggers a rapid programmed cell death response.In this study, we evaluated the evolution, population genetics and selective mechanisms operating at a nonself recognition complex consisting of two closely linked loci, het-c (NCU03493 and pin-c (NCU03494. The genomic position of pin-c next to het-c is unique to Neurospora/Sordaria species, and originated by gene duplication after divergence from other species within the Sordariaceae. The het-c pin-c alleles in N. crassa are in severe linkage disequilibrium and consist of three haplotypes, het-c1/pin-c1, het-c2/pin-c2 and het-c3/pin-c3, which are equally frequent in population samples and exhibit trans-species polymorphisms. The absence of recombinant haplotypes is correlated with divergence of the het-c/pin-c intergenic sequence. Tests for positive and balancing selection at het-c and pin-c support the conclusion that both of these loci are under non-neutral balancing selection; other regions of both genes appear to be under positive selection. Our data show that the het-c2/pin-c2 haplotype emerged by a recombination event between the het-c1/pin-c1 and het-c3/pin-c3 approximately 3-12 million years ago.These results support models by which loci that confer nonself discrimination form by the association of polymorphic genes with genes containing HET domains. Distinct allele classes can emerge by recombination and positive

Cryo-EM Structure of the TOM Core Complex from Neurospora crassa.

Science.gov (United States)

Bausewein, Thomas; Mills, Deryck J; Langer, Julian D; Nitschke, Beate; Nussberger, Stephan; Kühlbrandt, Werner

2017-08-10

The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the β-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. Our structure offers detailed insights into the molecular architecture of the mitochondrial preprotein import machinery. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of dietary supplementation with Rhizopus oryzae or Chrysonilia crassa on growth performance, blood profile, intestinal microbial population, and carcass traits in broilers exposed to heat stress

OpenAIRE

S. Sugiharto; T. Yudiarti; I. Isroli; E. Widiastuti; F. D. Putra

2017-01-01

Dietary supplementation of additives has recently been part of strategies to deal with the detrimental effects of heat stress (HS) on the performance and carcass traits in broiler chicks. This study aimed to investigate the effect of dietary supplementation with the fungi Rhizopus oryzae or Chrysonilia crassa on growth, blood profile, intestinal microbial population and carcass traits in broiler chicks subjected to HS. R. oryzae and C. crassa are filamentous fungi isolated from...

Automatic Detection of Wild-type Mouse Cranial Sutures

DEFF Research Database (Denmark)

Ólafsdóttir, Hildur; Darvann, Tron Andre; Hermann, Nuno V.

, automatic detection of the cranial sutures becomes important. We have previously built a craniofacial, wild-type mouse atlas from a set of 10 Micro CT scans using a B-spline-based nonrigid registration method by Rueckert et al. Subsequently, all volumes were registered nonrigidly to the atlas. Using......, the observer traced the sutures on each of the mouse volumes as well. The observer outperforms the automatic approach by approximately 0.1 mm. All mice have similar errors while the suture error plots reveal that suture 1 and 2 are cumbersome, both for the observer and the automatic approach. These sutures can...

Liver steatosis study_PFAA treated Wild type and PPAR KO mouse data

Data.gov (United States)

U.S. Environmental Protection Agency — Data set 1 consists of the experimental data for the Wild Type and PPAR KO animal study and includes data used to prepare Figures 1-4 and Table 1 of the Das et al,...

Dose selenomethionine have radio-protective effect on cell lines with wild type p53?

International Nuclear Information System (INIS)

Tsuji, K.; Hagihira, T.; Ohnishi, K.; Ohnishi, T.; Matsumoto, H.

2003-01-01

Full text: Selenium compounds are known to have cancer preventive effects. It is reported recently that selenium in the form of selenomethionine (SeMet) can protect cells with wild type p53 from UV-induced cell killing by activating the DNA repair mechanism of p53 tumor suppressor protein via redox factor Ref1 by reducing p53 cysteine residue 275 and 277. In contrast, SeMet has no protective effect on UV-induced cell killing in p53-null cells. If SeMet also has protective effect in cells with wild type p53 on cell killing by photon irradiation, SeMet can be used as normal tissue radio-protector. We examined the effect of SeMet on cell killing by X-ray irradiation in several cell lines with different p53 status at exponentially growing phase. Cell lines used in this experiment were as follows: H1299/neo; human lung cancer cell line of p53 null type tranfected with control vector with no p53, H1299/wp53; wild type p53 transfected counterpart. A172/neo; human glioblastoma cell line with wild type p53, A172/mp53-248; mp53-248 (248-mutant, ARG >TRP) transfected counterpart. SAS/neo; human tongue cancer cell line with wild type p53, and SAS/mp53-248; mp53-248 transfected counterpart. Cells were subcultured at monolayer in D-MEM containing 10% FBS. Survivals of the cells were determined by colony forming ability. Ten-MV linac X-ray was used to irradiate the cells. Exponentially growing cells were incubated with 20μM of SeMet for 15 hours before irradiation. After 24 hours exposure of SeMet, cells were incubated up to two weeks in growth medium for colony formation. Twenty-four hours exposure of 20μM of SeMet had no cytotoxicity on these cell lines. SeMet had no modification effect on cell killing by photon irradiation in H1299/neo, H1299/wp53, SAS/neo, SAS/mp53-248, and A172/mp53-248. On the other hand, SeMet sensitized A172/neo in radiation cell killing. The effects of p53 on interaction of SeMet and photon irradiation differ according to cell lines

ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

Science.gov (United States)

The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

Anthelmintic effect of Psidium guajava and Tagetes erecta on wild-type and Levamisole-resistant Caenorhabditis elegans strains.

Science.gov (United States)

Piña-Vázquez, Denia M; Mayoral-Peña, Zyanya; Gómez-Sánchez, Maricela; Salazar-Olivo, Luis A; Arellano-Carbajal, Fausto

2017-04-18

Psidium guajava and Tagetes erecta have been used traditionally to treat gastrointestinal parasites, but their active metabolites and mechanisms of action remain largely unknown. To evaluate the anthelmintic potential of Psidium guajava and Tagetes erecta extracts on Levamisole-sensitive and Levamisole-resistant strains of the model nematode Caenorhabditis elegans. Aqueous extracts of Psidium guajava (PGE) and Tagetes erecta (TEE) were assayed on locomotion and egg-laying behaviors of the wild-type (N2) and Levamisole-resistant (CB193) strains of Caenorhabditis elegans. Both extracts paralyzed wild-type and Levamisole-resistant nematodes in a dose-dependent manner. In wild-type worms, TEE 25mg/mL induced a 75% paralysis after 8h of treatment and PGE 25mg/mL induced a 100% paralysis after 4h of treatment. PGE exerted a similar paralyzing effect on N2 wild-type and CB193 Levamisole-resistant worms, while TEE only partially paralyzed CB193 worms. TEE 25mg/mL decreased N2 egg-laying by 65% with respect to the untreated control, while PGE did it by 40%. Psidium guajava leaves and Tagetes erecta flower-heads possess hydrosoluble compounds that block the motility of Caenorhabditis elegans by a mechanism different to that of the anthelmintic drug Levamisole. Effects are also observable on oviposition, which was diminished in the wild-type worms. The strong anthelmintic effects in crude extracts of these plants warrants future work to identify their active compounds and to elucidate their molecular mechanisms of action. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

The global nitrogen regulator, FNR1, regulates fungal nutrition-genes and fitness during Fusarium oxysporum pathogenesis.

Science.gov (United States)

Divon, Hege Hvattum; Ziv, Carmit; Davydov, Olga; Yarden, Oded; Fluhr, Robert

2006-11-01

SUMMARY Fusarium oxysporum is a soil-borne pathogen that infects plants through the roots and uses the vascular system for host ingress. Specialized for this route of infection, F. oxysporum is able to adapt to the scarce nutrient environment in the xylem vessels. Here we report the cloning of the F. oxysporum global nitrogen regulator, Fnr1, and show that it is one of the determinants for fungal fitness during in planta growth. The Fnr1 gene has a single conserved GATA-type zinc finger domain and is 96% and 48% identical to AREA-GF from Gibberella fujikuroi, and NIT2 from Neurospora crassa, respectively. Fnr1 cDNA, expressed under a constitutive promoter, was able to complement functionally an N. crassa nit-2(RIP) mutant, restoring the ability of the mutant to utilize nitrate. Fnr1 disruption mutants showed high tolerance to chlorate and reduced ability to utilize several secondary nitrogen sources such as amino acids, hypoxanthine and uric acid, whereas growth on favourable nitrogen sources was not affected. Fnr1 disruption also abolished in vitro expression of nutrition genes, normally induced during the early phase of infection. In an infection assay on tomato seedlings, infection rate of disruption mutants was significantly delayed in comparison with the parental strain. Our results indicate that FNR1 mediates adaptation to nitrogen-poor conditions in planta through the regulation of secondary nitrogen acquisition, and as such acts as a determinant for fungal fitness during infection.

Comparison of the nucleotide sequence of wild-type hepatitis - A virus and its attenuated candidate vaccine derivative

International Nuclear Information System (INIS)

Cohen, J.I.; Rosenblum, B.; Ticehurst, J.R.; Daemer, R.; Feinstone, S.; Purcell, R.H.

1987-01-01

Development of attenuated mutants for use as vaccines is in progress for other viruses, including influenza, rotavirus, varicella-zoster, cytomegalovirus, and hepatitis-A virus (HAV). Attenuated viruses may be derived from naturally occurring mutants that infect human or nonhuman hosts. Alternatively, attenuated mutants may be generated by passage of wild-type virus in cell culture. Production of attenuated viruses in cell culture is a laborious and empiric process. Despite previous empiric successes, understanding the molecular basis for attenuation of vaccine viruses could facilitate future development and use of live-virus vaccines. Comparison of the complete nucleotide sequences of wild-type (virulent) and vaccine (attenuated) viruses has been reported for polioviruses and yellow fever virus. Here, the authors compare the nucleotide sequence of wild-type HAV HM-175 with that of a candidate vaccine derivative

Histone variant H2A.Z antagonizes the positive effect of the transcriptional activator CPC1 to regulate catalase-3 expression under normal and oxidative stress conditions.

Science.gov (United States)

Dong, Qing; Wang, Yajun; Qi, Shaohua; Gai, Kexin; He, Qun; Wang, Ying

2018-05-05

In eukaryotes, deposition of the histone variant H2A.Z into nucleosomes through the chromatin remodeling complex, SWR1, is a crucial step in modulating gene transcription. Recently, H2A.Z has been shown to control the expression of responsive genes, but the underlying mechanism of how H2A.Z responds to physiological stimuli is not well understood. Here, we reveal that, in Neurospora crassa, H2A.Z is a negative regulator of catalase-3 gene, which is responsible for resistance to oxidative stress. H2A.Z represses cat-3 gene expression through direct incorporation at cat-3 locus in a SWR1 complex dependent pathway. Notably, loss of H2A.Z or SWR1 subunits leads to increased binding of a transcription factor, CPC1, at cat-3 locus. Additionally, introduction of plasmids containing gene encoding H2A.Z or SWR1 complex subunits into wild-type strains decreased CAT-3 expression, indicating that H2A.Z counteracts the positive effect of CPC1 to achieve low level cat-3 expression under non-inductive condition. Furthermore, upon oxidative stress, H2A.Z is rapidly evicted from cat-3 locus for the recruitment of CPC1, resulting in robust and full cat-3 gene expression in response to external stimuli. Collectively, this study strongly demonstrates that H2A.Z antagonizes the function of transcription factor to regulate responsive gene transcription under normal conditions and to poise for gene full activation under oxidative stress. Copyright © 2018. Published by Elsevier Inc.

Identification of Key Residues for Enzymatic Carboxylate Reduction

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Holly Stolterfoht

2018-02-01

Full Text Available Carboxylate reductases (CARs, E.C. 1.2.1.30 generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.

Mycelial pellet formation by edible ascomycete filamentous fungi, Neurospora intermedia.

Science.gov (United States)

Nair, Ramkumar B; Lennartsson, Patrik R; Taherzadeh, Mohammad J

2016-12-01

Pellet formation of filamentous fungi in submerged culture is an imperative topic of fermentation research. In this study, we report for the first time the growth of filamentous ascomycete fungus, Neurospora intermedia in its mycelial pellet form. In submerged culture, the growth morphology of the fungus was successfully manipulated into growing as pellets by modifying various cultivation conditions. Factors such as pH (2.0-10.0), agitation rate (100-150 rpm), carbon source (glucose, arabinose, sucrose, and galactose), the presence of additive agents (glycerol and calcium chloride) and trace metals were investigated for their effect on the pellet formation. Of the various factors screened, uniform pellets were formed only at pH range 3.0-4.0, signifying it as the most influential factor for N. intermedia pellet formation. The average pellet size ranged from 2.38 ± 0.12 to 2.86 ± 0.38 mm. The pellet formation remained unaffected by the inoculum type used and its size showed an inverse correlation with the agitation rate of the culture. Efficient glucose utilization was observed with fungal pellets, as opposed to the freely suspended mycelium, proving its viability for fast-fermentation processes. Scale up of the pelletization process was also carried out in bench-scale airlift and bubble column reactors (4.5 L).

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

International Nuclear Information System (INIS)

Plattet, Philippe; Rivals, Jean-Paul; Zuber, BenoIt; Brunner, Jean-Marc; Zurbriggen, Andreas; Wittek, Riccardo

2005-01-01

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F WT ) and attachment (H WT ) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H WT determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F WT reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

International Nuclear Information System (INIS)

Mitra, S.; Pal, B.C.; Foote, R.S.

1982-01-01

O 6 -Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3 H-labeled O 6 -methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase

Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

DEFF Research Database (Denmark)

Jensen, T G; Andresen, B S; Bross, P

1992-01-01

An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter...... and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild......-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild...

The correlation between the use of personal protective equipment and level wild-type p53 of dental technicians in Surabaya

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Puspa Dila Rohmaniar

2017-03-01

Full Text Available Background: Exposure of metals among dental technicians that come from the working environment can lead to the formation reactive oxygen species (ROS. ROS can cause mutations in the p53 gene (p53. The mutation is transversion mutation GuanineThymine. p53 mutations can lead to low expression of the wild-type p53 protein (p53. Wild-type p53 involved in many biological processes such as regulation of genes involved in cell cycle, cell growth after DNA damage, and apoptosis. However, exposure to metals among dental technicians can be prevented through the use of personal protective equipment (PPE during work. Purpose: The purpose of this study was to analyze the correlation between the use of personal protective equipment to wild-type p53 protein levels among dental technicians in Surabaya. Method: This study was observational analytic with cross sectional approach. 40 samples were taken by random sampling. Data were retrieved through interviews and observations. Wild-type p53 was analyzed from saliva with indirect ELISA method. Analysis of data used Kolmogorov Smirnov normality test and a Pearson correlation test. Value significance was p<0.05 (95% confidence level. Result: There was a significant association between the use of personal protective equipment with wild-type p53 levels with p=0.002 Conclusion: The use PPE properly is positively correlated with the wild-type p53 protein levels of dental technicians in Surabaya.

Massive Changes in Genome Architecture Accompany the Transition to Self-Fertility in the filamentous Fungus Neurospora tetrasperma

Energy Technology Data Exchange (ETDEWEB)

Ellison, Christoper; Stajich, Jason; Jacobson, David; Nativ, Donald; Lapidus, Alla; Foster, Brian; Aerts, Andrea; Riley, Robert; Lindquist, Erika; Grigoriev, Igor; Taylor, John

2011-05-16

A large region of suppressed recombination surrounds the sex-determining locus of the self-fertile fungus Neurospora tetrasperma. This region encompasses nearly one-fifth of the N. tetrasperma genome and suppression of recombination is necessary for self-fertility. The similarity of the N. tetrasperma mating chromosome to plant and animal sex chromosomes and its recent origin (5 MYA), combined with a long history of genetic and cytological research, make this fungus an ideal model for studying the evolutionary consequences of suppressed recombination. Here we compare genome sequences from two N. tetrasperma strains of opposite mating type to determine whether structural rearrangements are associated with the nonrecombining region and to examine the effect of suppressed recombination for the evolution of the genes within it. We find a series of three inversions encompassing the majority of the region of suppressed recombination and provide evidence for two different types of rearrangement mechanisms: the recently proposed mechanism of inversion via staggered single-strand breaks as well as ectopic recombination between transposable elements. In addition, we show that the N. tetrasperma mat a mating-type region appears to be accumulating deleterious substitutions at a faster rate than the other mating type (mat A) and thus may be in the early stages of degeneration.

Effect of uremia on HDL composition, vascular inflammation, and atherosclerosis in wild-type mice

DEFF Research Database (Denmark)

Bang, Christian A; Bro, Susanne; Bartels, Emil D

2007-01-01

Wild-type mice normally do not develop atherosclerosis, unless fed cholic acid. Uremia is proinflammatory and increases atherosclerosis 6- to 10-fold in apolipoprotein E-deficient mice. This study examined the effect of uremia on lipoproteins, vascular inflammation, and atherosclerosis in wild...... in cholic acid-fed sham mice. The results suggest that moderate uremia neither induces aortic inflammation nor atherosclerosis in C57BL/6J mice despite increased LDL/HDL cholesterol ratio and altered HDL composition....

BASAL MEDIA FORMULATION USING CANAVALIA ENSIFORMIS AS CARBON AND NITROGEN SOURCE FOR THE GROWTH OF SOME FUNGI SPECIES

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B.J. Akinyele2

2012-02-01

Full Text Available The possibility of developing alternative media to commercial potato dextrose agar was assessed using, Canavalia ensiformis (Linn (jack beans as carbon and nitrogen source. Six leguminous meal media were used as substitute for either carbon or nitrogen or both, while potato dextrose broth (PDB was used as a positive control and basal medium as a negative control. Six species of fungi Aspergillus flavus, A. niger, Meria coniospora, Mucor sp, Neurospora crassa and Rhizopus oryzae were aseptically inoculated into the formulated media and allowed to grow. Their mycelia dry weights were taken after 24, 48, 72, 96 and 120 hours. Growth of all fungal species was observed to be slightly lower, about the same or better in the formulated media relative to the control. Aspergillus flavus had its highest biomass of 1.70g in the media formulated with Canavalia ensiformis as the carbon source relative to 1.42g as the standard at the 120 hour. A. niger had a growth of 0.62g relative to 0.61g at 120 hours of the control. Meria coniospora had a growth of 0.27g relative to 0.38g at 120 hours. Mucor sp had a growth of 0.54g relative to 0.44g at 120 hours. Neurospora crassa had a growth of 1.05g relative to 0.24g at 120 hours. Rhizopus oryzae had a growth of 0.14g relative to 0.25g at 120 hours. The study revealed that Canavalia ensiformis contains minerals and nutrients that is able to provide the nutritional requirements of these fungi. Thus, it can be used as an alternative material in the preparation of culture media for in vitro cultivation of these fungi for teaching and research purposes.

Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity

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Ivaylo P. Ivanov

2017-06-01

Full Text Available Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs in its >700-nucleotide (nt 5′ leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5′ region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5′ conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro. In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression.

Wild-type minimal inhibitory concentration distributions in bacteria of animal origin in Argentina

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Florencia L Pantozzi

Full Text Available The aim of this study was to determine the antimicrobial resistance profiles of indicator bacteria isolated from domestic animal feces. Minimal inhibitory concentration (MIC was determined by agar dilution. Interpretative criteria on the basis of wild-type MIC distributions and epidemiological cutoff values (ECOFF or ECV were used according to the 'European Committee on Antimicrobial Susceptibility Testing' (EUCAST data. Results from 237 isolates of Escherichia coli showed reduced susceptibility for ampicillin, streptomycin and tetracycline, the antimicrobials commonly used in intensive breeding of pigs and hens. Regarding all the species of the genus Enterococcus spp., there are only ECOFF or ECV for vancomycin. Of the 173 Enterococcus spp. isolated, only one showed reduced susceptibility to vancomycin and was classified as 'non-wild-type' (NWT population. This is the first report in Argentina showing data of epidemiological cutoff values in animal bacteria.

A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

Science.gov (United States)

Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

2014-05-21

Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions. Copyright © 2014 the authors 0270-6474/14/347203-13$15.00/0.

Pediatric Glioblastoma Therapies Based on Patient-Derived Stem Cell Resources

Science.gov (United States)

2014-11-01

Homo sapiens (100) Drosophila melanogaster (65) Oryza sativa (45) Arabidopsis thaliana (42) Neurospora crassa (43) Nagnaporthe grisea (37) 0.2 Evidence...0 0 % W el ls w /o s p h er es 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 R el at iv e g ro w th NSC BTIC CX VM G179 12280131 G14G19 G26022TNHA G166CB660...G166 0 0.4 0.8 1.2 shControl shBUB1B-1 shBUB1B-2 R el at iv e m R N A ex p re ss io n ** ** ** ** BUB1B mRNAC

LILBID-mass spectrometry of the mitochondrial preprotein translocase TOM

International Nuclear Information System (INIS)

Mager, Frauke; Lintzel, Julia; Nussberger, Stephan; Sokolova, Lucie; Brutschy, Bernhard

2010-01-01

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

LILBID-mass spectrometry of the mitochondrial preprotein translocase TOM

Science.gov (United States)

Mager, Frauke; Sokolova, Lucie; Lintzel, Julia; Brutschy, Bernhard; Nussberger, Stephan

2010-11-01

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

La formación de anillos de crecimiento en Fissurella crassa en el norte de Chile

OpenAIRE

Marta Bretos

1980-01-01

Growth discontinuity in molluscs result in the formation of rings on their shells. Two kind of rings may be formed: disturbance and growth rings. Growth rings can be used to determine the age of molluscs with long life span. For this, it is necessary to know how many growth rings are formed per year. It has been demonstrated experimentally, using marked specimens, that F. crassa forms two growth rings per year at Huayquique, Northern Chile. They are formed during winter and summer. Disturbanc...

Interleukin-1 receptor type I gene-deficient mice are less susceptible to Staphylococcus epidermidis biomaterial-associated infection than are wild-type mice

NARCIS (Netherlands)

Boelens, J. J.; van der Poll, T.; Zaat, S. A.; Murk, J. L.; Weening, J. J.; Dankert, J.

2000-01-01

Elevated concentrations of interleukin-1 (IL-1) were found in tissue surrounding biomaterials infected with Staphylococcus epidermidis. To determine the role of IL-1 in biomaterial-associated infection (BAI), IL-1 receptor type I-deficient (IL-1R(-/-)) and wild-type mice received subcutaneous

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

Science.gov (United States)

Gause, K C; Homma, M K; Licciardi, K A; Seger, R; Ahn, N G; Peterson, M J; Krebs, E G; Meier, K E

1993-08-05

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.

Wild-type and mutated IDH1/2 enzymes and therapy responses.

Science.gov (United States)

Molenaar, Remco J; Maciejewski, Jaroslaw P; Wilmink, Johanna W; van Noorden, Cornelis J F

2018-04-01

Isocitrate dehydrogenase 1 and 2 (IDH1/2) are key enzymes in cellular metabolism, epigenetic regulation, redox states, and DNA repair. IDH1/2 mutations are causal in the development and/or progression of various types of cancer due to supraphysiological production of D-2-hydroxyglutarate. In various tumor types, IDH1/2-mutated cancers predict for improved responses to treatment with irradiation or chemotherapy. The present review discusses the molecular basis of the sensitivity of IDH1/2-mutated cancers with respect to the function of mutated IDH1/2 in cellular processes and their interactions with novel IDH1/2-mutant inhibitors. Finally, lessons learned from IDH1/2 mutations for future clinical applications in IDH1/2 wild-type cancers are discussed.

Phase II marker-driven trial of panitumumab and chemotherapy in KRAS wild-type biliary tract cancer

DEFF Research Database (Denmark)

Jensen, L H; Lindebjerg, J; Ploen, J

2012-01-01

BACKGROUND: Combination chemotherapy has proven beneficial in biliary tract cancer and further improvements may be achieved by individualizing treatment based on biomarkers and by adding biological agents. We report the effect of chemotherapy with panitumumab as first-line therapy for KRAS wild....... Combination chemotherapy with panitumumab in patients with KRAS wild-type tumors met the efficacy criteria for future testing in a randomized trial....

A ten-week biochemistry lab project studying wild-type and mutant bacterial alkaline phosphatase.

Science.gov (United States)

Witherow, D Scott

2016-11-12

This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important techniques, students acquire novel biochemical data in their kinetic analysis of mutant enzymes. The experiments are designed to build on students' work from week to week in a way that requires them to apply quantitative analysis and reasoning skills, reinforcing traditional textbook biochemical concepts. Students are assessed through lab reports focused on journal style writing, quantitative and conceptual question sheets, and traditional exams. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):555-564, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

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Cui Shang-jin

2010-05-01

Full Text Available Abstract A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV. A pair of primers (P1 and P4 specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV, canine parvovirus (CPV, canine coronavirus (CCV, rabies virus (RV, or canine adenovirus (CAV. The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus

Science.gov (United States)

2010-01-01

A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance. PMID:20433759

Mid-aged and aged wild-type and progestin receptor knockout (PRKO) mice demonstrate rapid progesterone and 3alpha,5alpha-THP-facilitated lordosis.

Science.gov (United States)

Frye, C A; Sumida, K; Lydon, J P; O'Malley, B W; Pfaff, D W

2006-05-01

Progesterone (P) and its 5alpha-reduced metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP), facilitate sexual behavior of rodents via agonist-like actions at intracellular progestin receptors (PRs) and membrane GABA(A)/benzodiazepine receptor complexes (GBRs), respectively. Given that ovarian secretion of progestins declines with aging, whether or not senescent mice are responsive to progestins was of interest. Homozygous PR knockout (PRKO) or wild-type mice that were between 10-12 (mid-aged) or 20-24 (aged) months of age were administered P or 3alpha,5alpha-THP, and the effect on lordosis were examined. Effects of a progestin-priming regimen that enhances PR-mediated (experiment 1) or more rapid, PR-independent effects of progestins (experiments 2 and 3) on sexual behavior were examined. Levels of P, 3alpha,5alpha-THP, and muscimol binding were examined in tissues from aged mice (experiment 4). Wild-type, but not PRKO, mice were responsive when primed with 17beta-estradiol (E(2); 0.5 microg) and administered P (500 microg, subcutaneously). Mid-aged wild-type mice demonstrated greater increases in lordosis 6 h later compared to their pre-P, baseline test than did aged wild-type mice (experiment 1). Lordosis of younger and older wild-type, but not PRKO, mice was significantly increased within 5 min of intravenous (IV) administration of P (100 ng), compared with E(2)-priming alone (experiment 2). However, wild-type and PRKO mice demonstrated significant increases in lordosis 5 min after IV administration of 3alpha,5alpha-THP, an effect which was more pronounced in mid-aged than in aged animals (100 ng-experiment 3). In tissues from aged wild-type and PRKO mice, levels of P, 3alpha,5alpha-THP, and muscimol binding were increased by P administration (experiment 4). PR binding was lower in the cortex of PRKO than that of wild-type mice. Mid-aged and aged PRKO and wild-type mice demonstrated rapid P or 3alpha,5alpha-THP-facilitated lordosis that may be

Changes in fatty acid content and composition between wild type and CsHMA3 overexpressing Camelina sativa under heavy-metal stress.

Science.gov (United States)

Park, Won; Feng, Yufeng; Kim, Hyojin; Suh, Mi Chung; Ahn, Sung-Ju

2015-09-01

Under heavy-metal stress, CsHMA3 overexpressing transgenic Camelina plants displayed not only a better quality, but also a higher quantity of unsaturated fatty acids in their seeds compared with wild type. Camelina sativa L. belongs to the Brassicaceae family and is frequently used as a natural vegetable oil source, as its seeds contain a high content of fatty acids. In this study, we observed that, when subjected to heavy metals (Cd, Co, Zn and Pb), the seeds of CsHMA3 (Heavy-Metal P1B-ATPase 3) transgenic lines retained their original golden yellow color and smooth outline, unlike wild-type seeds. Furthermore, we investigated the fatty acids content and composition of wild type and CsHMA3 transgenic lines after heavy metal treatments compared to the control. The results showed higher total fatty acid amounts in seeds of CsHMA3 transgenic lines compared with those in wild-type seeds under heavy-metal stresses. In addition, the compositions of unsaturated fatty acids-especially 18:1 (oleic acid), 18:2 (linoleic acid; only in case of Co treatment), 18:3 (linolenic acid) and 20:1 (eicosenoic acid)-in CsHMA3 overexpressing transgenic lines treated with heavy metals were higher than those of wild-type seeds under the same conditions. Furthermore, reactive oxygen species (ROS) contents in wild-type leaves and roots when treated with heavy metal were higher than in CsHMA3 overexpressing transgenic lines. These results indicate that overexpression of CsHMA3 affects fatty acid composition and content-factors that are responsible for the fuel properties of biodiesel-and can alleviate ROS accumulation caused by heavy-metal stresses in Camelina. Due to these factors, we propose that CsHMA3 transgenic Camelina can be used for phytoremediation of metal-contaminated soil as well as for oil production.

The Use of EGFR Tyrosine Kinase Inhibitors in EGFR Wild-Type Non-Small-Cell Lung Cancer.

Science.gov (United States)

Stinchcombe, Thomas E

2016-04-01

The objective response rate and progression-free survival observed with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in patients with metastatic epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) are modest. The adverse events associated with EGFR TKIs are manageable but they must be considered in the context of the limited efficacy. The development of anti-PD-1 immunotherapy as second-line therapy has reduced the role of EGFR TKIs in EGFR wild-type NSCLC. Recently, there has been increased recognition of the benefit of the earlier integration of palliative care and symptom management, and this is reasonable alternative to treatment with an EGFR TKI for many patients. My practice pattern for patients with EGFR wild-type NSCLC is platinum-based chemotherapy as first-line therapy, immunotherapy as second-line therapy, and single-agent chemotherapy as third-line therapy for patients with preserved performance status who want to pursue further therapy. Only a small proportion of patients are eligible for fourth-line therapy, and I prefer to enroll them in clinical trials rather than use EGFR TKIs. I suspect that the use of EGFR TKIs in clinical use and as a comparator arm for clinical trials will continue to decline over the next several years.

Establishment of new transmissible and drug-sensitive human immunodeficiency virus type 1 wild types due to transmission of nucleoside analogue-resistant virus.

Science.gov (United States)

de Ronde, A; van Dooren, M; van Der Hoek, L; Bouwhuis, D; de Rooij, E; van Gemen, B; de Boer, R; Goudsmit, J

2001-01-01

Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.

The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates.

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Busa, Veronica F; Rector, Maxwell J; Russell, Rick

2017-07-18

DEAD-box proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-box protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich "C-tail" that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (k cat /K M ) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or "threshold" number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the helicase core remained active in the mutants. In addition, changes in RNA unwinding efficiency of the mutants were mirrored by changes in functional RNA affinity, as determined from the RNA concentration dependence of ATPase activity, suggesting that the C-tail functions primarily to increase RNA affinity. Interestingly, the salt concentration dependence of RNA unwinding activity is unaffected by C-tail composition, suggesting that the C-tail uses primarily hydrogen bonding, not electrostatic interactions, to bind double-stranded RNA. Our results provide insights into how an unstructured C-tail contributes to DEAD-box protein activity and suggest parallels with other families of RNA- and DNA-binding proteins.

Black bear parathyroid hormone has greater anabolic effects on trabecular bone in dystrophin-deficient mice than in wild type mice.

Science.gov (United States)

Gray, Sarah K; McGee-Lawrence, Meghan E; Sanders, Jennifer L; Condon, Keith W; Tsai, Chung-Jui; Donahue, Seth W

2012-09-01

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease that has deleterious consequences in muscle and bone, leading to decreased mobility, progressive osteoporosis, and premature death. Patients with DMD experience a higher-than-average fracture rate, particularly in the proximal and distal femur and proximal tibia. The dystrophin-deficient mdx mouse is a model of DMD that demonstrates muscle degeneration and fibrosis and osteoporosis. Parathyroid hormone, an effective anabolic agent for post-menopausal and glucocorticoid-induced osteoporosis, has not been explored for DMD. Black bear parathyroid hormone (bbPTH) has been implicated in the maintenance of bone properties during extended periods of disuse (hibernation). We cloned bbPTH and found 9 amino acid residue differences from human PTH. Apoptosis was mitigated and cAMP was activated by bbPTH in osteoblast cultures. We administered 28nmol/kg of bbPTH 1-84 to 4-week old male mdx and wild type mice via daily (5×/week) subcutaneous injection for 6 weeks. Vehicle-treated mdx mice had 44% lower trabecular bone volume fraction than wild type mice. No changes were found in femoral cortical bone geometry or mechanical properties with bbPTH treatment in wild type mice, and only medio-lateral moment of inertia changed with bbPTH treatment in mdx femurs. However, μCT analyses of the trabecular regions of the distal femur and proximal tibia showed marked increases in bone volume fraction with bbPTH treatment, with a greater anabolic response (7-fold increase) in mdx mice than wild type mice (2-fold increase). Trabecular number increased in mdx long bone, but not wild type bone. Additionally, greater osteoblast area and decreased osteoclast area were observed with bbPTH treatment in mdx mice. The heightened response to PTH in mdx bone compared to wild type suggests a link between dystrophin deficiency, altered calcium signaling, and bone. These findings support further investigation of PTH as an anabolic

Effects of age and liquid holding on the UV-radiation sensitivities of wild-type and mutant Caenorhabditis elegans dauer larvae

Energy Technology Data Exchange (ETDEWEB)

Hartman, P S

1984-01-01

The dauer larva is a facultative developmental stage in the life cycle of the nematode Caenorhabditis elegans. Dauer larvae, which can survive under starvation for over 60 days, resume normal development when feeding is resumed. Wild-type (N2) and 4 radiation-sensitive (rad) mutant dauer larvae were tested for their abilities to develop into adults after UV-irradiation. The rad-3 mutant was over 30 times as sensitive as N2; rad-1, rad-2 and rad-7 mutants were not hypersensitive. Irradiation also delayed development in survivors. Wild-type dauer larvae did not differ in radiation sensitivity from 0 through 50 days of age. There was no liquid holding recovery (LHR); that is, survival did not increase when wild-type dauer larvae were held in buffer after irradiation. (orig.). 28 refs.; 4 figs.

PBI creams: a spontaneously mutated mouse strain showing wild animal-type reactivity.

Science.gov (United States)

Hendrie, C A; Van Driel, K S; Talling, J C; Inglis, I R

2001-01-01

PBI creams are mice derived from warfarin-resistant wild stock that has been maintained under laboratory conditions since the 1970s. This study compares their behaviour to that of laboratory mice and wild house and wood mice. Animals were tested in a black/white box and a 2.64x1.4 m runway. In the black/white box, the behaviour of PBI creams was not significantly different from that of house mice and differed most from that of laboratory mice. Notably, the PBI creams showed the greatest activity and escape-orientated behaviours. When animals were approached by the experimenter in the open runway test, the PBI creams had higher flight speeds than both house and wood mice, whilst laboratory mice failed to respond. In the closed runway test where the animals could not escape, the PBI creams, house mice and wood mice all turned and attempted to run past the approaching experimenter, whilst the laboratory mice again failed to react. At the end of this test session, the time taken to catch each animal was recorded. It took less than 5 s to catch laboratory mice but significantly longer to catch the wild strains and the PBI creams (90-100 s for the latter). In these tests, the PBI creams showed wild animal-type reactivity, and as this behaviour has been retained in the laboratory colony for over 30 years, these animals may be useful in the study of the physiological and genetic basis of fear/anxiety in mice.

Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine.

Directory of Open Access Journals (Sweden)

Victoria Matyushenko

Full Text Available Live attenuated influenza vaccines (LAIVs are considered as safe and effective tool to control influenza in different age groups, especially in young children. An important part of the LAIV safety evaluation is the detection of vaccine virus replication in the nasopharynx of the vaccinees, with special attention to a potential virus transmission to the unvaccinated close contacts. Conducting LAIV clinical trials in some geographical regions with year-round circulation of influenza viruses warrants the development of robust and reliable tools for differentiating vaccine viruses from wild-type influenza viruses in nasal pharyngeal wash (NPW specimens of vaccinated subjects. Here we report the development of genotyping assay for the detection of wild-type and vaccine-type influenza virus genes in NPW specimens of young children immunized with Russian-backbone seasonal trivalent LAIV using Sanger sequencing from newly designed universal primers. The new primer set allowed amplification and sequencing of short fragments of viral genes in NPW specimens and appeared to be more sensitive than conventional real-time RT-PCR protocols routinely used for the detection and typing/subtyping of influenza virus in humans. Furthermore, the new assay is capable of defining the origin of wild-type influenza virus through BLAST search with the generated sequences of viral genes fragments.

Progenesis in Proctoeces lintoni (Fellodistomidae), a parasite of Fissurella crassa (Archaeogastropoda) in a latitudinal gradient in the Pacific Coast of South America.

Science.gov (United States)

Oliva, M E; Huaquin, L G

2000-08-01

The fellodistomid Proctoeces lintoni is a common parasite of the gonads of key-hole limpets Fissurella spp. (Archaeogastropoda). It has also been found in the mantle of Octopus vulgaris and as an intestinal parasite of haemulid and gobiesocid fishes. Fissurella crassa, a host for progenetic P. lintoni, can be found from Huarmey, Peni (10 degrees S) to Chiloé, Chile (42 degrees S). Proctoeces lintoni has been found parasitizing fishes and molluscs from Callao, Peni (12 degrees S) to Valdivia, Chile (39 degrees S). Progenesis is thought to be a latitude-dependent phenomenon, and high progenesis is expected at higher latitude. In the present article, the association between latitude and progenesis was examined over a latitudinal gradient of about 3,000 km. Data suggest that progenesis of P. lintoni infecting F. crassa was not associated with latitude. Low levels of progenesis found in the Peruvian population could be a consequence of parasite-induced mortality rather than of low latitude, as would be predicted by the latitude dependence hypothesis.

Unique Safety Issues Associated with Virus Vectored Vaccines: Potential for and Theoretical Consequences of Recombination with Wild Type Virus Strains

Science.gov (United States)

Condit, Richard C.; Williamson, Anna-Lise; Sheets, Rebecca; Seligman, Stephen J.; Monath, Thomas P.; Excler, Jean-Louis; Gurwith, Marc; Bok, Karin; Robertson, James S.; Kim, Denny; Hendry, Michael; Singh, Vidisha; Mac, Lisa M.; Chen, Robert T.

2016-01-01

In 2003 and 2013, the World Health Organization convened informal consultations on characterization and quality aspects of vaccines based on live virus vectors. In the resulting reports, one of several issues raised for future study was the potential for recombination of virus-vectored vaccines with wild type pathogenic virus strains. This paper presents an assessment of this issue formulated by the Brighton Collaboration. To provide an appropriate context for understanding the potential for recombination of virus-vectored vaccines, we review briefly the current status of virus vectored vaccines, mechanisms of recombination between viruses, experience with recombination involving live attenuated vaccines in the field, and concerns raised previously in the literature regarding recombination of virus-vectored vaccines with wild type virus strains. We then present a discussion of the major variables that could influence recombination between a virus-vectored vaccine and circulating wild type virus and the consequences of such recombination, including intrinsic recombination properties of the parent virus used as a vector; sequence relatedness of vector and wild virus; virus host range, pathogenesis and transmission; replication competency of vector in target host; mechanism of vector attenuation; additional factors potentially affecting virulence; and circulation of multiple recombinant vectors in the same target population. Finally, we present some guiding principles for vector design and testing intended to anticipate and mitigate the potential for and consequences of recombination of virus-vectored vaccines with wild type pathogenic virus strains. PMID:27346303

Exploratory biomarker analysis for treatment response in KRAS wild type metastatic colorectal cancer patients who received cetuximab plus irinotecan

International Nuclear Information System (INIS)

Kim, Seung Tae; Ahn, Tae Jin; Lee, Eunjin; Do, In-Gu; Lee, Su Jin; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Kim, Suk Hyeong; Lee, Jeeyun; Kim, Hee Cheol

2015-01-01

More than half of the patients selected based on KRAS mutation status fail to respond to the treatment with cetuximab in metastatic colorectal cancer (mCRC). We designed a study to identify additional biomarkers that could act as indicators for cetuximab treatment in mCRC. We investigated 58 tumor samples from wild type KRAS CRC patients treated with cetuximab plus irinotecan (CI). We conducted the genotyping for mutations in either BRAF or PIK3CA and profiled comprehensively the expression of 522 kinase genes. BRAF mutation was detected in 5.1 % (3/58) of patients. All 50 patients showed wild type PIK3CA. Gene expression patterns that categorized patients with or without the disease control to CI were compared by supervised classification analysis. PSKH1, TLK2 and PHKG2 were overexpressed significantly in patients with the disease control to IC. The higher expression value of PSKH1 (r = 0.462, p < 0.001) and TLK2 (r = 0.361, p = 0.005) had the significant correlation to prolonged PFS. The result of this work demonstrated that expression nature of kinase genes such as PSKH1, TLK2 and PHKG2 may be informative to predict the efficacy of CI in wild type KRAS CRC. Mutations in either BRAF or PIK3CA were rare subsets in wild type KRAS CRC

MLH1-deficient Colorectal Carcinoma With Wild-type BRAF and MLH1 Promoter Hypermethylation Harbor KRAS Mutations and Arise From Conventional Adenomas.

Science.gov (United States)

Farchoukh, Lama; Kuan, Shih-Fan; Dudley, Beth; Brand, Randall; Nikiforova, Marina; Pai, Reetesh K

2016-10-01

Between 10% and 15% of colorectal carcinomas demonstrate sporadic DNA mismatch-repair protein deficiency as a result of MLH1 promoter methylation and are thought to arise from sessile serrated adenomas, termed the serrated neoplasia pathway. Although the presence of the BRAF V600E mutation is indicative of a sporadic cancer, up to 30% to 50% of colorectal carcinomas with MLH1 promoter hypermethylation will lack a BRAF mutation. We report the clinicopathologic and molecular features of MLH1-deficient colorectal carcinoma with wild-type BRAF and MLH1 promoter hypermethylation (referred to as MLH1-hypermethylated BRAF wild-type colorectal carcinoma, n=36) in comparison with MLH1-deficient BRAF-mutated colorectal carcinoma (n=113) and Lynch syndrome-associated colorectal carcinoma (n=36). KRAS mutations were identified in 31% of MLH1-hypermethylated BRAF wild-type colorectal carcinomas compared with 0% of MLH1-deficient BRAF-mutated colorectal carcinomas and 37% of Lynch syndrome-associated colorectal carcinomas. When a precursor polyp was identified, MLH1-hypermethylated BRAF wild-type colorectal carcinomas arose from precursor polyps resembling conventional tubular/tubulovillous adenomas in contrast to MLH1-deficient BRAF-mutated colorectal carcinomas, which arose from precursor sessile serrated adenomas (PMLH1-hypermethylated BRAF wild-type colorectal carcinoma and MLH1-deficient BRAF-mutated colorectal carcinoma had a predilection for the right colon compared with Lynch syndrome-associated colorectal carcinoma (86% vs. 92% vs. 49%, P0.05). In conclusion, our results indicate that MLH1-hypermethylated BRAF wild-type colorectal carcinomas can harbor KRAS mutations and arise from precursor polyps resembling conventional tubular/tubulovillous adenomas.

Mineral phosphate solubilization by wild type and radiation induced mutants of pantoea dispersa and pantoea terrae

International Nuclear Information System (INIS)

Murugesan, Senthilkumar; Lee, Young Keun; Kim, Jung Hun

2009-01-01

Three mineral phosphate solubilizing (MPS) bacteria where isolated from rhizosphere soil samples of common bean and weed plants. 16S rDNA analysis indicated that the isolate P2 and P3 are closely related to Pantoea dispersa while isolate P4 is closely related to Pantoea terrae. Islates P2 and P3 recorded 381.60 μg ml -1 of tricalcium phosphate (TCP) solubilization respectively on 3 days incubation. Isolate P4 recorded the TCP solubilization of 215.85 μg ml -1 and the pH was dropped to 4.44 on 24 h incubation. Further incubation of P4 sharply decreased the available phosphorous to 28.94 μg ml -1 and pH level was raised to 6.32. Gamma radiation induced mutagenesis was carried out at LD 99 dose of the wild type strains. The total of 14 mutant clones with enhanced MPS activity and 4 clones with decreased activity were selected based on solubilization index (SI) and phosphate solubilization assay. Mutant P2-M1 recorded the highest P-solubilizing potential among any other wild or mutnat clones by releasing 504.21 μg ml -1 of phosphorous i.e. 35% higher than its wild type by the end of day 5. A comparative evaluation of TCP solubilization by wild type isolates of Pantoea and their mutants, led to select three MPS mutant clones such as P2-M1, P3-M2 and P3-M4 with a potential to release >471.67 μg ml 1 of phosphorous from TCP. These over expressing mutant clones are considered as suitable candidates for biofertilization

Fitness of crop-wild hybrid sunflower under competitive conditions: implications for crop-to-wild introgression.

Science.gov (United States)

Mercer, Kristin L; Emry, D Jason; Snow, Allison A; Kost, Matthew A; Pace, Brian A; Alexander, Helen M

2014-01-01

Understanding the likelihood and extent of introgression of novel alleles in hybrid zones requires comparison of lifetime fitness of parents and hybrid progeny. However, fitness differences among cross types can vary depending on biotic conditions, thereby influencing introgression patterns. Based on past work, we predicted that increased competition would enhance introgression between cultivated and wild sunflower (Helianthus annuus) by reducing fitness advantages of wild plants. To test this prediction, we established a factorial field experiment in Kansas, USA where we monitored the fitness of four cross types (Wild, F1, F2, and BCw hybrids) under different levels of interspecific and intraspecific competition. Intraspecific manipulations consisted both of density of competitors and of frequency of crop-wild hybrids. We recorded emergence of overwintered seeds, survival to reproduction, and numbers of seeds produced per reproductive plant. We also calculated two compound fitness measures: seeds produced per emerged seedling and seeds produced per planted seed. Cross type and intraspecific competition affected emergence and survival to reproduction, respectively. Further, cross type interacted with competitive treatments to influence all other fitness traits. More intense competition treatments, especially related to density of intraspecific competitors, repeatedly reduced the fitness advantage of wild plants when considering seeds produced per reproductive plant and per emerged seedling, and F2 plants often became indistinguishable from the wilds. Wild fitness remained superior when seedling emergence was also considered as part of fitness, but the fitness of F2 hybrids relative to wild plants more than quadrupled with the addition of interspecific competitors and high densities of intraspecific competitors. Meanwhile, contrary to prediction, lower hybrid frequency reduced wild fitness advantage. These results emphasize the importance of taking a full life cycle

Fitness of crop-wild hybrid sunflower under competitive conditions: implications for crop-to-wild introgression.

Directory of Open Access Journals (Sweden)

Kristin L Mercer

Full Text Available Understanding the likelihood and extent of introgression of novel alleles in hybrid zones requires comparison of lifetime fitness of parents and hybrid progeny. However, fitness differences among cross types can vary depending on biotic conditions, thereby influencing introgression patterns. Based on past work, we predicted that increased competition would enhance introgression between cultivated and wild sunflower (Helianthus annuus by reducing fitness advantages of wild plants. To test this prediction, we established a factorial field experiment in Kansas, USA where we monitored the fitness of four cross types (Wild, F1, F2, and BCw hybrids under different levels of interspecific and intraspecific competition. Intraspecific manipulations consisted both of density of competitors and of frequency of crop-wild hybrids. We recorded emergence of overwintered seeds, survival to reproduction, and numbers of seeds produced per reproductive plant. We also calculated two compound fitness measures: seeds produced per emerged seedling and seeds produced per planted seed. Cross type and intraspecific competition affected emergence and survival to reproduction, respectively. Further, cross type interacted with competitive treatments to influence all other fitness traits. More intense competition treatments, especially related to density of intraspecific competitors, repeatedly reduced the fitness advantage of wild plants when considering seeds produced per reproductive plant and per emerged seedling, and F2 plants often became indistinguishable from the wilds. Wild fitness remained superior when seedling emergence was also considered as part of fitness, but the fitness of F2 hybrids relative to wild plants more than quadrupled with the addition of interspecific competitors and high densities of intraspecific competitors. Meanwhile, contrary to prediction, lower hybrid frequency reduced wild fitness advantage. These results emphasize the importance of taking

Fitness of Crop-Wild Hybrid Sunflower under Competitive Conditions: Implications for Crop-to-Wild Introgression

Science.gov (United States)

Mercer, Kristin L.; Emry, D. Jason; Snow, Allison A.; Kost, Matthew A.; Pace, Brian A.; Alexander, Helen M.

2014-01-01

Understanding the likelihood and extent of introgression of novel alleles in hybrid zones requires comparison of lifetime fitness of parents and hybrid progeny. However, fitness differences among cross types can vary depending on biotic conditions, thereby influencing introgression patterns. Based on past work, we predicted that increased competition would enhance introgression between cultivated and wild sunflower (Helianthus annuus) by reducing fitness advantages of wild plants. To test this prediction, we established a factorial field experiment in Kansas, USA where we monitored the fitness of four cross types (Wild, F1, F2, and BCw hybrids) under different levels of interspecific and intraspecific competition. Intraspecific manipulations consisted both of density of competitors and of frequency of crop-wild hybrids. We recorded emergence of overwintered seeds, survival to reproduction, and numbers of seeds produced per reproductive plant. We also calculated two compound fitness measures: seeds produced per emerged seedling and seeds produced per planted seed. Cross type and intraspecific competition affected emergence and survival to reproduction, respectively. Further, cross type interacted with competitive treatments to influence all other fitness traits. More intense competition treatments, especially related to density of intraspecific competitors, repeatedly reduced the fitness advantage of wild plants when considering seeds produced per reproductive plant and per emerged seedling, and F2 plants often became indistinguishable from the wilds. Wild fitness remained superior when seedling emergence was also considered as part of fitness, but the fitness of F2 hybrids relative to wild plants more than quadrupled with the addition of interspecific competitors and high densities of intraspecific competitors. Meanwhile, contrary to prediction, lower hybrid frequency reduced wild fitness advantage. These results emphasize the importance of taking a full life cycle

Mathematical model of the SOS response regulation in wild-type Escherichia coli

International Nuclear Information System (INIS)

Aksenov, S.V.

1997-01-01

Regulation of the SOS response in Escherichia coli, which is a set of inducible cellular reactions introduced after DNA damage, is due to specific interaction of LexA and RecA proteins. LexA protein is a common repressor of the genes of the SOS system, and RecA protein, once transiently activated by the so-called SOS-inducing signal, promotes LexA protein destruction. We have described the SOS regulation by means of differential equations with regard to LexA and RecA concentrations elsewhere. The 'input' function for model equations is the level of the SOS-inducing signal against time. Here we present a means for calculating the concentration of single-stranded DNA (SOS-inducing signal) as a function of time in wild-type cells after ultraviolet irradiation. With model equations one can simulate kinetic curves of SOS regulatory proteins after DNA damage to survey the SOS response kinetics. Simulation of LexA protein kinetics agrees with experimental data. We compare simulated LexA kinetic curves in wild-type and uνr - mutant bacteria, which is useful in investigating the way uνrABC-dependent excision repair modulates the SOS response kinetics. Possible applications of the model to investigating various aspects of the SOS induction are discussed

Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

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Eliton da Silva Vasconcelos

2013-12-01

Full Text Available Clavulanic acid (CA is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064. The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

Science.gov (United States)

da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

2013-12-01

Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

Craniofacial Statistical Deformation Models of Wild-type mice and Crouzon mice

DEFF Research Database (Denmark)

Ólafsdóttir, Hildur; Darvann, Tron Andre; Ersbøll, Bjarne Kjær

2007-01-01

Crouzon syndrome is characterised by the premature fusion of cranial sutures and synchondroses leading to craniofacial growth disturbances. The gene causing the syndrome was discovered approximately a decade ago and recently the first mouse model of the syndrome was generated. In this study, a set...... of Micro CT scannings of the heads of wild-type (normal) mice and Crouzon mice were investigated. Statistical deformation models were built to assess the anatomical differences between the groups, as well as the within-group anatomical variation. Following the approach by Rueckert et al. we built an atlas...

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

Science.gov (United States)

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Optimization of the HyPer sensor for robust real-time detection of hydrogen peroxide in the rice blast fungus.

Science.gov (United States)

Huang, Kun; Caplan, Jeff; Sweigard, James A; Czymmek, Kirk J; Donofrio, Nicole M

2017-02-01

Reactive oxygen species (ROS) production and breakdown have been studied in detail in plant-pathogenic fungi, including the rice blast fungus, Magnaporthe oryzae; however, the examination of the dynamic process of ROS production in real time has proven to be challenging. We resynthesized an existing ROS sensor, called HyPer, to exhibit optimized codon bias for fungi, specifically Neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct could detect changes in fungal ROS during the plant infection process. Using confocal microscopy, we were able to visualize fluctuating ROS levels during the formation of an appressorium on an artificial hydrophobic surface, as well as during infection on host leaves. Using the plate reader, we were able to ascertain measurements of hydrogen peroxide (H 2 O 2 ) levels in conidia as detected by the MoHyPer sensor. Overall, by the optimization of codon usage for N. crassa and related fungal genomes, the MoHyPer sensor can be used as a robust, dynamic and powerful tool to both monitor and quantify H 2 O 2 dynamics in real time during important stages of the plant infection process. © 2016 BSPP AND JOHN WILEY & SONS LTD.

The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

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Logan M. Decker

2017-04-01

Full Text Available In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD, which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC. Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute of the perinuclear meiotic silencing complex (MSC, directly linking the two cellular factors.

Phosphorylated EGFR expression may predict outcome of EGFR-TKIs therapy for the advanced NSCLC patients with wild-type EGFR

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Wang Fen

2012-08-01

Full Text Available Abstract Background EGFR mutation is a strong predictive factor of EGFR-TKIs therapy. However, at least 10% of patients with EGFR wild-type are responsive to TKIs, suggesting that other determinants of outcome besides EGFR mutation might exist. We hypothesized that activation of phosphorylated EGFR could be a potential predictive biomarker to EGFR-TKIs treatment among patients in wild-type EGFR. Method Total of 205 stage IIIb and IV NSCLC patients, tissue samples of whom were available for molecular analysis, were enrolled in this study. The phosphorylation of EGFR at tyrosine 1068 (pTyr1068 and 1173 (pTyr1173 were assessed by immunohistochemistry, and EGFR mutations were detected by denaturing high performance liquid chromatograph (DHPLC. Results Among 205 patients assessable for EGFR mutation and phosphorylation analysis, 92 (44.9% were EGFR mutant and 165 patients (57.6% had pTyr1173 expression. Superior progression-free survival (PFS was seen after EGFR-TKIs therapy in patients with pTyr1068 expression compared to pTyr1068 negative ones (median PFS 7.0 months vs. 1.2 months, P P = 0.016. In subgroup of patients with wild-type EGFR, pTyr1068 expression positive ones had a significantly prolonged PFS (4.2 months vs.1.2 months P  Conclusion pTyr1068 may be a predictive biomarker for screening the population for clinical response to EGFR-TKIs treatment; especially for patients with wild-type EGFR.

Differentiation between probiotic and wild-type Bacillus cereus isolates by antibiotic susceptibility test and Fourier transform infrared spectroscopy (FT-IR).

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Mietke, Henriette; Beer, W; Schleif, Julia; Schabert, G; Reissbrodt, R

2010-05-30

Animal feed often contains probiotic Bacillus strains used as feed additives. Spores of the non-pathogenic B. cereus var. toyoi (product name Toyocerin) are used. Distinguishing between toxic wild-type Bacillus cereus strains and this probiotic strain is essential for evaluating the quality and risk of feed. Bacillus cereus CIP 5832 (product name Paciflor was used as probiotic strain until 2001. The properties of the two probiotic strains are quite similar. Differentiating between probiotic strains and wild-type B. cereus strains is not easy. ss-lactam antibiotics such as penicillin and cefamandole exhibit an inhibition zone in the agar diffusion test of probiotic B. cereus strains which are not seen for wild-type strains. Therefore, performing the agar diffusion test first may make sense before FT-IR testing. When randomly checking these strains by Fourier transform infrared spectroscopy (FT-IR), the probiotic B. cereus strains were separated from wild-type B. cereus/B. thuringiensis/B. mycoides/B. weihenstephanensis strains by means of hierarchical cluster analysis. The discriminatory information was contained in the spectral windows 3000-2800 cm(-1) ("fatty acid region"), 1200-900 cm(-1) ("carbohydrate region") and 900-700 cm(-1) ("fingerprint region"). It is concluded that FT-IR spectroscopy can be used for the rapid quality control and risk analysis of animal feed containing probiotic B. cereus strains. (c) 2010 Elsevier B.V. All rights reserved.

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

International Nuclear Information System (INIS)

Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L.

1989-01-01

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

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Nowrousian, Minou; Stajich, Jason E; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D; Pöggeler, Stefanie; Read, Nick D; Seiler, Stephan; Smith, Kristina M; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-04-08

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

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Minou Nowrousian

2010-04-01

Full Text Available Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data

Genome Sequences of Three Vaccine Strains and Two Wild-Type Canine Distemper Virus Strains from a Recent Disease Outbreak in South Africa.

Science.gov (United States)

Loots, Angelika K; Du Plessis, Morné; Dalton, Desiré Lee; Mitchell, Emily; Venter, Estelle H

2017-07-06

Canine distemper virus causes global multihost infectious disease. This report details complete genome sequences of three vaccine and two new wild-type strains. The wild-type strains belong to the South African lineage, and all three vaccine strains to the America 1 lineage. This constitutes the first genomic sequences of this virus from South Africa. Copyright © 2017 Loots et al.

Characterization of two second-site mutations preventing wild type protein aggregation caused by a dominant negative PMA1 mutant.

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Pilar Eraso

Full Text Available The correct biogenesis and localization of Pma1 at the plasma membrane is essential for yeast growth. A subset of PMA1 mutations behave as dominant negative because they produce aberrantly folded proteins that form protein aggregates, which in turn provoke the aggregation of the wild type protein. One approach to understand this dominant negative effect is to identify second-site mutations able to suppress the dominant lethal phenotype caused by those mutant alleles. We isolated and characterized two intragenic second-site suppressors of the PMA1-D378T dominant negative mutation. We present here the analysis of these new mutations that are located along the amino-terminal half of the protein and include a missense mutation, L151F, and an in-frame 12bp deletion that eliminates four residues from Cys409 to Ala412. The results show that the suppressor mutations disrupt the interaction between the mutant and wild type enzymes, and this enables the wild type Pma1 to reach the plasma membrane.

Parasitic castration in Fissurella crassa (Archaeogastropoda due to an adult Digenea, Proctoeces lintoni (Fellodistomidae

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Marcelo E. Oliva

1992-03-01

Full Text Available Specimens of Fissurella crassa (Archaeogastropoda from Ilo, southern Perú, are infected with the adult stage of the digenetic trematode Proctoeces lintoni (Fellodistomidae. The histopatological analysis of the male and female gonads show a strong effect of the parasite on the structure and function of these organs. P. lintoni live unencysted in the gonads, and the main mechanical damage is originated by the action of a well developed acetabulum. Chemical actions of parasitic secretions may also be involved. The infected gonads show altered structure and the gametogenic processes is aborted. There is no evidence of hemocytic response, but leucocite infiltration is evident at least in male infected gonads. An increased content of polysaccarides is evident in infected gonads.

Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

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Dong, X Y; Li, W H; Zhu, J L; Liu, W J; Zhao, M Q; Luo, Y W; Chen, J D

2015-01-01

Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance.

Comparative Study on Growth Performance of Transgenic (Over-Expressed OsNHX1 and Wild-Type Nipponbare under Different Salinity Regimes

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Nurul Kahrani ISHAK

2015-11-01

Full Text Available Transgenic Nipponbare which over-expressed a Na+/H+ antiporter gene OsNHX1 was used to compare its growth performance, water status and photosynthetic efficiency with its wild type under varying salinity regimes. Chlorophyll content, quantum yield and photosynthetic rate were measured to assess the impact of salinity stress on photosynthetic efficiency for transgenic and wild-type Nipponbare. Effects of salinity on water status and gas exchange to both lines were studied by measuring water use efficiency, instantaneous transpiration rate and stomatal conductance. Dry shoot weight and leaf area were determined after three months of growth to assess the impacts of salinity on the growth of those two lines. Our study showed that both lines were affected by salinity stress, however, the transgenic line showed higher photosynthetic efficiency, better utilization of water, and better growth due to low transpiration rate and stomatal conductance. Reduction of photosynthetic efficiency exhibited by the wild-type Nipponbare was correlated to its poor growth under salinity stress.

Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

DEFF Research Database (Denmark)

Hakim-Weber, Robab; Krogsdam, Anne-M; Jørgensen, Claus

2011-01-01

Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate...... this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs...... of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis...

Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts

OpenAIRE

Tang, Ning; Zhang, Qianqian; Fang, Shu; Han, Xiao; Wang, Zhehai

2016-01-01

Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and record...

Functional characterization of the Woronin body protein WscA of the pathogenic mold Aspergillus fumigatus.

Science.gov (United States)

Leonhardt, Yannik; Beck, Julia; Ebel, Frank

2016-05-01

Woronin bodies are fungal-specific organelles that seal damaged hyphal compartments and thereby contribute to the stress resistance and virulence of filamentous fungi. In this study, we have characterized the Aspergillus fumigatus Woronin body protein WscA. WscA is homologous to Neurospora crassa WSC, a protein that was shown to be important for biogenesis, segregation and positioning of Woronin bodies. WscA and WSC both belong to the Mpv17/PMP22 family of peroxisomal membrane proteins. An A. fumigatus ΔwscA mutant is unable to form Woronin bodies, and HexA, the protein that forms the crystal-like core of Woronin bodies, accumulates in large peroxisomes instead. The ΔwscA mutant showed no defect in segregation of HexA containing organelles, as has been reported for the corresponding N. crassa mutant. In the peroxisomes of the A. fumigatus mutant, HexA assembles into compact, donut-shaped structures. Experiments with GFP fusion proteins revealed that WscA function is highly sensitive to these modifications, in particular to an N-terminal fusion of GFP. In N. crassa, WSC was shown to be essentially required for Woronin body positioning, but the respective domain is not conserved in most other Pezizomycotina, including A. fumigatus. We have recently found evidence that HexA may have a direct role in WB positioning, since a HexA-GFP fusion protein, lacking a functional PTS1 motif, is efficiently recruited to the septal pore. In the current study we show that this targeting of HexA-GFP is independent of WscA. Copyright © 2016 Elsevier GmbH. All rights reserved.

Annotating and quantifying pri-miRNA transcripts using RNA-Seq data of wild type and serrate-1 globular stage embryos of Arabidopsis thaliana

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Daniel Lepe-Soltero

2017-12-01

Full Text Available The genome annotation for the model plant Arabidopsis thaliana does not include the primary transcripts from which MIRNAs are processed. Here we present and analyze the raw mRNA sequencing data from wild type and serrate-1 globular stage embryos of A. thaliana, ecotype Columbia. Because SERRATE is required for pri-miRNA processing, these precursors accumulate in serrate-1 mutants, facilitating their detection using standard RNA-Seq protocols. We first use the mapping of the RNA-Seq reads to the reference genome to annotate the potential primary transcripts of MIRNAs expressed in the embryo. We then quantify these pri-miRNAs in wild type and serrate-1 mutants. Finally, we use differential expression analysis to determine which are up-regulated in serrate-1 compared to wild type, to select the best candidates for bona fide pri-miRNAs expressed in the globular stage embryos. In addition, we analyze a previously published RNA-Seq dataset of wild type and dicer-like 1 mutant embryos at the globular stage [1]. Our data are interpreted and discussed in a separate article [2].

Annotating and quantifying pri-miRNA transcripts using RNA-Seq data of wild type and serrate-1 globular stage embryos of Arabidopsis thaliana.

Science.gov (United States)

Lepe-Soltero, Daniel; Armenta-Medina, Alma; Xiang, Daoquan; Datla, Raju; Gillmor, C Stewart; Abreu-Goodger, Cei

2017-12-01

The genome annotation for the model plant Arabidopsis thaliana does not include the primary transcripts from which MIRNAs are processed. Here we present and analyze the raw mRNA sequencing data from wild type and serrate-1 globular stage embryos of A. thaliana , ecotype Columbia. Because SERRATE is required for pri-miRNA processing, these precursors accumulate in serrate-1 mutants, facilitating their detection using standard RNA-Seq protocols. We first use the mapping of the RNA-Seq reads to the reference genome to annotate the potential primary transcripts of MIRNAs expressed in the embryo. We then quantify these pri-miRNAs in wild type and serrate-1 mutants. Finally, we use differential expression analysis to determine which are up-regulated in serrate-1 compared to wild type, to select the best candidates for bona fide pri-miRNAs expressed in the globular stage embryos. In addition, we analyze a previously published RNA-Seq dataset of wild type and dicer-like 1 mutant embryos at the globular stage [1]. Our data are interpreted and discussed in a separate article [2].

Genotype-temperature interaction in the regulation of development, growth, and morphometrics in wild-type, and growth-hormone transgenic coho salmon.

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Mare Lõhmus

2010-04-01

Full Text Available The neuroendocrine system is an important modulator of phenotype, directing cellular genetic responses to external cues such as temperature. Behavioural and physiological processes in poikilothermic organisms (e.g. most fishes, are particularly influenced by surrounding temperatures.By comparing the development and growth of two genotypes of coho salmon (wild-type and transgenic with greatly enhanced growth hormone production at six different temperatures, ranging between 8 degrees and 18 degrees C, we observed a genotype-temperature interaction and possible trend in directed neuroendocrine selection. Differences in growth patterns of the two genotypes were compared by using mathematical models, and morphometric analyses of juvenile salmon were performed to detect differences in body shape. The maximum hatching and alevin survival rates of both genotypes occurred at 12 degrees C. At lower temperatures, eggs containing embryos with enhanced GH production hatched after a shorter incubation period than wild-type eggs, but this difference was not apparent at and above 16 degrees C. GH transgenesis led to lower body weights at the time when the yolk sack was completely absorbed compared to the wild genotype. The growth of juvenile GH-enhanced salmon was to a greater extent stimulated by higher temperatures than the growth of the wild-type. Increased GH production significantly influenced the shape of the salmon growth curves.Growth hormone overexpression by transgenesis is able to stimulate the growth of coho salmon over a wide range of temperatures. Temperature was found to affect growth rate, survival, and body morphology between GH transgenic and wild genotype coho salmon, and differential responses to temperature observed between the genotypes suggests they would experience different selective forces should they ever enter natural ecosystems. Thus, GH transgenic fish would be expected to differentially respond and adapt to shifts in environmental

Sex-Specific Diurnal Immobility Induced by Forced Swim Test in Wild Type and Clock Gene Deficient Mice

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Ningyue Li

2015-03-01

Full Text Available Objective: The link between alterations in circadian rhythms and depression are well established, but the underlying mechanisms are far less elucidated. We investigated the circadian characteristics of immobility behavior in wild type (WT mice and mice with mutations in core Clock genes. Methods: All mice were tested with forced swim test (FST at 4 h intervals. Results: These experiments revealed significant diurnal rhythms associated with immobility behavior in both male and female WT mice with sex-different circadian properties. In addition, male mice showed significantly less immobility during the night phase in comparison to female mice. Female Per1Brdm1 mice also showed significant rhythmicity. However, the timing of rhythmicity was very different from that observed in female wild type mice. Male Per1Brdm1 mice showed a pattern of rhythmicity similar to that of wild type mice. Furthermore, female Per1Brdm1 mice showed higher duration of immobility in comparison to male Per1Brdm1 mice in both daytime and early night phases. Neither Per2Brdm1 nor ClockΔ19 mice showed significant rhythmicity, but both female Per2Brdm1 and ClockΔ19 mice had lower levels of immobility, compared to males. Conclusions: This study highlights the differences in the circadian characteristics of immobility induced by FST in WT, ClockΔ19, Per1, and Per2 deficient mice.

Multilocus sequence typing of commensal and enteropathogenic Escherichia coli from domestic and wild lagomorphs in Italy

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Giorgia Dotto

2015-12-01

Full Text Available The aim of the study was to determine the multilocus sequence types of Escherichia coli from diseased farm rabbits and apparently healthy wild lagomorphs, and the genetic relatedness among them. Fifty-five enteropathogenic E. coli from reared rabbits and 32 from wild rabbits and hares were characterised by multilocus sequence typing (MLST according to the Michigan State University EcMLST scheme. Isolates were differentiated into 37 sequence types (STs, which were grouped into 8 clonal complexes (CCs. The most common ST was ST140 (CC31, followed by ST238 and ST119 (CC17. MLST analysis revealed 22 novel STs. Phylogenetic analyses showed a heterogeneous distribution of STs into 3 clusters of genetically related strains. The genetic relationship among STs of different origin and the detection of new, as well as previously described STs as human pathogens, indicate a widespread distribution and adaptability of particular lineages to different hosts. These findings highlight the need for further research to improve the knowledge about E. coli populations colonising the gut of lagomorphs and their zoonotic potential.

Maintenance erlotinib in advanced nonsmall cell lung cancer: cost-effectiveness in EGFR wild-type across Europe

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Walleser S

2012-09-01

Full Text Available Silke Walleser,1 Joshua Ray,2 Helge Bischoff,3 Alain Vergnenègre,4 Hubertus Rosery,5 Christos Chouaid,6 David Heigener,7 Javier de Castro Carpeño,8 Marcello Tiseo,9 Stefan Walzer21Health Economic Consultancy, Renens, Switzerland; 2F Hoffmann-La Roche Pharmaceuticals AG, Basel, Switzerland; 3Thoracic Hospital of Heidelberg, Heidelberg, Germany; 4Limoges University Hospital, Limoges, France; 5Assessment-in-Medicine GmbH, Loerrach, Germany; 6Hospital Saint Antoine, Paris, France; 7Hospital Grosshansdorf, Grosshansdorf, Germany; 8University Hospital La Paz, Madrid, Spain; 9University Hospital of Parma, Parma, ItalyBackground: First-line maintenance erlotinib in patients with locally advanced or metastatic nonsmall cell lung cancer (NSCLC has demonstrated significant overall survival and progression-free survival benefits compared with best supportive care plus placebo, irrespective of epidermal growth factor receptor (EGFR status (SATURN trial. The cost-effectiveness of first-line maintenance erlotinib in the overall SATURN population has been assessed and published recently, but analyses according to EGFR mutation status have not been performed yet, which was the rationale for assessing the cost-effectiveness of first-line maintenance erlotinib specifically in EGFR wild-type metastatic NSCLC.Methods: The incremental cost per life-year gained of first-line maintenance erlotinib compared with best supportive care in patients with EGFR wild-type stable metastatic NSCLC was assessed for five European countries (the United Kingdom, Germany, France, Spain, and Italy with an area-under-the-curve model consisting of three health states (progression-free survival, progressive disease, death. Log-logistic survival functions were fitted to Phase III patient-level data (SATURN to model progression-free survival and overall survival. The first-line maintenance erlotinib therapy cost (modeled for time to treatment cessation, medication cost in later lines, and

Proteomics of Arabidopsis Seed Germination : a Comparative Study of Wild-Type and Gibberellin-Deficient Seeds

NARCIS (Netherlands)

Gallardo, K.; Job, C.; Groot, S.P.C.; Puype, M.; Vandekerckhove, J.; Job, D.

2002-01-01

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA

Modelling biological control with wild-type and genetically modified baculoviruses in the Helicoverpa armigera-cotton system

NARCIS (Netherlands)

Sun, X.; Werf, van der W.; Bianchi, F.J.J.A.; Hu, Z.; Vlak, J.M.

2006-01-01

A comprehensive model was developed to simulate virus epizootics in a stage structured insect population and analyse scenarios for the biological control of cotton bollworm (CBW), Helicoverpa armigera, in cotton, using wild-type or genetically modified baculoviruses. In simulations on dosage and

CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequence of epigenetic regulation

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Drexler Hans G

2010-04-01

Full Text Available Abstract Background CD7 is a negative prognostic marker in myeloid malignancies. In acute myeloid leukemia (AML, an inverse correlation exists between expression of wild-type CEBPA and CD7. Aim of this study was to find out whether C/EBPα is a negative regulator of CD7 and which other regulatory mechanisms might be involved. Results As already described for primary AML cells, the majority of AML cell lines tested were either C/EBPα+/CD7- or C/EBPα-/CD7+. However, the existence of isolated CD7+ cell lines expressing wild-type C/EBPα challenges the notion that C/EBPα acts as a unique repressor of CD7. Furthermore, ectopic expression of CEBPA did not reduce CD7 in CD7+ cells and knock-down of C/EBPα failed to induce CD7 in CD7- cells. In contrast, the DNA demethylating agent Aza-2'deoxycytidine triggered CD7 expression in CD7- AML and in T-cell lines suggesting epigenetic regulation of CD7. Bisulfite sequencing data confirmed that CpGs in the CD7 exon1 region are methylated in CD7- cell lines, and unmethylated in CD7+ cell lines. Conclusion We confirmed an inverse correlation between the expression of wild-type CEBPA and of CD7 in AML cells. Our results contradict the hypothesis that C/EBPα acts as repressor for CD7, and instead show that epigenetic mechanisms are responsible for CD7 regulation, in AML cells as well as in T-cells, the typical CD7 expressing cell type.

Partially dissecting the steady-state electron fluxes in Photosystem I in wild-type and pgr5 and ndh mutants of Arabidopsis

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Jiancun eKou

2015-09-01

Full Text Available Cyclic electron flux (CEF around Photosystem I (PS I is difficult to quantify. We obtained the linear electron flux (LEFO2 through both photosystems and the total electron flux through PS I (ETR1 in Arabidopsis in CO2-enriched air. DeltaFlux = ETR1 – LEFO2 is an upper estimate of CEF, which consists of two components, an antimycin A-sensitive, PGR5 (proton gradient regulation 5 protein-dependent component and an insensitive component facilitated by a chloroplastic nicotinamide adenine dinucleotide dehydrogenase-like complex (NDH. Using wild type as well as pgr5 and ndh mutants, we observed that (1 40% of the absorbed light was partitioned to PS I; (2 at high irradiance a substantial antimycin A-sensitive CEF occurred in the wild type and the ndh mutant; (3 at low irradiance a sizable antimycin A-sensitive CEF occurred in the wild type but not in the ndh mutant, suggesting an enhancing effect of NDH in low light; and (4 in the pgr5 mutant, and the wild type and ndh mutant treated with antimycin A, a residual DeltaFlux existed at high irradiance, attributable to charge recombination and/or pseudo-cyclic electron flow. Therefore, in low-light-acclimated plants exposed to high light, DeltaFlux has contributions from various paths of electron flow through PS I.

Hepcidin regulation in wild-type and Hfe knockout mice in response to alcohol consumption: evidence for an alcohol-induced hypoxic response.

Science.gov (United States)

Heritage, Mandy L; Murphy, Therese L; Bridle, Kim R; Anderson, Gregory J; Crawford, Darrell H G; Fletcher, Linda M

2009-08-01

Expression of Hamp1, the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice (Hfe(-/-)). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe(-/-) mice. Hfe(-/-) and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was measured by western blot. Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe(-/-) mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1alpha protein levels were elevated in alcohol-fed wild-type animals compared with controls. Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.

Antibody Prevalence to Influenza Type A in Wild Boar of Northern Ukraine.

Science.gov (United States)

Kovalenko, Ganna; Molozhanova, Alona; Halka, Ihor; Nychyk, Serhiy

2017-12-01

A preliminary serological survey was carried out to assess the likelihood of influenza A (IA) infection in wild boar and begin to characterize the role of wild boar in the epidemiology of the IA virus (IAV). Sera collected from 120 wild boar that were hunted in 2014 were tested. To detect antibodies to IA, a blocking the enzyme-linked immunosorbent assay (ELISA) was used. Thirty boar were collected from each of four oblasts in the north central and northwestern regions of Ukraine. Antibodies against IAV were detected in 27 samples (22.5%; 95% confidence interval 16.0-30.8) and in at least some of the wild boar from all of the four oblasts. This preliminary survey of IA antibodies in wild boar populations of northern Ukraine indicates a substantial frequency of exposure to IAV throughout the region. Infection of wild boar populations could provide an alternative or additional route for spillover from wild populations to domestic animals and humans.

Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport.

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Ming Liu

2010-10-01

Full Text Available Recently, a syndrome of Mutant INS-gene-induced Diabetes of Youth (MIDY, derived from one of 26 distinct mutations has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inheritance of even one MIDY allele, causes diabetes. Although a favored explanation for the onset of diabetes is that insurmountable ER stress and ER stress response from the mutant proinsulin causes a net loss of beta cells, in this report we present three surprising and interlinked discoveries. First, in the presence of MIDY mutants, an increased fraction of wild-type proinsulin becomes recruited into nonnative disulfide-linked protein complexes. Second, regardless of whether MIDY mutations result in the loss, or creation, of an extra unpaired cysteine within proinsulin, Cys residues in the mutant protein are nevertheless essential in causing intracellular entrapment of co-expressed wild-type proinsulin, blocking insulin production. Third, while each of the MIDY mutants induces ER stress and ER stress response; ER stress and ER stress response alone appear insufficient to account for blockade of wild-type proinsulin. While there is general agreement that ultimately, as diabetes progresses, a significant loss of beta cell mass occurs, the early events described herein precede cell death and loss of beta cell mass. We conclude that the molecular pathogenesis of MIDY is initiated by perturbation of the disulfide-coupled folding pathway of wild-type proinsulin.

STUDY DATA OF KRAS- AND RAS-UNMUTATED (WILD TYPE OF COLORECTAL CANCER

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V. A. Gorbunova

2015-01-01

Full Text Available  Analysis of latest trials, comparing treatment schemes including chemotherapy with anti-EGFR monoclonal antibodies or bevacizumab is presented in this article. The data in these trials is inconsistent, but detailed analysis of FIRE-3 trial allows to distinguish a wild-type RAS patient group that benefits most from chemotherapy with cetuximab or panitumumab as 1st line metastatic colorectal cancer treatment. A final analysis of this patient group in CALGB/SWOG 80 405 trial is pending. The RAS analysis is pivotal for choice of 1st line chemotherapy.

Effect of dietary supplementation with Rhizopus oryzae or Chrysonilia crassa on growth performance, blood profile, intestinal microbial population, and carcass traits in broilers exposed to heat stress

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S. Sugiharto

2017-09-01

Full Text Available Dietary supplementation of additives has recently been part of strategies to deal with the detrimental effects of heat stress (HS on the performance and carcass traits in broiler chicks. This study aimed to investigate the effect of dietary supplementation with the fungi Rhizopus oryzae or Chrysonilia crassa on growth, blood profile, intestinal microbial population and carcass traits in broiler chicks subjected to HS. R. oryzae and C. crassa are filamentous fungi isolated from the ileum of indigenous Indonesian chickens which exhibited probiotic and antioxidant properties. Two hundred and forty 21-day-old male broiler chicks were randomly allotted into six groups, including birds reared under normal temperature (28 ± 2 °C (CONT, birds reared under HS conditions (35 ± 2 °C (HS-CONT, birds reared under HS and provided with commercial anti-stress formula (HS-VIT, birds reared under HS and provided with R. oryzae (HS-RO, birds reared under HS and provided with C. crassa (HS-CC and birds reared under HS and provided with rice bran (HS-RB. Body weight gain was highest (P < 0. 01 and lowest (P < 0. 01 in CONT and HS-CONT birds, respectively. The heart was heavier (P < 0. 05 in CONT than in HS-CONT and HS-VIT birds. CONT birds had heavier duodenum (P < 0. 05 and jejunum (P < 0. 01 than other birds. Eosinophils was higher (P < 0. 05 in HS-CC than in other birds. Low-density lipoprotein (LDL was higher (P < 0. 05 in HS-CONT than in CONT, HS-VIT and HS-CC birds. Total triglyceride was highest (P < 0. 05 and lowest (P < 0. 05 in HS-RB and HS-RO birds, respectively. Alanine aminotransferase (ALT was higher (P < 0. 05 in HS-CONT than in other HS birds. Total protein was lowest and highest (P < 0. 05 in CONT and HS-CONT birds, respectively. Albumin was higher (P < 0. 05 in HS-CONT and HS-VIT than in HS-RO birds. Globulin was lower (P < 0. 05 in CONT than in HS

lambda. -prophage induction in repair-deficient and wild type E. coli strains by. gamma. -rays and heavy ions

Energy Technology Data Exchange (ETDEWEB)

Bonev, M.N.; Kozubek, S.; Krasavin, E.A.; Amirtajev, K.G. (Joint Inst. for Nuclear Research, Dubna (USSR))

1990-05-01

{lambda}-prophage induction in repair-deficient and wild-type E. coli strains by heavy ions and {gamma}-rays was investigated. The dose dependence of the fraction of induced cells has been measured and its initial slope ({lambda}-induction potency) determined. Induction by {gamma}-rays was found to be more efficient in a polA-repair-deficient strain; the value of {lambda}-induction potency is zero in lexA{sup -} and recA{sup -} strains. The {lambda}-induction potency potency increased with LET for wild-type cells but remained constant in polA{sup -} mutant cells. It is suggested that DNA damage triggering the {lambda}-prophage induction in the case of ionizing radiation could be a type of DNA single-strand break with complex structures which cannot be repaired by fast repair processes, and requires a substantial level of energy deposition for induction in a DNA molecule. (author).

La formación de anillos de crecimiento en Fissurella crassa en el norte de Chile

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Marta Bretos

1980-12-01

Full Text Available Growth discontinuity in molluscs result in the formation of rings on their shells. Two kind of rings may be formed: disturbance and growth rings. Growth rings can be used to determine the age of molluscs with long life span. For this, it is necessary to know how many growth rings are formed per year. It has been demonstrated experimentally, using marked specimens, that F. crassa forms two growth rings per year at Huayquique, Northern Chile. They are formed during winter and summer. Disturbance ring formation has also been observed as a result of sawing marks on their shells. A growth curve for this species is proposed on the basis of Walford's line.

Effects of animal type (wild vs. domestic) and diet alfalfa level on intake and digestibility of European adult rabbits (Oryctolagus cuniculus)

NARCIS (Netherlands)

Pinheiro, Victor; Outor-Monteiro, D.; Mourão, J.L.; Cone, J.W.; Guimaraes Dias Lourenco, Ana

2018-01-01

The aim of this study was to evaluate the effect of the level of alfalfa in the diet on feed intake and digestibility of two types of rabbits, wild (Oryctolagus cuniculus algirus) vs. domestic (O. cuniculus cuniculus). Ten wild (W; mean LW = 927 g) and 10 domestic (D; mean LW = 4,645 g) adult rabbit

The In Vivo Granulopoietic Response to Dexamethasone Injection Is Abolished in Perforin-Deficient Mutant Mice and Corrected by Lymphocyte Transfer from Nonsensitized Wild-Type Donors

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Pedro Xavier-Elsas

2015-01-01

Full Text Available Exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone-marrow. A hematological response dependent on endogenous glucocorticoids underlies bone-marrow eosinophilia induced by trauma or allergic sensitization/challenge. We detected a defect in granulopoiesis in nonsensitized, perforin-deficient mice. In steady-state conditions, perforin- (Pfp- deficient mice showed significantly decreased bone-marrow and blood eosinophil and neutrophil counts, and colony formation in response to GM-CSF, relative to wild-type controls of comparable age and/or weight. By contrast, peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency. Dexamethasone enhanced colony formation by GM-CSF-stimulated progenitors from wild-type controls, but not Pfp mice. Dexamethasone injection increased bone-marrow eosinophil and neutrophil counts in wild-type controls, but not Pfp mice. Because perforin is expressed in effector lymphocytes, we examined whether this defect would be corrected by transferring wild-type lymphocytes into perforin-deficient recipients. Short-term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild-type donors. Transfer of the same amount of splenic lymphocytes from perforin-deficient donors was ineffective. This demonstrates that the perforin-dependent, granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations.

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

International Nuclear Information System (INIS)

Sedliakova, M.; Slezarikova, V.; Brozmanova, J.; Masek, F.; Bayerova, V.

1980-01-01

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7, proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival. We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP + ). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells given with potentially lethal doses of UV irradiation. (orig.)

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

Science.gov (United States)

Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

Near-Infrared Spectroscopy, a Rapid Method for Predicting the Age of Male and Female Wild-Type and Wolbachia Infected Aedes aegypti.

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Maggy T Sikulu-Lord

2016-10-01

Full Text Available Estimating the age distribution of mosquito populations is crucial for assessing their capacity to transmit disease and for evaluating the efficacy of available vector control programs. This study reports on the capacity of the near-infrared spectroscopy (NIRS technique to rapidly predict the ages of the principal dengue and Zika vector, Aedes aegypti. The age of wild-type males and females, and males and females infected with wMel and wMelPop strains of Wolbachia pipientis were characterized using this method. Calibrations were developed using spectra collected from their heads and thoraces using partial least squares (PLS regression. A highly significant correlation was found between the true and predicted ages of mosquitoes. The coefficients of determination for wild-type females and males across all age groups were R2 = 0.84 and 0.78, respectively. The coefficients of determination for the age of wMel and wMelPop infected females were 0.71 and 0.80, respectively (P< 0.001 in both instances. The age of wild-type female Ae. aegypti could be identified as < or ≥ 8 days old with an accuracy of 91% (N = 501, whereas female Ae. aegypti infected with wMel and wMelPop were differentiated into the two age groups with an accuracy of 83% (N = 284 and 78% (N = 229, respectively. Our results also indicate NIRS can distinguish between young and old male wild-type, wMel and wMelPop infected Ae. aegypti with accuracies of 87% (N = 253, 83% (N = 277 and 78% (N = 234, respectively. We have demonstrated the potential of NIRS as a predictor of the age of female and male wild-type and Wolbachia infected Ae. aegypti mosquitoes under laboratory conditions. After field validation, the tool has the potential to offer a cheap and rapid alternative for surveillance of dengue and Zika vector control programs.

Pigment Production by the Edible Filamentous Fungus Neurospora Intermedia

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Rebecca Gmoser

2018-02-01

Full Text Available The production of pigments by edible filamentous fungi is gaining attention as a result of the increased interest in natural sources with added functionality in the food, feed, cosmetic, pharmaceutical and textile industries. The filamentous fungus Neurospora intermedia, used for production of the Indonesian food “oncom”, is one potential source of pigments. The objective of the study was to evaluate the fungus’ pigment production. The joint effect from different factors (carbon and nitrogen source, ZnCl2, MgCl2 and MnCl2 on pigment production by N. intermedia is reported for the first time. The scale-up to 4.5 L bubble column bioreactors was also performed to investigate the effect of pH and aeration. Pigment production of the fungus was successfully manipulated by varying several factors. The results showed that the formation of pigments was strongly influenced by light, carbon, pH, the co-factor Zn2+ and first- to fourth-order interactions between factors. The highest pigmentation (1.19 ± 0.08 mg carotenoids/g dry weight biomass was achieved in a bubble column reactor. This study provides important insights into pigmentation of this biotechnologically important fungus and lays a foundation for future utilizations of N. intermedia for pigment production.

Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.

Science.gov (United States)

Lee, Shauna A; Roques, Céline; Magwood, Alissa C; Masson, Jean-Yves; Baker, Mark D

2009-02-01

The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.

EFFECT OF FERMENTED Sauropus androgynus LEAVES ON BLOOD LIPID FRACTION AND HAEMATOLOGICAL PROFILE IN BROILER CHICKENS

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U. Santoso

2015-12-01

Full Text Available This study was conducted to evaluate effect of fermented Sauropus androgynus leaves on blood lipid fractions and haematological profiles in broilers. One hundred and twelve broilers were distributed to 7 treatment groups. One group was fed diets without Sauropus androgynus leaves as the control, and other six groups were fed Sauropus androgynus leaves fermented by Neurospora crassa, Lactobacillus sp. or Saccharomyces cerevisiae at level of 25 g or 50 g/kg diet. Experimental results showed that the treatments had no effect on cholesterol, high-density lipoprotein cholesterol (HDL-c, low-density lipoprotein cholesterol (LDL-c and atherogenic index, very low-density lipoprotein cholesterol (VLDL-c and triglyceride concentration (P>0.05. It was shown that fermented Sauropus androgynus leaves significantly affected red blood count (RBC, white blood count (WBC, packed cell volume (PCV, trombosit dan erythrocyte sedimentation rate (ESR (P

Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation

DEFF Research Database (Denmark)

Agger, Jane W.; Isaksen, Trine; Várnai, Anikó

2014-01-01

of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades...... walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting...... on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect...

Inorganic phosphate uptake in unicellular eukaryotes.

Science.gov (United States)

Dick, Claudia F; Dos-Santos, André L A; Meyer-Fernandes, José R

2014-07-01

Inorganic phosphate (Pi) is an essential nutrient for all organisms. The route of Pi utilization begins with Pi transport across the plasma membrane. Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of Pi transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding Pi uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum. Pi uptake is the key step of Pi homeostasis and in the subsequent signaling event in eukaryotic microorganisms. Biochemical and structural studies are important for clarifying mechanisms of Pi homeostasis, as well as Pi sensor and downstream pathways, and raise possibilities for future studies in this field. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of social stress and intrauterine position on sexual phenotype in wild-type house mice (Mus musculus)

Science.gov (United States)

William J. Zielinski; John G. Vandenbergh; Monica M. Montano

1991-01-01

Wild-type house mice were used to test the effect of intrauterine position on anogenital distance (AGD) and to verify whether crowding stress would masculinize female pups, developing at all intrauterine positions, as has been demonstrated in CF-1 mice stressed by restraint, heat, and...

Characterization of a sensitive mouse Aβ40 PD biomarker assay for Alzheimer's disease drug development in wild-type mice.

Science.gov (United States)

Lu, Yanmei; Hoyte, Kwame; Montgomery, William H; Luk, Wilman; He, Dongping; Meilandt, William J; Zuchero, Y Joy Yu; Atwal, Jasvinder K; Scearce-Levie, Kimberly; Watts, Ryan J; DeForge, Laura E

2016-05-01

Transgenic mice that overexpress human amyloid precursor protein with Swedish or London (APPswe or APPlon) mutations have been widely used for preclinical Alzheimer's disease (AD) drug development. AD patients, however, rarely possess these mutations or overexpress APP. We developed a sensitive ELISA that specifically and accurately measures low levels of endogenous Aβ40 in mouse plasma, brain and CSF. In wild-type mice treated with a bispecific anti-TfR/BACE1 antibody, significant Aβ reductions were observed in the periphery and the brain. APPlon transgenic mice showed a slightly less reduction, whereas APPswe mice did not have any decrease. This sensitive and well-characterized mouse Aβ40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.

A Caenorhabditis elegans wild type defies the temperature-size rule owing to a single nucleotide polymorphism in tra-3.

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Jan E Kammenga

2007-03-01

Full Text Available Ectotherms rely for their body heat on surrounding temperatures. A key question in biology is why most ectotherms mature at a larger size at lower temperatures, a phenomenon known as the temperature-size rule. Since temperature affects virtually all processes in a living organism, current theories to explain this phenomenon are diverse and complex and assert often from opposing assumptions. Although widely studied, the molecular genetic control of the temperature-size rule is unknown. We found that the Caenorhabditis elegans wild-type N2 complied with the temperature-size rule, whereas wild-type CB4856 defied it. Using a candidate gene approach based on an N2 x CB4856 recombinant inbred panel in combination with mutant analysis, complementation, and transgenic studies, we show that a single nucleotide polymorphism in tra-3 leads to mutation F96L in the encoded calpain-like protease. This mutation attenuates the ability of CB4856 to grow larger at low temperature. Homology modelling predicts that F96L reduces TRA-3 activity by destabilizing the DII-A domain. The data show that size adaptation of ectotherms to temperature changes may be less complex than previously thought because a subtle wild-type polymorphism modulates the temperature responsiveness of body size. These findings provide a novel step toward the molecular understanding of the temperature-size rule, which has puzzled biologists for decades.

Control of acute, chronic, and constitutive hyperammonemia by wild-type and genetically engineered Lactobacillus plantarum in rodents.

Science.gov (United States)

Nicaise, Charles; Prozzi, Deborah; Viaene, Eric; Moreno, Christophe; Gustot, Thierry; Quertinmont, Eric; Demetter, Pieter; Suain, Valérie; Goffin, Philippe; Devière, Jacques; Hols, Pascal

2008-10-01

Hyperammonemia is a common complication of acute and chronic liver diseases. Often accompanied with side effects, therapeutic interventions such as antibiotics or lactulose are generally targeted to decrease the intestinal production and absorption of ammonia. In this study, we aimed to modulate hyperammonemia in three rodent models by administration of wild-type Lactobacillus plantarum, a genetically engineered ammonia hyperconsuming strain, and a strain deficient for the ammonia transporter. Wild-type and metabolically engineered L. plantarum strains were administered in ornithine transcarbamoylase-deficient Sparse-fur mice, a model of constitutive hyperammonemia, in a carbon tetrachloride rat model of chronic liver insufficiency and in a thioacetamide-induced acute liver failure mice model. Constitutive hyperammonemia in Sparse-fur mice and hyperammonemia in a rat model of chronic hepatic insufficiency were efficiently decreased by Lactobacillus administration. In a murine thioacetamide-induced model of acute liver failure, administration of probiotics significantly increased survival and decreased blood and fecal ammonia. The ammonia hyperconsuming strain exhibited a beneficial effect at a lower dose than its wild-type counterpart. Improved survival in the acute liver failure mice model was associated with lower blood ammonia levels but also with a decrease of astrocyte swelling in the brain cortex. Modulation of ammonia was abolished after administration of the strain deficient in the ammonium transporter. Intestinal pH was clearly lowered for all strains and no changes in gut flora were observed. Hyperammonemia in constitutive model or after acute or chronic induced liver failure can be controlled by the administration of L. plantarum with a significant effect on survival. The mechanism involved in this ammonia decrease implicates direct ammonia consumption in the gut.

General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels.

Science.gov (United States)

Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

2004-03-01

1. Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl(-) channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl(-) channel activity of wild-type CFTR and delF508-CFTR mutant. 2. The effects of n-alkanols like octanol on CFTR activity were measured by iodide ((125)I) efflux and patch-clamp techniques on three distinct cellular models: (1). CFTR-expressing Chinese hamster ovary cells, (2). human airway Calu-3 epithelial cells and (3). human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. 3. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated (125)I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. (125)I efflux and Cl(-) currents induced by octanol were blocked by glibenclamide but insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, as expected for a CFTR Cl(-) current. 4. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. 5. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanoloctanoloctanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF.

Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

Science.gov (United States)

Pratter, S M; Eixelsberger, T; Nidetzky, B

2015-12-01

A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dietary Calcium and Dairy Modulation of Oxidative Stress and Mortality in aP2-Agouti and Wild-type Mice

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Antje Bruckbauer

2009-08-01

Full Text Available Oxidative and inflammatory stress have been implicated as major contributors to the aging process. Dietary Ca reduced both factors in short-term interventions, while milk exerted a greater effect than supplemental Ca. In this work, we examined the effects of life-long supplemental and dairy calcium on lifespan and life-span related biomarkers in aP2-agouti transgenic (model of diet-induced obesity and wild-type mice fed obesigenic diets until their death. These data demonstrate that dairy Ca exerts sustained effects resulting in attenuated adiposity, protection against age-related muscle loss and reduction of oxidative and inflammatory stress in both mouse strains. Although these effects did not alter maximum lifespan, they did suppress early mortality in wild-type mice, but not in aP2-agouti transgenic mice.

Comparison of the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303

DEFF Research Database (Denmark)

Rogowska-Wrzesinska, A.; Mose Larsen, P.; Blomberg, A.

2001-01-01

Yeast deletion strains created during gene function analysis projects very often show drastic phenotypic differences depending on the genetic background used. These results indicate the existence of important molecular differences between the CEN.PK2, FY1679 and W303 wild type strains...

Lymphotropism and host responses during acute wild-type canine distemper virus infections in a highly susceptible natural host

DEFF Research Database (Denmark)

Nielsen, Line; Søgaard, Mette; Jensen, Trine Hammer

2009-01-01

The mechanisms behind the in vivo virulence of immunosuppressive wild-type Morbillivirus infections are still not fully understood. To investigate lymphotropism and host responses we have selected the natural host model of canine distemper virus (CDV) infection in mink. This model displays...

Effect of deuterium on the circadian period and metabolism in wild-type and tau mutant Syrian hamsters

NARCIS (Netherlands)

Oklejewicz, M; Hut, RA; Daan, S

2000-01-01

Homozygous tau mutant Syrian hamsters (tau-/-) have a free-running circadian period (tau) around 20 h and a proportionally higher metabolic rate compared with wild-type hamsters (tau+/+) with a period of circa 24 h. In this study, we applied deuterium oxide (D2O) to hamsters to test whether

Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae)

Energy Technology Data Exchange (ETDEWEB)

Torricelli, Elena; Gorbi, Gessica; Pawlik-Skowronska, Barbara; Di Toppi, Luigi Sanita; Corradi, Maria Grazia

2004-07-14

Two strains of the unicellular green alga Scenedesmus acutus with different sensitivity to hexavalent chromium were compared for their tolerance of cadmium, by means of growth and recovery tests, and determination of cysteine, reduced glutathione and phytochelatin content, after short-term exposure to various cadmium concentrations (from 1.125 to 27 {mu}M). Growth experiments showed that, after 7-day treatments with cadmium, the chromium-tolerant strain reached a significantly higher cell density and, after 24-h exposure to Cd, was able to resume growth significantly better than the wild type. Constitutive level of cysteine was higher in the chromium-tolerant strain, while glutathione levels were similar in the two strains. The higher content of cysteine and the maintenance of both reduced glutathione and phytochelatin high levels in the presence of cadmium, support the higher cadmium co-tolerance of the chromium-tolerant strain in comparison with the wild type one.

Cadmium tolerance, cysteine and thiol peptide levels in wild type and chromium-tolerant strains of Scenedesmus acutus (Chlorophyceae)

International Nuclear Information System (INIS)

Torricelli, Elena; Gorbi, Gessica; Pawlik-Skowronska, Barbara; Di Toppi, Luigi Sanita; Corradi, Maria Grazia

2004-01-01

Two strains of the unicellular green alga Scenedesmus acutus with different sensitivity to hexavalent chromium were compared for their tolerance of cadmium, by means of growth and recovery tests, and determination of cysteine, reduced glutathione and phytochelatin content, after short-term exposure to various cadmium concentrations (from 1.125 to 27 μM). Growth experiments showed that, after 7-day treatments with cadmium, the chromium-tolerant strain reached a significantly higher cell density and, after 24-h exposure to Cd, was able to resume growth significantly better than the wild type. Constitutive level of cysteine was higher in the chromium-tolerant strain, while glutathione levels were similar in the two strains. The higher content of cysteine and the maintenance of both reduced glutathione and phytochelatin high levels in the presence of cadmium, support the higher cadmium co-tolerance of the chromium-tolerant strain in comparison with the wild type one

Identifying the Integrated Neural Networks Involved in Capsaicin-Induced Pain Using fMRI in Awake TRPV1 Knockout and Wild-Type Rats

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Jason Richard Yee

2015-02-01

Full Text Available In the present study, we used functional MRI in awake rats to investigate the pain response that accompanies intradermal injection of capsaicin into the hindpaw. To this end, we used BOLD imaging together with a 3D segmented, annotated rat atlas and computational analysis to identify the integrated neural circuits involved in capsaicin-induced pain. The specificity of the pain response to capsaicin was tested in a transgenic model that contains a biallelic deletion of the gene encoding for the transient receptor potential cation channel subfamily V member 1 (TRPV1. Capsaicin is an exogenous ligand for the TRPV1 receptor, and in wild-type rats, activated the putative pain neural circuit. In addition, capsaicin-treated wild-type rats exhibited activation in brain regions comprising the Papez circuit and habenular system, systems that play important roles in the integration of emotional information, and learning and memory of aversive information, respectively. As expected, capsaicin administration to TRPV1-KO rats failed to elicit the robust BOLD activation pattern observed in wild-type controls. However, the intradermal injection of formalin elicited a significant activation of the putative pain pathway as represented by such areas as the anterior cingulate, somatosensory cortex, parabrachial nucleus, and periaqueductal gray. Notably, comparison of neural responses to capsaicin in wild-type versus knock-out rats uncovered evidence that capsaicin may function in an antinociceptive capacity independent of TRPV1 signaling. Our data suggest that neuroimaging of pain in awake, conscious animals has the potential to inform the neurobiological basis of full and integrated perceptions of pain.

Role of wild-type p53 in apoptotic and non-apoptotic cell death induced by X-irradiation and heat treatment in p53-mutated mouse M10 cells

International Nuclear Information System (INIS)

Ito, Atsushi; Nakano, Hisako; Shinohara, Kunio

2010-01-01

The sensitizing effects of wild-type p53 on X-ray-induced cell death and on heat-induced apoptosis in M10, a radiosensitive and Trp53 (mouse p53 gene)-mutated lymphoma cell line which dies through necrosis by X-irradiation, were investigated using three M10 derived transfectants with wild-type TP53 (human p53 gene). Cell death was determined by colony formation and/or dye exclusion test, and apoptosis was detected as the changes in nuclear morphology by Giemsa staining. Expression of wild-type p53 protein increased radiosensitivity of cell death as determined by both clonogenic and dye exclusion assays. This increase in radiosensitivity was attributable largely to apoptosis induction in addition to a small enhancement of necrosis. Interestingly neither pathway to cell death was accompanied by caspase-3 activation. On the other hand, heat-induced caspase-3 dependent apoptotic cell death without transfection was further increased by the transfection of wild-type p53. In conclusion, the introduction of wild-type p53 enhanced apoptotic cell death by X-rays or heat via different mechanisms that do or do not activate caspase-3, respectively. In addition, p53 also enhanced the X-ray-induced necrosis in M10 cells. (author)

Spatial encoding in spinal sensorimotor circuits differs in different wild type mice strains

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Schouenborg Jens

2008-05-01

Full Text Available Abstract Background Previous studies in the rat have shown that the spatial organisation of the receptive fields of nociceptive withdrawal reflex (NWR system are functionally adapted through experience dependent mechanisms, termed somatosensory imprinting, during postnatal development. Here we wanted to clarify 1 if mice exhibit a similar spatial encoding of sensory input to NWR as previously found in the rat and 2 if mice strains with a poor learning capacity in various behavioural tests, associated with deficient long term potention, also exhibit poor adaptation of NWR. The organisation of the NWR system in two adult wild type mouse strains with normal long term potentiation (LTP in hippocampus and two adult wild type mouse strains exhibiting deficiencies in corresponding LTP were used and compared to previous results in the rat. Receptive fields of reflexes in single hindlimb muscles were mapped with CO2 laser heat pulses. Results While the spatial organisation of the nociceptive receptive fields in mice with normal LTP were very similar to those in rats, the LTP impaired strains exhibited receptive fields of NWRs with aberrant sensitivity distributions. However, no difference was found in NWR thresholds or onset C-fibre latencies suggesting that the mechanisms determining general reflex sensitivity and somatosensory imprinting are different. Conclusion Our results thus confirm that sensory encoding in mice and rat NWR is similar, provided that mice strains with a good learning capability are studied and raise the possibility that LTP like mechanisms are involved in somatosensory imprinting.

Explaining the resurgent popularity of the wild: motivations for wild plant gathering in the Biosphere Reserve Grosses Walsertal, Austria.

Science.gov (United States)

Schunko, Christoph; Grasser, Susanne; Vogl, Christian R

2015-06-30

Wild plant gathering becomes again a popular and fashionable activity in Europe after gathering practices have been increasingly abandoned over the last decades. Recent ethnobotanical research documented a diversity of gathering practices from people of diverse socio-economic and cultural backgrounds who gather in urban and rural areas. Few efforts were though made to study the motivations for gathering wild plants and to understand the resurgent popularity of wild plant gathering. This paper addresses the following research questions: (1) which motivations activate wild plant gatherers? (2) which motivation-types of gatherers exist in the Grosses Walsertal? (3) how do the motivations for gathering relate to the socio-demographic background of gatherers? Field research was conducted in the Grosses Walsertal, Austria in the years 2008 and 2009 in two field research periods. Thirty-six local farmers were first interviewed with semi-structured interviews. The motivations identified in these interviews were then included in a structured questionnaire, which was used to interview 353 residents of the valley. Pupils of local schools participated in the data collection as interviewers. Principal Component Analysis was used to categorize the motivations and to identify motivation-types of wild plant gatherers. Generalized Linear Models were calculated to identify relations between motivations and the socio-demographic background of gatherers. The respondents listed 13 different motivations for gathering wild plants and four motivations for not gathering. These 17 motivations were grouped in five motivation-types of wild plant gatherers, which are in decreasing importance: product quality, fun, tradition, not-gathering, income. Women, older respondents and homegardeners gather wild plants more often for fun; older respondents gather more often for maintaining traditions; non-homegardeners more frequently mention motivations for not gathering. The resurgent popularity of

Photomorphogenic responses to UV radiation III: a comparative study of UVB effects on anthocyanin and flavonoid accumulation in wild-type and aurea mutant of tomato (Lycopersicon esculentum Mill.)

International Nuclear Information System (INIS)

Brandt, K.; Giannini, A.; Lercari, B.

1995-01-01

The UV-mediated induction of anthocyanin and UV-absorbing compounds was characterized in etiolated hypocotyls of wild-type and aurea (au) mutant tomato seedlings. Ultraviolet radiation induced significant increases of anthocyanin and UV-absorbing compounds in hypocotyls of the au mutant and of its isogenic wild-type, but the differences in the time courses of UV-induced pigment accumulation indicate that different photoregulatory mechanisms are involved for each of these two groups of pigments. It appears that prolonged presence of adequate levels of UVB (290-320 nm) energy and consequently the action of a specific UVB photoreceptor are indispensable for the photoinduction of anthocyanin accumulation in UV-irradiated hypocotyl of the au mutant that is missing the labile phytochrome pool. The large difference found between the wild-type and the au mutant strongly indicate the involvement of labile phytochrome as the primary functional photoreceptor for the photoinduction of anthocyanin accumulation in wild-type tomato hypocotyls. (author)

Extracellular enzyme activities during lignocellulose degradation by Streptomyces spp.: a comparative study of wild-type and genetically manipulated strains

International Nuclear Information System (INIS)

Ramachandra, M.; Crawford, D.L.; Pometto, A.L. III

1987-01-01

The wild-type ligninolytic actinomycete Streptomyces viridosporus T7A and two genetically manipulated strains with enhanced abilities to produce a water-soluble lignin degradation intermediate, an acid-precipitable polymeric lignin (APPL), were grown on lignocellulose in solid-state fermentation cultures. Culture filtrates were periodically collected, analyzed for APPL, and assayed for extracellular lignocellulose-catabolizing enzyme activities. Two APPL-overproducing strains, UV irradiation mutant T7A-81 and protoplast fusion recombinant SR-10, had higher and longer persisting peroxidase, esterase, and endoglucanase activities than did the wild-type strain T7A. Results implicated one or more of these enzymes in lignin solubilization. Only mutant T7A-81 had higher xylanase activity than the wild type. The peroxidase was induced by both lignocellulose and APPL. This extracellular enzyme has some similarities to previously described ligninases in fungi. This is the first report of such an enzyme in Streptomyces spp. Four peroxidase isozymes were present, and all catalyzed the oxidation of 3,4-dihydroxyphenylalanine, while one also catalyzed hydrogen peroxide-dependent oxidation of homoprotocatechuic acid and caffeic acid. Three constitutive esterase isozymes were produced which differed in substrate specificity toward α-naphthyl acetate and α-naphthyl butyrate. Three endoglucanase bands, which also exhibited a low level of xylanase activity, were identified on polyacrylamide gels as was one xylanase-specific band. There were no major differences in the isoenzymes produced by the different strains. The probable role of each enzyme in lignocellulose degradation is discussed

Biodegradation of diuron by an endophytic fungus Neurospora intermedia DP8-1 isolated from sugarcane and its potential for remediating diuron-contaminated soils

OpenAIRE

Wang, Yanhui; Li, Honghong; Feng, Guojun; Du, Liangwei; Zeng, Dongqiang

2017-01-01

A diuron-degrading endophyte DP8-1 was isolated from sugarcane root grown in diuron-treated soil in the present study. The endophyte was identified as Neurospora intermedia based on the morphological characteristics and sequence analysis. The fermentation parameters including temperature, pH, inoculation size, carbon source, and initial diuron concentration were also investigated for the optimization of degradation efficiency. The results indicated that strain DP8-1 was capable of degrading u...

Characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses

International Nuclear Information System (INIS)

Burke, Crystal W.; Gardner, Christina L.; Steffan, Joshua J.; Ryman, Kate D.; Klimstra, William B.

2009-01-01

We examined the characteristics of interferon alpha/beta (IFN-α/β) induction after alphavirus or control Sendai virus (SeV) infection of murine fibroblasts (MEFs). As expected, SeV infection of wild-type (wt) MEFs resulted in strong dimerization of IRF3 and the production of high levels of IFN-α/β. In contrast, infection of MEFs with multiple alphaviruses failed to elicit detectable IFN-α/β. In more detailed studies, Sindbis virus (SINV) infection caused dimerization and nuclear migration of IRF3, but minimal IFN-β promoter activity, although surprisingly, the infected cells were competent for IFN production by other stimuli early after infection. A SINV mutant defective in host macromolecular synthesis shutoff induced IFN-α/β in the MEF cultures dependent upon the activities of the TBK1 IRF3 activating kinase and host pattern recognition receptors (PRRs) PKR and MDA5 but not RIG-I. These results suggest that wild-type alphaviruses antagonize IFN induction after IRF3 activation but also may avoid detection by host PRRs early after infection.

Commentary

Indian Academy of Sciences (India)

PRAKASH

all known Neurospora mutants, chromosome rearrangements, wild and .... Robert Metzenberg and colleagues, including David's associate N B Raju, ... tetrasperma was Dodge's favorite species and he tried (without success) to get his visitors interested in ... They are survived by their daughter Susan and her husband John.

EvoWild: a demosimulator about wild life

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Eduardo Palacio Gayoso

2008-12-01

Full Text Available During the last years we can see how AI (Artificial Intelligence is reappearing because of technological improvements. These improvements make possible the management of large groups of information with acceptable reply times.On the other hand, cost reductions in technology make possible that an investigation field like AI becomes to an inversion field closer to scale economies, that’s why it’ll be economically profitable to invert in this type of applications.One of the fastest consequences is the AI implantation in a big amount of devices of our environment, cell telephones, palms and of course, in the video game industry.This is the reason that took us to develop EvoWild, a simulation about wild life that has video game format and tools but at the same time implements AI algorithms like genetic algorithms and reasoning based in cases.

A STE12 homologue of the homothallic ascomycete Sordaria macrospora interacts with the MADS box protein MCM1 and is required for ascosporogenesis.

Science.gov (United States)

Nolting, Nicole; Pöggeler, Stefanie

2006-11-01

The MADS box protein MCM1 controls diverse developmental processes and is essential for fruiting body formation in the homothallic ascomycete Sordaria macrospora. MADS box proteins derive their regulatory specificity from a wide range of different protein interactions. We have recently shown that the S. macrospora MCM1 is able to interact with the alpha-domain mating-type protein SMTA-1. To further evaluate the functional roles of MCM1, we used the yeast two-hybrid approach to identify MCM1-interacting proteins. From this screen, we isolated a protein with a putative N-terminal homeodomain and C-terminal C2/H2-Zn2+ finger domains. The protein is a member of the highly conserved fungal STE12 transcription factor family of proteins and was therefore termed STE12. Furthermore, we demonstrate by means of two-hybrid and far western analysis that in addition to MCM1, the S. macrospora STE12 protein is able to interact with the mating-type protein SMTA-1. Unlike the situation in the closely related heterothallic ascomycete Neurospora crassa, deletion (Delta) of the ste12 gene in S. macrospora neither affects vegetative growth nor fruiting body formation. However, ascus and ascospore development are highly impaired by the Deltaste12 mutation. Our data provide another example of the functional divergence within the fungal STE12 transcription factor family.

Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

Science.gov (United States)

Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

2015-11-01

Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Beyond T and DHT - novel steroid derivatives capable of wild type androgen receptor activation.

Science.gov (United States)

Mostaghel, Elahe A

2014-01-01

While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa), castration does not eliminate androgens from the prostate tumor microenvironment, and residual intratumoral androgens are implicated in nearly every mechanism by which androgen receptor (AR)-mediated signaling promotes castration-resistant disease. The uptake and intratumoral (intracrine) conversion of circulating adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S) to steroids capable of activating the wild type AR is a recognized driver of castration resistant prostate cancer (CRPC). However, less well-characterized adrenal steroids, including 11-deoxcorticosterone (DOC) and 11beta-hydroxyandrostenedione (11OH-AED) may also play a previously unrecognized role in promoting AR activation. In particular, recent data demonstrate that the 5α-reduced metabolites of DOC and 11OH-AED are activators of the wild type AR. Given the well-recognized presence of SRD5A activity in CRPC tissue, these observations suggest that in the low androgen environment of CRPC, alternative sources of 5α-reduced ligands may supplement AR activation normally mediated by the canonical 5α-reduced agonist, 5α-DHT. Herein we review the emerging data that suggests a role for these alternative steroids of adrenal origin in activating the AR, and discuss the enzymatic pathways and novel downstream metabolites mediating these effects. We conclude by discussing the potential implications of these findings for CRPC progression, particularly in context of new agents such as abiraterone and enzalutamide which target the AR-axis for prostate cancer therapy.

Action potentials and ion conductances in wild-type and CALHM1-knockout type II taste cells

Science.gov (United States)

Saung, Wint Thu; Foskett, J. Kevin

2017-01-01

Taste bud type II cells fire action potentials in response to tastants, triggering nonvesicular ATP release to gustatory neurons via voltage-gated CALHM1-associated ion channels. Whereas CALHM1 regulates mouse cortical neuron excitability, its roles in regulating type II cell excitability are unknown. In this study, we compared membrane conductances and action potentials in single identified TRPM5-GFP-expressing circumvallate papillae type II cells acutely isolated from wild-type (WT) and Calhm1 knockout (KO) mice. The activation kinetics of large voltage-gated outward currents were accelerated in cells from Calhm1 KO mice, and their associated nonselective tail currents, previously shown to be highly correlated with ATP release, were completely absent in Calhm1 KO cells, suggesting that CALHM1 contributes to all of these currents. Calhm1 deletion did not significantly alter resting membrane potential or input resistance, the amplitudes and kinetics of Na+ currents either estimated from action potentials or recorded from steady-state voltage pulses, or action potential threshold, overshoot peak, afterhyperpolarization, and firing frequency. However, Calhm1 deletion reduced the half-widths of action potentials and accelerated the deactivation kinetics of transient outward currents, suggesting that the CALHM1-associated conductance becomes activated during the repolarization phase of action potentials. NEW & NOTEWORTHY CALHM1 is an essential ion channel component of the ATP neurotransmitter release mechanism in type II taste bud cells. Its contribution to type II cell resting membrane properties and excitability is unknown. Nonselective voltage-gated currents, previously associated with ATP release, were absent in cells lacking CALHM1. Calhm1 deletion was without effects on resting membrane properties or voltage-gated Na+ and K+ channels but contributed modestly to the kinetics of action potentials. PMID:28202574

Identification of enzymes and quantification of metabolic fluxes in the wild type and in a recombinant Aspergillus oryzae strain

DEFF Research Database (Denmark)

Pedersen, Henrik; Carlsen, Morten; Nielsen, Jens Bredal

1999-01-01

Two alpha-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the alpha-amylase gene, were characterized,vith respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition...

Paternal spatial training enhances offspring's cognitive performance and synaptic plasticity in wild-type but not improve memory deficit in Alzheimer's mice.

Science.gov (United States)

Zhang, Shujuan; Li, Xiaoguang; Wang, Zhouyi; Liu, Yanchao; Gao, Yuan; Tan, Lu; Liu, Enjie; Zhou, Qiuzhi; Xu, Cheng; Wang, Xin; Liu, Gongping; Chen, Haote; Wang, Jian-Zhi

2017-05-08

Recent studies suggest that spatial training can maintain associative memory capacity in Tg2576 mice, but it is not known whether the beneficial effects can be inherited from the trained fathers to their offspring. Here, we exposed male wild-type and male 3XTg Alzheimer disease (AD) mice (3-m old) respectively to spatial training for one week and assessed the transgenerational effects in the F1 offspring when they were grown to 7-m old. We found that the paternal spatial training significantly enhanced progeny's spatial cognitive performance and synaptic transmission in wild-type mice. Among several synapse- or memory-associated proteins, we observed that the expression level of synaptotagmin 1 (SYT1) was significantly increased in the hippocampus of the paternally trained-offspring. Paternal training increased histone acetylation at the promoter of SYT1 in both fathers' and the offspring's hippocampus, and as well as in the fathers' sperm. Finally, paternal spatial training for one week did not improve memory and synaptic plasticity in 3XTg AD F1 offspring. Our findings suggest paternal spatial training for one week benefits the offspring's cognitive performance in wild-type mice with the mechanisms involving an enhanced transgenerational histone acetylation at SYT1 promoter.

Differential inhibition of ex-vivo tumor kinase activity by vemurafenib in BRAF(V600E and BRAF wild-type metastatic malignant melanoma.

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Andliena Tahiri

Full Text Available Treatment of metastatic malignant melanoma patients harboring BRAF(V600E has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.

Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells.

Science.gov (United States)

Cawood, Ryan; Chen, Hannah H; Carroll, Fionnadh; Bazan-Peregrino, Miriam; van Rooijen, Nico; Seymour, Leonard W

2009-05-01

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

Nothing special in the specialist? Draft genome sequence of Cryomyces antarcticus, the most extremophilic fungus from Antarctica.

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Katja Sterflinger

Full Text Available The draft genome of the Antarctic endemic fungus Cryomyces antarcticus is presented. This rock inhabiting, microcolonial fungus is extremely stress tolerant and it is a model organism for exobiology and studies on stress resistance in Eukaryots. Since this fungus is a specialist in the most extreme environment of the Earth, the analysis of its genome is of important value for the understanding of fungal genome evolution and stress adaptation. A comparison with Neurospora crassa as well as with other microcolonial fungi shows that the fungus has a genome size of 24 Mbp, which is the average in the fungal kingdom. Although sexual reproduction was never observed in this fungus, 34 mating genes are present with protein homologs in the classes Eurotiomycetes, Sordariomycetes and Dothideomycetes. The first analysis of the draft genome did not reveal any significant deviations of this genome from comparative species and mesophilic hyphomycetes.

Myco-fluidics: The fluid dynamics of fungal chimerism

Science.gov (United States)

Roper, Marcus; Hickey, Patrick; Dressaire, Emilie; Roch, Sebastien

2012-11-01

Chimeras-fantastical creatures formed as amalgams of many animals-have captured the human imagination since Ancient times. But they are also surprisingly common in Nature. The syncytial cells of filamentous fungi harbor large numbers of nuclei bathed in a single cytoplasm. As a fungus grows these nuclei become genetically diverse, either from mutation or from exchange of nuclei between different fungal individuals, a process that is known to increase the virulence of the fungus and its adaptability. By directly measuring nuclear movement in the model ascomycete fungus Neurospora crassa, we show that the fungus' tolerance for internal genetic diversity is enabled by hydrodynamic mixing of nuclei acting at all length scales within the fungal mycelium. Mathematical modeling and experiments in a mutant with altered mycelial morphology reveal some of the exquisite hydraulic engineering necessary to create these mixing flows from spatially coarse pressure gradients.

Identification and functional analysis of endogenous nitric oxide in a filamentous fungus.

Science.gov (United States)

Pengkit, Anchalee; Jeon, Seong Sil; Son, Soo Ji; Shin, Jae Ho; Baik, Ku Yeon; Choi, Eun Ha; Park, Gyungsoon

2016-07-18

In spite of its prevalence in animals and plants, endogenous nitric oxide (NO) has been rarely reported in fungi. We present here our observations on production of intracellular NO and its possible roles during development of Neurospora crassa, a model filamentous fungus. Intracellular NO was detected in hypha 8-16 hours after incubation in Vogel's minimal liquid media and conidiophores during conidiation using a fluorescent indicator (DAF-FM diacetate). Treatment with cPTIO, an NO scavenger, significantly reduced fluorescence levels and hindered hyphal growth in liquid media and conidiation, whereas exogenous NO enhanced hyphal extension on VM agar media and conidia formation. NO scavenging also dramatically diminished transcription of con-10 and con-13, genes preferentially expressed during conidiation. Our results suggest that intracellular NO is generated in young hypha growing in submerged culture and during conidia development and regulate mycelial development and conidia formation.

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS

Science.gov (United States)

Estes, Patricia S.; Boehringer, Ashley; Zwick, Rebecca; Tang, Jonathan E.; Grigsby, Brianna; Zarnescu, Daniela C.

2011-01-01

The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders. PMID:21441568

Gene genealogies indicates abundant gene conversions and independent evolutionary histories of the mating-type chromosomes in the evolutionary history of Neurospora tetrasperma

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Whittle Carrie A

2010-07-01

Full Text Available Abstract Background The self-fertile filamentous ascomycete Neurospora tetrasperma contains a large (~7 Mbp and young (mat chromosomes. The objective of the present study is to reveal the evolutionary history, including key genomic events, associated with the various regions of the mat chromosomes among ten strains representing all the nine known species (lineages contained within the N. tetrasperma species complex. Results Comparative analysis of sequence divergence among alleles of 24 mat-linked genes (mat A and mat a indicates that a large region of suppressed recombination exists within the mat chromosome for each of nine lineages of N. tetrasperma sensu latu. The recombinationally suppressed region varies in size and gene composition among lineages, and is flanked on both ends by normally recombining regions. Genealogical analyses among lineages reveals that eight gene conversion events have occurred between homologous mat A and mat a-linked alleles of genes located within the region of restricted recombination during the evolutionary history of N. tetrasperma. Conclusions We conclude that the region of suppressed recombination in the mat chromosomes has likely been subjected to independent contraction and/or expansion during the evolutionary history of the N. tetrasperma species complex. Furthermore, we infer that gene conversion events are likely a common phenomenon within this recombinationally suppressed genomic region. We argue that gene conversions might provide an efficient mechanism of adaptive editing of functional genes, including the removal of deleterious mutations, within the young recombinationally suppressed region of the mat chromosomes.

Biological activities of extracts from Chenopodium ambrosioides Lineu and Kielmeyera neglecta Saddi

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Sousa Zulane

2012-07-01

Full Text Available Abstract Background Chenopodium ambrosioides and Kielmeyera neglecta are plants traditionally used in Brazil to treat various infectious diseases. The study of the biological activities of these plants is of great importance for the detection of biologically active compounds. Methods Extracts from these plants were extracted with hexane (Hex, dichloromethane (DCM, ethyl acetate (EtOAc and ethanol (EtOH and assessed for their antimicrobial properties, bioactivity against Artemia salina Leach and antifungal action on the cell wall of Neurospora crassa. Results Extracts from C. ambrosioides (Hex, DCM and EtOH and K. neglecta (EtOAc and EtOH showed high bioactivity against A. salina (LD50 C. ambrosioides Hex and DCM showed specific activity against yeasts, highlighting the activity of hexanic extract against Candida krusei (MIC = 100 μg/mL. By comparing the inhibitory concentration of 50% growth (IC 50% with the growth control, extracts from K. neglecta EtOAc and EtOH have shown activities against multidrug-resistant bacteria (Enterococcus faecalis ATCC 51299 and Staphylococcus aureus ATCC 43300, with IC 50% of 12.5 μg/mL The assay carried out on N. crassa allowed defining that extracts with antifungal activity do not have action through inhibition of cell wall synthesis. Conclusions Generally speaking, extracts from C. ambrosioides and K. neglecta showed biological activities that have made the search for bioactive substances in these plants more attractive, illustrating the success of their use in the Brazilian folk medicine.

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

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Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

2012-01-01

Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

An Epichloë festucae homologue of MOB3, a component of the STRIPAK complex, is required for the establishment of a mutualistic symbiotic interaction with Lolium perenne

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Green, Kimberly A.; Becker, Yvonne; Fitzsimons, Helen L.

2016-01-01

Summary In both Sordaria macrospora and Neurospora crassa, components of the conserved STRIPAK (striatin‐interacting phosphatase and kinase) complex regulate cell–cell fusion, hyphal network development and fruiting body formation. Interestingly, a number of Epichloë festucae genes that are required for hyphal cell–cell fusion, such as noxA, noxR, proA, mpkA and mkkA, are also required for the establishment of a mutualistic symbiotic interaction with Lolium perenne. To determine whether MobC, a homologue of the STRIPAK complex component MOB3 in S. macrospora and N. crassa, is required for E. festucae hyphal fusion and symbiosis, a mobC deletion strain was generated. The ΔmobC mutant showed reduced rates of hyphal cell–cell fusion, formed intrahyphal hyphae and exhibited enhanced conidiation. Plants infected with ΔmobC were severely stunted. Hyphae of ΔmobC showed a proliferative pattern of growth within the leaves of Lolium perenne with increased colonization of the intercellular spaces and vascular bundles. Although hyphae were still able to form expressoria, structures allowing the colonization of the leaf surface, the frequency of formation was significantly reduced. Collectively, these results show that the STRIPAK component MobC is required for the establishment of a mutualistic symbiotic association between E. festucae and L. perenne, and plays an accessory role in the regulation of hyphal cell–cell fusion and expressorium development in E. festucae. PMID:27277141

MT-PCR panel detection of canine parvovirus (CPV-2): Vaccine and wild-type CPV-2 can be difficult to differentiate in canine diagnostic fecal samples.

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Meggiolaro, Maira N; Ly, Anna; Rysnik-Steck, Benjamin; Silva, Carolina; Zhang, Joshua; Higgins, Damien P; Muscatello, Gary; Norris, Jacqueline M; Krockenberger, Mark; Šlapeta, Jan

2017-06-01

Canine parvovirus (CPV-2) remains an important cause of devastating enteritis in young dogs. It can be successfully prevented with live attenuated CPV-2 vaccines when given at the appropriate age and in the absence of maternal antibody interference. Rapid diagnosis of parvoviral enteritis in young dogs is essential to ensuring suitable barrier nursing protocols within veterinary hospitals. The current diagnostic trend is to use multiplexed PCR panels to detect an array of pathogens commonly responsible for diarrhea in dogs. The multiplexed PCR assays do not distinguish wild from vaccine CPV-2. They are highly sensitive and detect even a low level of virus shedding, such as those caused by the CPV-2 vaccine. The aim of this study was to identify the CPV-2 subtypes detected in diagnostic specimens and rule out occult shedding of CPV-2 vaccine strains. For a total of 21 samples that tested positive for CPV-2 in a small animal fecal pathogens diagnostic multiplexed tandem PCR (MT-PCR) panel during 2014-2016 we partially characterized the VP2 gene of CPV-2. Vaccine CPV-2 strain, wild type CPV-2a subtypes and vaccine-like CPV-2b subtypes were detected. High copy number was indicative of wild-type CPV-2a presence, but presence of vaccine-like CPV-2b had a variable copy number in fecal samples. A yardstick approach to a copy number or C t -value to discriminate vaccine strain from a wild type virus of CPV-2 can be, in some cases, potentially misleading. Therefore, discriminating vaccine strain from a wild type subtype of CPV-2 remains ambitious. Copyright © 2017 Elsevier Ltd. All rights reserved.

Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars

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Abiri, Rambod; Kalhori, Nahid; Atabaki, Narges

2017-01-01

Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties. PMID:28191468

Screening and Expression of a Silicon Transporter Gene (Lsi1 in Wild-Type Indica Rice Cultivars

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Mahbod Sahebi

2017-01-01

Full Text Available Silicon (Si is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.

Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars.

Science.gov (United States)

Sahebi, Mahbod; Hanafi, Mohamed M; Rafii, M Y; Azizi, Parisa; Abiri, Rambod; Kalhori, Nahid; Atabaki, Narges

2017-01-01

Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.

Lysogenic Streptococcus suis isolate SS2-4 containing prophage SMP showed increased mortality in zebra fish compared to the wild-type isolate.

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Fang Tang

Full Text Available Streptococcus suis (S. suis infection is considered to be a major problem in the swine industry worldwide. Based on the capsular type, 33 serotypes of S. suis have been described, with serotype 2 (SS2 being the most frequently isolated from diseased piglets. Little is known, however, about the pathogenesis and virulence factors of S. suis. Research on bacteriophages highlights a new area in S. suis research. A S. suis serotype 2 bacteriophage, designated SMP, has been previously isolated in our laboratory. Here, we selected a lysogenic isolate in which the SMP phage was integrated into the chromosome of strain SS2-4. Compared to the wild-type isolate, the lysogenic strain showed increased mortality in zebra fish. Moreover the sensitivity of the lysogenic strain to lysozyme was seven times higher than that of the wild-type.

Environmental Surveillance System To Track Wild Poliovirus Transmission

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Deshpande, Jagadish M.; Shetty, Sushmitha J.; Siddiqui, Zaeem A.

2003-01-01

Eradication of poliomyelitis from large metropolis cities in India has been difficult due to high population density and the presence of large urban slums. Three paralytic poliomyelitis cases were reported in Mumbai, India, in 1999 and 2000 in spite of high immunization coverage and good-quality supplementary immunization activities. We therefore established a systematic environmental surveillance study by weekly screening of sewage samples from three high-risk slum areas to detect the silent transmission of wild poliovirus. In 2001, from among the 137 sewage samples tested, wild poliovirus type 1 was isolated from 35 and wild poliovirus type 3 was isolated from 1. Acute flaccid paralysis (AFP) surveillance indicated one case of paralytic poliomyelitis from the city. Phylogenetic analysis with complete VP1 sequences revealed that the isolates from environmental samples belonged to four lineages of wild polioviruses recently isolated from poliomyelitis cases in Uttar Pradesh and not to those previously isolated from AFP cases in Mumbai. Wild poliovirus thus introduced caused one case of paralytic poliomyelitis. The virus was detected in environmental samples 3 months before. It was found that wild polioviruses introduced several times during the year circulated in Mumbai for a limited period before being eliminated. Environmental surveillance was found to be sensitive for the detection of wild poliovirus silent transmission. Nucleotide sequence analysis helped identify wild poliovirus reservoir areas. PMID:12732567

Single-molecule study of oxidative enzymatic deconstruction of cellulose.

Science.gov (United States)

Eibinger, Manuel; Sattelkow, Jürgen; Ganner, Thomas; Plank, Harald; Nidetzky, Bernd

2017-10-12

LPMO (lytic polysaccharide monooxygenase) represents a unique paradigm of cellulosic biomass degradation by an oxidative mechanism. Understanding the role of LPMO in deconstructing crystalline cellulose is fundamental to the enzyme's biological function and will help to specify the use of LPMO in biorefinery applications. Here we show with real-time atomic force microscopy that C1 and C4 oxidizing types of LPMO from Neurospora crassa (NcLPMO9F, NcLPMO9C) bind to nanocrystalline cellulose with high preference for the very same substrate surfaces that are also used by a processive cellulase (Trichoderma reesei CBH I) to move along during hydrolytic cellulose degradation. The bound LPMOs, however, are immobile during their adsorbed residence time ( ~ 1.0 min for NcLPMO9F) on cellulose. Treatment with LPMO resulted in fibrillation of crystalline cellulose and strongly ( ≥ 2-fold) enhanced the cellulase adsorption. It also increased enzyme turnover on the cellulose surface, thus boosting the hydrolytic conversion.Understanding the role of enzymes in biomass depolymerization is essential for the development of more efficient biorefineries. Here, the authors show by atomic force microscopy the real-time mechanism of cellulose deconstruction by lytic polysaccharide monooxygenases.

Biochemical characterization of the plasma membrane H+ - ATPase from red beet (Beta vulgaris) hypocotyl tissue

International Nuclear Information System (INIS)

Oleski, N.A.

1986-01-01

Several biochemical techniques including selective solubilization followed by gel filtration or various types of affinity chromatography, and antibody production were employed in an attempt to purify the plasma membrane H + - ATPase from red beet hypocotyl tissue. While the enzyme could not be purified using any of these methods, it was possible to successfully conduct a more detailed biochemical analysis of the H + - ATPase. The molecular weight and isoelectric point of the enzyme were determined using N,N'dicyclohexylcarbodiimide (DCCD) and a H + - ATPase antibody. When plasma membrane vesicles were incubated with 20 μM [ 14 C]-DCCD at 0 C, a single 97,000 dalton protein was apparent on a fluorograph. A close correlation between [ 14 C]-DCCD labelling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentrations suggests that this 97,000 dalton protein is a component of the plasma membrane H + - ATPase. An antibody raised against the plasma membrane H + - ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the enzyme to be pH 6.5

Wild and domesticated mushroom consumption in Nigeria ...

African Journals Online (AJOL)

African Crop Science Journal ... On the other hand, if nutrition analysis reveals different nutrition parameters for both types of mushrooms, 43.3% opted for cultivated mushroom, 42.2%, wild; 12.2% both; while 2.2% would eat ... Keywords: Consumption pattern, Lentinus squarrosulus, nutrition, perception, wild mushroom ...

Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells.

Science.gov (United States)

Shi, Ming-Zhu; Xie, De-Yu

2011-04-01

We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC-MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1-GL3/TT8-PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.

Pulmonary hypertension in wild type mice and animals with genetic deficit in KCa2.3 and KCa3.1 channels.

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Christine Wandall-Frostholm

Full Text Available In vascular biology, endothelial KCa2.3 and KCa3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of KCa2.3 and KCa3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of KCa2.3 and KCa3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.Male wild type and KCa3.1-/-/KCa2.3T/T(+DOX mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The KCa3.1-/-/KCa2.3T/T(+DOX mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the KCa2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the KCa2.3 and KCa3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of KCa2.3 and KCa3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.Despite the deficits of the KCa2.3 and KCa3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of KCa2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary

Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands

International Nuclear Information System (INIS)

Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S.; Damborsky, Jiri; Kuta Smatanova, Ivana

2011-01-01

Crystals of the wild-type haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 and of its catalytically inactive variant DhaA13 were grown in the presence of various ligands and diffraction data were collected to high and atomic resolution. Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon–halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2 1 2 1 2 1 as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively

Panitumumab and pegylated liposomal doxorubicin in platinum-resistant epithelial ovarian cancer with KRAS wild-type

DEFF Research Database (Denmark)

Dahl Steffensen, Karina; Waldstrøm, Marianne; Lund, Bente

, and head and neck cancer. No previous studies have evaluated the effect of panitumumab in OC based on KRAS mutation status. Methods: Eligibility criteria are confirmed stage I-IV primary epithelial ovarian/fallopian/peritoneal cancer patients with progression either during or within 6 months after end...... to a total of 33 patients. At present, 15 patients have been enrolled. The primary endpoint is to investigate the response rate in platinum-resistant, KRAS wild- type OC patients treated with PLD supplemented with panitumumab. Translational research is included as a secondary endpoint and tumor tissue...

Detection of multiple cocirculating wild poliovirus type 1 lineages through environmental surveillance: impact and progress during 2011-2013 in Pakistan.

Science.gov (United States)

Alam, Muhammad Masroor; Shaukat, Shahzad; Sharif, Salmaan; Angez, Mehar; Khurshid, Adnan; Malik, Farzana; Rehman, Lubna; Zaidi, Syed Sohail Zahoor

2014-11-01

The environmental surveillance has proven to be a useful tool to identify poliovirus circulation in different countries and was started in Pakistan during July 2009 to support the acute flaccid paralysis (AFP) surveillance system. Sewage samples were collected from 27 environmental sampling (ENV) sites and processed for poliovirus isolation through 2-phase separation method. Poliovirus isolates were identified as Sabin-like or wild type through real-time polymerase chain reaction (PCR). Wild-type strains were subjected to VP1 gene sequencing and phylogenetic analysis performed using MEGA 5.0. During 2011-2013, a total of 668 samples were collected from 4 provinces that resulted in 40% of samples positive for wild poliovirus type-1 (WPV-1). None of the samples were positive for WPV-3. The areas with high frequency of WPV-1 detection were Karachi-Gadap (69%), Peshawar (82%), and Rawalpindi (65%), whereas the samples from Quetta and Sukkur remained negative for WPV during 2013. Phylogenetic analysis revealed 3 major clusters with multiple poliovirus lineages circulating across different country areas as well as in bordering areas of Afghanistan. Environmental surveillance in Pakistan has been proven to be a powerful tool to detect WPV circulation in the absence of poliomyelitis cases in many communities. Our findings emphasize the need to continue and expand such surveillance activities to other high-risk areas in the country. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

First report of wild boar susceptibility to Porcine circovirus type 3: High prevalence in the Colli Euganei Regional Park (Italy) in the absence of clinical signs.

Science.gov (United States)

Franzo, Giovanni; Tucciarone, Claudia Maria; Drigo, Michele; Cecchinato, Mattia; Martini, Marco; Mondin, Alessandra; Menandro, Maria Luisa

2018-05-18

The genus Circovirus includes one of the most relevant infectious agents affecting domestic pigs, Porcine circovirus type 2 (PCV-2). The wild boar susceptibility to this pathogen has also been demonstrated although the actual epidemiological role of wild populations is still debated. In recent times, a new circovirus, Porcine circovirus type 3 (PCV-3), has been discovered and reported in the presence of several clinical conditions. However, no information is currently available about PCV-3 circulation and prevalence in wild boar. To fill this gap, 187 wild boar serum samples were collected in the Colli Euganei Regional Park (Northern Italy) and screened for PCV-3, demonstrating a high viral prevalence (approximately 30%). No gender differences were demonstrated while a lower infection prevalence was observed in animals younger than 12 months compared to older ones, differently from what described in commercial pigs. Almost all sampled animals were in good health conditions and no association was proven between PCV-3 status and clinical syndromes in wild animals. The genetic characterization of selected strains enlightened a relevant variability and the absence of closely related strains originating from domestic pigs. Therefore, the observed scenario is suggestive of multiple introductions from other wild or domestic swine populations followed by prolonged circulation and independent evolution. Worldwide, this study reports for the first time the high susceptibility of the wild boar to PCV-3 infection. The high prevalence and the absence of association with clinical signs support the marginal role of this virus in the wild boar population ecology. However, its epidemiological role as reservoir endangering commercial swine cannot be excluded and will require further investigations. © 2018 Blackwell Verlag GmbH.

Phenylbutyrate Sensitizes Human Glioblastoma Cells Lacking Wild-Type P53 Function to Ionizing Radiation

International Nuclear Information System (INIS)

Lopez, Carlos A.; Feng, Felix Y.; Herman, Joseph M.; Nyati, Mukesh K.; Lawrence, Theodore S.; Ljungman, Mats

2007-01-01

Purpose: Histone deacetylase (HDAC) inhibitors induce growth arrest, differentiation, and apoptosis in cancer cells. Phenylbutyrate (PB) is a HDAC inhibitor used clinically for treatment of urea cycle disorders. Because of its low cytotoxicity, cerebrospinal fluid penetration, and high oral bioavailability, we investigated PB as a potential radiation sensitizer in human glioblastoma cell lines. Methods and Materials: Four glioblastoma cell lines were selected for this study. Phenylbutyrate was used at a concentration of 2 mM, which is achievable in humans. Western blots were used to assess levels of acetylated histone H3 in tumor cells after treatment with PB. Flow cytometry was used for cell cycle analysis. Clonogenic assays were performed to assess the effect of PB on radiation sensitivity. We used shRNA against p53 to study the role of p53 in radiosensitization. Results: Treatment with PB alone resulted in hyperacetylation of histones, confirmed by Western blot analysis. The PB alone resulted in cytostatic effects in three cell lines. There was no evidence of G 1 arrest, increase in sub-G 1 fraction or p21 protein induction. Clonogenic assays showed radiosensitization in two lines harboring p53 mutations, with enhancement ratios (± SE) of 1.5 (± 0.2) and 1.3 (± 0.1), respectively. There was no radiopotentiating effect in two cell lines with wild-type p53, but knockdown of wild-type p53 resulted in radiosensitization by PB. Conclusions: Phenylbutyrate can produce p21-independent cytostasis, and enhances radiation sensitivity in p53 mutant human glioblastoma cells in vitro. This suggests the potential application of combined PB and radiotherapy in glioblastoma harboring mutant p53

Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

Energy Technology Data Exchange (ETDEWEB)

O; Dell, William B.; Swartz, Paul D.; Weiss, Kevin L.; Meilleur, Flora (ORNL); (NCSU)

2017-01-19

Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.

Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels

Science.gov (United States)

Li, Xin; Yu, Vivian Yaci; Lin, Yuping; Chomvong, Kulika; Estrela, Raíssa; Park, Annsea; Liang, Julie M; Znameroski, Elizabeth A; Feehan, Joanna; Kim, Soo Rin; Jin, Yong-Su; Glass, N Louise; Cate, Jamie HD

2015-01-01

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production. DOI: http://dx.doi.org/10.7554/eLife.05896.001 PMID:25647728

Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing.

Science.gov (United States)

Silar, Philippe; Barreau, Christian; Debuchy, Robert; Kicka, Sébastien; Turcq, Béatrice; Sainsard-Chanet, Annie; Sellem, Carole H; Billault, Alain; Cattolico, Laurence; Duprat, Simone; Weissenbach, Jean

2003-08-01

A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species.

Epigenetics as an emerging tool for improvement of fungal strains used in biotechnology.

Science.gov (United States)

Aghcheh, Razieh Karimi; Kubicek, Christian P

2015-08-01

Filamentous fungi are today a major source of industrial biotechnology for the production of primary and secondary metabolites, as well as enzymes and recombinant proteins. All of them have undergone extensive improvement strain programs, initially by classical mutagenesis and later on by genetic manipulation. Thereby, strategies to overcome rate-limiting or yield-reducing reactions included manipulating the expression of individual genes, their regulatory genes, and also their function. Yet, research of the last decade clearly showed that cells can also undergo heritable changes in gene expression that do not involve changes in the underlying DNA sequences (=epigenetics). This involves three levels of regulation: (i) DNA methylation, (ii) chromatin remodeling by histone modification, and (iii) RNA interference. The demonstration of the occurrence of these processes in fungal model organisms such as Aspergillus nidulans and Neurospora crassa has stimulated its recent investigation as a tool for strain improvement in industrially used fungi. This review describes the progress that has thereby been obtained.

H+-ATPase activity from storage tissue of Beta vulgaris. IV. N,N'-dicyclohexylcarbodiimide binding and inhibition of the plasma membrane H+-ATPase

International Nuclear Information System (INIS)

Oleski, N.A.; Bennett, A.B.

1987-01-01

The molecular weight and isoelectric point of the plasma membrane H + -ATPase from red beet storage tissue were determined using N,N'-dicyclohexylcarbodiimide (DCCD) and a H + -ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [ 14 C]-DCCD at 0 0 C, a single 97,000 dalton protein was visualized on a fluorography of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [ 14 C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H + -ATPase. An antibody raised against the plasma membrane H + -ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H + -ATPase to be 6.5

Three-dimensional image analysis of plugging at the septal pore by Woronin body during hypotonic shock inducing hyphal tip bursting in the filamentous fungus Aspergillus oryzae

International Nuclear Information System (INIS)

Maruyama, Jun-ichi; Juvvadi, Praveen Rao; Ishi, Kazutomo; Kitamoto, Katsuhiko

2005-01-01

We observed that the filamentous fungus, Aspergillus oryzae, grown on agar media burst out cytoplasmic constituents from the hyphal tip soon after flooding with water. Woronin body is a specialized organelle known to plug the septal pore adjacent to the lysed compartment to prevent extensive loss of cytoplasm. A. oryzae Aohex1 gene homologous to Neurospora crassa HEX1 gene encoding a major protein in Woronin body was expressed as a fusion with DsRed2, resulting in visualization of Woronin body. Confocal microscopy and three-dimensional reconstruction of images visualized the septal pore as a dark region surrounded by green fluorescence of EGFP-fused secretory protein, RNase T1, on the septum. Dual fluorescent labeling revealed the plugging of the septal pores adjacent to the lysed apical compartments by Woronin bodies during hypotonic shock. Disruption of Aohex1 gene caused disappearance of Woronin bodies and the defect to prevent extensive loss of cytoplasm during hypotonic shock

Single-Agent Panitumumab in Frail Elderly Patients With Advanced RAS and BRAF Wild-Type Colorectal Cancer: Challenging Drug Label to Light Up New Hope

Science.gov (United States)

Cremolini, Chiara; Aprile, Giuseppe; Lonardi, Sara; Orlandi, Armando; Mennitto, Alessia; Berenato, Rosa; Antoniotti, Carlotta; Casagrande, Mariaelena; Marsico, Valentina; Marmorino, Federica; Cardellino, Giovanni Gerardo; Bergamo, Francesca; Tomasello, Gianluca; Formica, Vincenzo; Longarini, Raffaella; Giommoni, Elisa; Caporale, Marta; Di Bartolomeo, Maria; Loupakis, Fotios; de Braud, Filippo

2015-01-01

Background. No prospective trials have specifically addressed the efficacy and safety of panitumumab in elderly patients with metastatic colorectal cancer (CRC). We aimed at assessing the efficacy and safety of single agent panitumumab in “frail” elderly patients diagnosed with metastatic RAS and BRAF wild-type CRC. Materials and Methods. Forty elderly patients (aged ≥75 years) with metastatic RAS-BRAF wild-type CRC received off-label prescriptions of single-agent panitumumab at seven Italian institutions. Treatment was administered as first line in patients with absolute contraindication to any chemotherapy or as second-line treatment after failure of a fluoropyrimidine-based treatment, in the presence of contraindication to irinotecan. The outcome measures included objective response rate (ORR), as well as progression-free survival (PFS), disease control rate (DCR), overall survival (OS), and safety. Results. The median PFS and OS were 6.4 months (95% confidence interval [CI]: 4.9–8 months) and 14.3 months (95% CI: 10.9–17.7 months), respectively. ORR was 32.5%, and DCR was 72.5%. Dose reductions related to adverse events (AEs) were reported in 9 (23%) patients, but no permanent treatment discontinuation caused by was reported. The most frequent grade 3 AE was skin rash, with an incidence of 20%. Conclusion. Panitumumab is effective and well-tolerated in frail elderly patients with RAS-BRAF wild-type metastatic CRC and deemed unfit for chemotherapy. A randomized study is needed to confirm these data. Implications for Practice: Treatment of elderly patients with metastatic colorectal cancer represents a difficult challenge in clinical practice. A significant proportion of frail elderly patients do not receive treatment, reflecting ongoing uncertainty of clinical benefit and toxicity of chemotherapy. Unfit condition in this cohort of patients further limits antineoplastic prescription and consequently patient survival. RAS and BRAF wild-type status could

Tenofovir alafenamide demonstrates broad cross-genotype activity against wild-type HBV clinical isolates and maintains susceptibility to drug-resistant HBV isolates in vitro.

Science.gov (United States)

Liu, Yang; Miller, Michael D; Kitrinos, Kathryn M

2017-03-01

Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV). This study evaluated the antiviral activity of TAF against wild-type genotype A-H HBV clinical isolates as well as adefovir-resistant, lamivudine-resistant, and entecavir-resistant HBV isolates. Full length HBV genomes or the polymerase/reverse transcriptase (pol/RT) region from treatment-naïve patients infected with HBV genotypes A-H were amplified and cloned into an expression vector under the control of a CMV promoter. In addition, 11 drug resistant HBV constructs were created by site-directed mutagenesis of a full length genotype D construct. Activity of TAF was measured by transfection of each construct into HepG2 cells and assessment of HBV DNA levels following treatment across a range of TAF concentrations. TAF activity in vitro was similar against wild-type genotype A-H HBV clinical isolates. All lamivudine- and entecavir-resistant isolates and 4/5 adefovir-resistant isolates were found to be sensitive to inhibition by TAF in vitro as compared to the wild-type isolate. The adefovir-resistant isolate rtA181V + rtN236T exhibited low-level reduced susceptibility to TAF. TAF is similarly active in vitro against wild-type genotype A-H HBV clinical isolates. The TAF sensitivity results for all drug-resistant isolates are consistent with what has been observed with the parent drug TFV. The in vitro cell-based HBV phenotyping assay results support the use of TAF in treatment of HBV infected subjects with diverse HBV genotypes, in both treatment-naive and treatment-experienced HBV infected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Phase II Study of the Dual EGFR/HER3 Inhibitor Duligotuzumab (MEHD7945A) versus Cetuximab in Combination with FOLFIRI in Second-Line RAS Wild-Type Metastatic Colorectal Cancer.

Science.gov (United States)

Hill, Andrew G; Findlay, Michael P; Burge, Matthew E; Jackson, Christopher; Alfonso, Pilar Garcia; Samuel, Leslie; Ganju, Vinod; Karthaus, Meinolf; Amatu, Alessio; Jeffery, Mark; Bartolomeo, Maria Di; Bridgewater, John; Coveler, Andrew L; Hidalgo, Manuel; Kapp, Amy V; Sufan, Roxana I; McCall, Bruce B; Hanley, William D; Penuel, Elicia M; Pirzkall, Andrea; Tabernero, Josep

2018-05-15

Purpose: Duligotuzumab is a dual-action antibody directed against EGFR and HER3. Experimental Design: Metastatic colorectal cancer (mCRC) patients with KRAS ex2 wild-type received duligotuzumab or cetuximab and FOLFIRI until progression or intolerable toxicity. Mandatory tumor samples underwent mutation and biomarker analysis. Efficacy analysis was conducted in patients with RAS exon 2/3 wild-type tumors. Results: Of 134 randomly assigned patients, 98 had RAS ex2/3 wild-type. Duligotuzumab provided no progression-free survival (PFS) or overall survival (OS) benefit compared with cetuximab, although there was a trend for a lower objective response rate (ORR) in the duligotuzumab arm. No relationship was seen between PFS or ORR and ERBB3, NRG1, or AREG expression. There were fewer skin rash events for duligotuzumab but more diarrhea. Although the incidence of grade ≥3 AEs was similar, the frequency of serious AEs was higher for duligotuzumab. Conclusions: Duligotuzumab plus FOLFIRI did not appear to improve the outcomes in patients with RAS exon 2/3 wild-type mCRC compared with cetuximab + FOLFIRI. Clin Cancer Res; 24(10); 2276-84. ©2018 AACR . ©2018 American Association for Cancer Research.

Brucella abortus ΔrpoE1 confers protective immunity against wild type challenge in a mouse model of brucellosis.

Science.gov (United States)

Willett, Jonathan W; Herrou, Julien; Czyz, Daniel M; Cheng, Jason X; Crosson, Sean

2016-09-30

The Brucella abortus general stress response (GSR) system regulates activity of the alternative sigma factor, σ(E1), which controls transcription of approximately 100 genes and is required for persistence in a BALB/c mouse chronic infection model. We evaluated the host response to infection by a B. abortus strain lacking σ(E1) (ΔrpoE1), and identified pathological and immunological features that distinguish ΔrpoE1-infected mice from wild-type (WT), and that correspond with clearance of ΔrpoE1 from the host. ΔrpoE1 infection was indistinguishable from WT in terms of splenic bacterial burden, inflammation and histopathology up to 6weeks post-infection. However, Brucella-specific serum IgG levels in ΔrpoE1-infected mice were 5 times higher than WT by 4weeks post-infection, and remained significantly higher throughout the course of a 12-week infection. Total IgG and Brucella-specific IgG levels peaked strongly in ΔrpoE1-infected mice at 6weeks, which correlated with reduced splenomegaly and bacterial burden relative to WT-infected mice. Given the difference in immune response to infection with wild-type and ΔrpoE1, we tested whether ΔrpoE1 confers protective immunity to wild-type challenge. Mice immunized with ΔrpoE1 completely resisted WT infection and had significantly higher serum titers of Brucella-specific IgG, IgG2a and IFN-γ after WT challenge relative to age-matched naïve mice. We conclude that immunization of BALB/c mice with the B. abortus GSR pathway mutant, ΔrpoE1, elicits an adaptive immune response that confers significant protective immunity against WT infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preclinical efficacy of the MDM2 inhibitor RG7112 in MDM2 amplified and TP53 wild-type glioblastomas

Science.gov (United States)

Verreault, Maite; Schmitt, Charlotte; Goldwirt, Lauriane; Pelton, Kristine; Haidar, Samer; Levasseur, Camille; Guehennec, Jeremy; Knoff, David; Labussiere, Marianne; Marie, Yannick; Ligon, Azra H.; Mokhtari, Karima; Hoang-Xuan, Khe; Sanson, Marc; Alexander, Brian M; Wen, Patrick Y.; Delattre, Jean-Yves; Ligon, Keith L.; Idbaih, Ahmed

2016-01-01

Rationale p53 pathway alterations are key molecular events in glioblastoma (GBM). MDM2 inhibitors increase expression and stability of p53 and are presumed to be most efficacious in patients with TP53 wild-type and MDM2-amplified cancers. However, this biomarker hypothesis has not been tested in patients or patient-derived models for GBM. Methods We performed a preclinical evaluation of RG7112 MDM2 inhibitor, across a panel of 36 patient-derived GBM cell lines (PDCLs), each genetically characterized according to their P53 pathway status. We then performed a pharmacokinetic (PK) profiling of RG7112 distribution in mice and evaluated the therapeutic activity of RG7112 in orthotopic and subcutaneous GBM models. Results MDM2-amplified PDCLs were 44 times more sensitive than TP53 mutated lines that showed complete resistance at therapeutically attainable concentrations (avg. IC50 of 0.52 μM vs 21.9 μM). MDM4 amplified PDCLs were highly sensitive but showed intermediate response (avg. IC50 of 1.2 μM), whereas response was heterogeneous in TP53 wild-type PDCLs with normal MDM2/4 levels (avg. IC50 of 7.7 μM). In MDM2-amplified lines, RG7112 restored p53 activity inducing robust p21 expression and apoptosis. PK profiling of RG7112-treated PDCL intracranial xenografts demonstrated that the compound significantly crosses the blood-brain and the blood-tumor barriers. Most importantly, treatment of MDM2-amplified/TP53 wild-type PDCL-derived model (subcutaneous and orthotopic) reduced tumor growth, was cytotoxic, and significantly increased survival. Conclusion These data strongly support development of MDM2 inhibitors for clinical testing in MDM2-amplified GBM patients. Moreover, significant efficacy in a subset of non-MDM2 amplified models suggests that additional markers of response to MDM2 inhibitors must be identified. PMID:26482041

Molecular Evolution of a Type 1 Wild-Vaccine Poliovirus Recombinant during Widespread Circulation in China

Science.gov (United States)

Liu, Hong-Mei; Zheng, Du-Ping; Zhang, Li-Bi; Oberste, M. Steven; Pallansch, Mark A.; Kew, Olen M.

2000-01-01

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3′-terminal sequences of VP1 (115 nt) and the 5′ half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3′ half of 2A were more distantly related (polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of ∼3.7 × 10−2 substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection. PMID:11070012

Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

Science.gov (United States)

Citrus paradisi Macf. cv. Duncan was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from CTV genome and used to gener...

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli

NARCIS (Netherlands)

Schulze, Egbert; Westphal, Adrie H.; Veenhuis, Marten; Kok, Arie de

1992-01-01

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been

Genetic variability in Cynara cardunculus L. domestic and wild types for grain oil production and fatty acids composition

International Nuclear Information System (INIS)

Raccuia, Salvatore Antonino; Piscioneri, Ilario; Sharma, Neeta; Melilli, Maria Grazia

2011-01-01

This paper aimed to study the genetic variability within different types of Cynara cardunculus L., domestic and wild types, for their grain oil amount and oil fatty acid composition. The grain oils were extracted from 8 domestic cardoons and 4 wild cardoons, by Soxhlet method, and obtained oils were characterized for palmitic, stearic, oleic and linoleic acids by gas chromatography. The oil amount, resulted on average of accessions 216 g kg -1 DM with a good range of variability (CV = 11.7%). Unsaturated acids (oleic and linoleic) predominated over saturated ones (stearic and palmitic acids), the chemical characterization of extracted oil, showed the main compound (as % of analysed fatty acids), averaged for all populations, was linoleic acid (44.5%), followed by oleic acid (42.6%), palmitic acid (9.8%) and stearic acid (3.1%). In particular referring the oleic acid wild cardoon populations showed a mean value of 289 g kg -1 oil, against a mean value of 472 g kg -1 oil showed by domestic cardoon accessions. Three of the studied domestic cardoon ('DC1', 'DC3' and 'DC7') showed values higher than 795 g kg -1 oil, while all the other accessions had concentration lower than 370 g kg -1 oil. The three types of domestic cardoon 'DC1', 'DC3' and 'DC7' showed a fatty acids profile similar to genetic modified sunflower oil, representing new genetic material that potentially could be used for high quality biodiesel production, characterised by a low Iodine Number. -- Highlights: → The grain oils from 12 cardoons were characterized for fatty acids composition. → The oil amount, resulted on average of accessions 216 g kg -1 DM. → Oleic and linoleic acids predominated over stearic and palmitic acids. → Three domestic cardoons grain oil showed high oleic acid content (795 g kg -1 oil). → This oil could be used for high quality biodiesel production, with a low IN.

Cetuximab Plus Various Chemotherapy Regimens for Patients with KRAS Wild-Type Metastatic Colorectal Cancer.

Science.gov (United States)

Azadeh, Payam; Mortazavi, Nafiseh; Tahmasebi, Arezoo; Hosseini Kamal, Farnaz; Novin, Kambiz

2016-01-01

The aim of this study was to compare the efficacy and hematologic toxicity of cetuximab combined with various types of chemotherapy regimens in patients with KRAS wild-type metastatic colorectal cancer (mCRC). The response rate, progression-free survival (PFS) and overall survival of the patients were analyzed. In total, 45 patients were included in the study. The overall response rate for the combination of cetuximab and FOLFOX, FOLFIRI and CAPOX was 20, 46 and 30%, respectively, but the differences were not statistically significant. The median PFS for the three groups were 8, 6 and 3.5 months, respectively, but again these differences were not significant. All-grade leukopenia and anemia for the cetuximab plus FOLFOX group were significantly higher than for the other chemotherapy regimens. Our findings suggest that the combination of cetuximab and the three standard chemotherapy regimens resulted in the same outcomes in our patient population of mCRC, with higher hematologic toxicities among the FOLFOX subgroup. © 2015 S. Karger AG, Basel.

Innate immune responses to obesity in cloned and wild-type domestic pig

DEFF Research Database (Denmark)

Højbøge, Tina Rødgaard; Skovgaard, Kerstin; Stagsted, Jan

as a refined pig model for obesity-induced innate host responses by reducing pig-to-pig biological variation compared to wild-type (WT) pigs (n=19). Pigs were fed ad libitum with a high fat/high sucrose diet to induce obesity or kept lean on a restricted diet (60% of ad libitum intake) beginning at three...... months of age. mRNA expression levels were determined for 39 innate immune factors on a high-throughput qPCR system in samples from liver, abdominal fat, mesenteric fat and subcutaneous fat. Previous findings have suggested that cloning may affect certain phenotypic traits of pigs including basic...... concentrations and responsiveness of components of the innate immune system. Terminal body weights at 7½ - 9½ months of age were significantly higher for both (WT and cloned) obese groups compared to the lean groups. However, obese WT pigs weighed significantly more than obese cloned pigs (P

Identification of proteins that regulate radiation-induced apoptosis in murine tumors with wild type p53

Energy Technology Data Exchange (ETDEWEB)

Seong, Jinsil; Oh, Hae Jin; Kim, Jiyoung; An, Jeung Hee; Kim, Wonwoo [Dept. of Radiation Oncology, Yonsei Univ. Medical College, Seoul (Korea, Republic of)

2007-09-15

In this study, we investigated the molecular factors determining the induction of apoptosis by radiation. Two murine tumors syngeneic to C3H/HeJ mice were used: an ovarian carcinoma OCa-I, and a hepatocarcinoma HCa-I. Both have wild type p53, but display distinctly different radiosensitivity in terms of specific growth delay (12.7 d in OCa-I and 0.3 d in HCa-I) and tumor cure dose 50% (52.6 Gy in OCa-I and >80 Gy in HCa-I). Eight-mm tumors on the thighs of mice were irradiated with 25 Gy and tumor samples were collected at regular time intervals after irradiation. The peak levels of apoptosis were 16.1{+-}0.6% in OCa-I and 0.2{+-}0.0% in HCa-I at 4 h after radiation, and this time point was used for subsequent proteomics analysis. Protein spots were identified by peptide mass fingerprinting with a focus on those related to apoptosis. In OCa-I tumors, radiation increased the expression of cytochrome c oxidase and Bcl2/adenovirus E1B-interacting 2 (Nip 2) protein higher than 3-fold. However in HCa-I, these two proteins showed no significant change. The results suggest that radiosensitivity in tumors with wild type p53 is regulated by a complex mechanism. Furthermore, these proteins could be molecular targets for a novel therapeutic strategy involving the regulation of radiosensitivity. (author)

Identification of proteins that regulate radiation-induced apoptosis in murine tumors with wild type p53

International Nuclear Information System (INIS)

Seong, Jinsil; Oh, Hae Jin; Kim, Jiyoung; An, Jeung Hee; Kim, Wonwoo

2007-01-01

In this study, we investigated the molecular factors determining the induction of apoptosis by radiation. Two murine tumors syngeneic to C3H/HeJ mice were used: an ovarian carcinoma OCa-I, and a hepatocarcinoma HCa-I. Both have wild type p53, but display distinctly different radiosensitivity in terms of specific growth delay (12.7 d in OCa-I and 0.3 d in HCa-I) and tumor cure dose 50% (52.6 Gy in OCa-I and >80 Gy in HCa-I). Eight-mm tumors on the thighs of mice were irradiated with 25 Gy and tumor samples were collected at regular time intervals after irradiation. The peak levels of apoptosis were 16.1±0.6% in OCa-I and 0.2±0.0% in HCa-I at 4 h after radiation, and this time point was used for subsequent proteomics analysis. Protein spots were identified by peptide mass fingerprinting with a focus on those related to apoptosis. In OCa-I tumors, radiation increased the expression of cytochrome c oxidase and Bcl2/adenovirus E1B-interacting 2 (Nip 2) protein higher than 3-fold. However in HCa-I, these two proteins showed no significant change. The results suggest that radiosensitivity in tumors with wild type p53 is regulated by a complex mechanism. Furthermore, these proteins could be molecular targets for a novel therapeutic strategy involving the regulation of radiosensitivity. (author)

The effect of dietary folic acid deficiency on the cytotoxic and mutagenic responses to methyl methanesulfonate in wild-type and in 3-methyladenine DNA glycosylase-deficient Aag null mice.

Science.gov (United States)

Branda, Richard F; O'Neill, J Patrick; Brooks, Elice M; Powden, Cheryl; Naud, Shelly J; Nicklas, Janice A

2007-02-03

Folic acid deficiency (FA-) augments DNA damage caused by alkylating agents. The role of DNA repair in modulating this damage was investigated in mice. Weanling wild-type or 3-methyladenine glycosylase (Aag) null mice were maintained on a FA- diet or the same diet supplemented with folic acid (FA+) for 4 weeks. They were then treated with methyl methanesulfonate (MMS), 100mg/kg i.p. Six weeks later, spleen cells were collected for assays of non-selected and 6-thioguanine (TG) selected cloning efficiency to measure the mutant frequency at the Hprt locus. In wild-type mice, there was no significant effect of either MMS treatment or folate dietary content on splenocyte non-selected cloning efficiency. In contrast, non-selected cloning efficiency was significantly higher in MMS-treated Aag null mice than in saline treated controls (diet-gene interaction variable, p=0.04). The non-selected cloning efficiency was significantly higher in the FA+ diet than in the FA- diet group after MMS treatment of Aag null mice. Mutant frequency after MMS treatment was significantly higher in FA- wild-type and Aag null mice and in FA+ Aag null mice, but not in FA+ wild-type mice. For the Aag null mice, mutant frequency was higher in the FA+ mice than in the FA- mice after either saline or MMS treatment. These studies indicate that in wild-type mice treated with MMS, dietary folate content (FA+ or FA-) had no effect on cytotoxicity, but FA- diet increased DNA mutation frequency compared to FA+ diet. In Aag null mice, FA- diet increased the cytotoxic effects of alkylating agents but decreased the risk of DNA mutation.

Labeled Azospirillum brasilense wild type and excretion-ammonium strains in association with barley roots.

Science.gov (United States)

Santos, Adrian Richard Schenberger; Etto, Rafael Mazer; Furmam, Rafaela Wiegand; Freitas, Denis Leandro de; Santos, Karina Freire d'Eça Nogueira; Souza, Emanuel Maltempi de; Pedrosa, Fábio de Oliveira; Ayub, Ricardo Antônio; Steffens, Maria Berenice Reynaud; Galvão, Carolina Weigert

2017-09-01

Soil bacteria colonization in plants is a complex process, which involves interaction between many bacterial characters and plant responses. In this work, we labeled Azospirillum brasilense FP2 (wild type) and HM053 (excretion-ammonium) strains by insertion of the reporter gene gusA-kanamycin into the dinitrogenase reductase coding gene, nifH, and evaluated bacteria colonization in barley (Hordeum vulgare). In addition, we determined inoculation effect based on growth promotion parameters. We report an uncommon endophytic behavior of A. brasilense Sp7 derivative inside the root hair cells of barley and highlight the promising use of A. brasilense HM053 as plant growth-promoting bacterium. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

Science.gov (United States)

Li, Yafan; Wheeler, Deric L; Ananthaswamy, Honnavara N; Verma, Ajit K; Oberley, Terry D

2007-12-01

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

Gastric Medullary Carcinoma with Sporadic Mismatch Repair Deficiency and a TP53 R273C Mutation: An Unusual Case with Wild-Type BRAF

Directory of Open Access Journals (Sweden)

Brett M. Lowenthal

2017-01-01

Full Text Available Medullary carcinoma has long been recognized as a subtype of colorectal cancer associated with microsatellite instability and Lynch syndrome. Gastric medullary carcinoma is a very rare neoplasm. We report a 67-year-old male who presented with a solitary gastric mass. Total gastrectomy revealed a well-demarcated, poorly differentiated carcinoma with an organoid growth pattern, pushing borders, and abundant peritumoral lymphocytic response. The prior cytology was cellular with immunohistochemical panel consistent with upper gastrointestinal/pancreaticobiliary origin. Overall, the histopathologic findings were consistent with gastric medullary carcinoma. A mismatch repair panel revealed a mismatch repair protein deficient tumor with loss of MLH1 and PMS2 expression. BRAF V600E immunostain (VE1 and BRAF molecular testing were negative, indicating a wild-type gene. Tumor sequencing of MLH1 demonstrated a wild-type gene, while our molecular panel identified TP53 c.817C>T (p.R273C mutation. These findings were compatible with a sporadic tumor. Given that morphologically identical medullary tumors often occur in Lynch syndrome, it is possible that mismatch repair loss is an early event in sporadic tumors with p53 mutation being a late event. Despite having wild-type BRAF, this tumor is sporadic and unrelated to Lynch syndrome. This case report demonstrates that coordinate ancillary studies are needed to resolve sporadic versus hereditary rare tumors.

IFN-{gamma} enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer's disease

DEFF Research Database (Denmark)

Baron, Rona; Nemirovsky, Anna; Harpaz, Idan

2008-01-01

the spatial learning and memory performance of the animals. In older mice, the effect of IFN-gamma is more pronounced in both wild-type mice and mice with Alzheimer's-like disease and is associated with neuroprotection. In addition, IFN-gamma reverses the increase in oligodendrogenesis observed in a mouse...... mechanisms can generate immunity to such deficits in neuronal repair. We demonstrate that in contrast to primarily innate immunity cytokines, such as interleukin-6 and tumor necrosis factor-alpha, the adaptive immunity cytokine IFN-gamma enhances neurogenesis in the dentate gyrus of adult mice and improves...

A PCR-based genotyping method to distinguish between wild-type and ornamental varieties of Imperata cylindrica.

Science.gov (United States)

Cseke, Leland J; Talley, Sharon M

2012-02-20

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas(1-2). Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety(4) (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed. Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce

Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs.

Science.gov (United States)

Bolado-Martínez, E; Acedo-Félix, E; Peregrino-Uriarte, A B; Yepiz-Plascencia, G

2012-01-01

Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.

General anesthetic octanol and related compounds activate wild-type and delF508 cystic fibrosis chloride channels

OpenAIRE

Marcet, Brice; Becq, Frédéric; Norez, Caroline; Delmas, Patrick; Verrier, Bernard

2004-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl− channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl− channel activity of wild-type CFTR and delF508-CFTR mutant.The effects of n-alkanols like octanol on CFTR activity were measured by iodide (125I) efflux and patch-clamp techniques o...

Dietary Supplementation of Hericium erinaceus Increases Mossy Fiber-CA3 Hippocampal Neurotransmission and Recognition Memory in Wild-Type Mice

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Federico Brandalise

2017-01-01

Full Text Available Hericium erinaceus (Bull. Pers. is a medicinal mushroom capable of inducing a large number of modulatory effects on human physiology ranging from the strengthening of the immune system to the improvement of cognitive functions. In mice, dietary supplementation with H. erinaceus prevents the impairment of spatial short-term and visual recognition memory in an Alzheimer model. Intriguingly other neurobiological effects have recently been reported like the effect on neurite outgrowth and differentiation in PC12 cells. Until now no investigations have been conducted to assess the impact of this dietary supplementation on brain function in healthy subjects. Therefore, we have faced the problem by considering the effect on cognitive skills and on hippocampal neurotransmission in wild-type mice. In wild-type mice the oral supplementation with H. erinaceus induces, in behaviour test, a significant improvement in the recognition memory and, in hippocampal slices, an increase in spontaneous and evoked excitatory synaptic current in mossy fiber-CA3 synapse. In conclusion, we have produced a series of findings in support of the concept that H. erinaceus induces a boost effect onto neuronal functions also in nonpathological conditions.

Dietary Supplementation of Hericium erinaceus Increases Mossy Fiber-CA3 Hippocampal Neurotransmission and Recognition Memory in Wild-Type Mice.

Science.gov (United States)

Brandalise, Federico; Cesaroni, Valentina; Gregori, Andrej; Repetti, Margherita; Romano, Chiara; Orrù, Germano; Botta, Laura; Girometta, Carolina; Guglielminetti, Maria Lidia; Savino, Elena; Rossi, Paola

2017-01-01

Hericium erinaceus (Bull.) Pers. is a medicinal mushroom capable of inducing a large number of modulatory effects on human physiology ranging from the strengthening of the immune system to the improvement of cognitive functions. In mice, dietary supplementation with H. erinaceus prevents the impairment of spatial short-term and visual recognition memory in an Alzheimer model. Intriguingly other neurobiological effects have recently been reported like the effect on neurite outgrowth and differentiation in PC12 cells. Until now no investigations have been conducted to assess the impact of this dietary supplementation on brain function in healthy subjects. Therefore, we have faced the problem by considering the effect on cognitive skills and on hippocampal neurotransmission in wild-type mice. In wild-type mice the oral supplementation with H. erinaceus induces, in behaviour test, a significant improvement in the recognition memory and, in hippocampal slices, an increase in spontaneous and evoked excitatory synaptic current in mossy fiber-CA3 synapse. In conclusion, we have produced a series of findings in support of the concept that H. erinaceus induces a boost effect onto neuronal functions also in nonpathological conditions.

Molecular analysis of mutant and wild type alcohol dehydrogenase alleles from Drosophila

International Nuclear Information System (INIS)

Batzer, M.A.

1988-01-01

Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray-induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray-induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from

Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands.

Science.gov (United States)

Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S; Damborsky, Jiri; Smatanova, Ivana Kuta

2011-02-01

Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.

Location of Primary Tumor and Benefit From Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies in Patients With RAS and BRAF Wild-Type Metastatic Colorectal Cancer

Science.gov (United States)

Moretto, Roberto; Cremolini, Chiara; Rossini, Daniele; Pietrantonio, Filippo; Battaglin, Francesca; Mennitto, Alessia; Bergamo, Francesca; Loupakis, Fotios; Marmorino, Federica; Berenato, Rosa; Marsico, Valentina Angela; Caporale, Marta; Antoniotti, Carlotta; Masi, Gianluca; Salvatore, Lisa; Borelli, Beatrice; Fontanini, Gabriella; Lonardi, Sara; De Braud, Filippo

2016-01-01

Introduction. Right- and left-sided colorectal cancers (CRCs) differ in clinical and molecular characteristics. Some retrospective analyses suggested that patients with right-sided tumors derive less benefit from anti-epidermal growth factor receptor (EGFR) antibodies; however, molecular selection in those studies was not extensive. Patients and Methods. Patients with RAS and BRAF wild-type metastatic CRC (mCRC) who were treated with single-agent anti-EGFRs or with cetuximab-irinotecan (if refractory to previous irinotecan) were included in the study. Differences in outcome between patients with right- and left-sided tumors were investigated. Results. Of 75 patients, 14 and 61 had right- and left-sided tumors, respectively. None of the right-sided tumors responded according to RECIST, compared with 24 left-sided tumors (overall response rate: 0% vs. 41%; p = .0032), and only 2 patients with right-sided tumors (15%) versus 47 patients with left-sided tumors (80%) achieved disease control (p < .0001). The median duration of progression-free survival was 2.3 and 6.6 months in patients with right-sided and left-sided tumors, respectively (hazard ratio: 3.97; 95% confidence interval: 2.09–7.53; p < .0001). Conclusion. Patients with right-sided RAS and BRAF wild-type mCRC seemed to derive no benefit from single-agent anti-EGFRs. Implications for Practice: Right- and left-sided colorectal tumors have peculiar epidemiological and clinicopathological characteristics, distinct gene expression profiles and genetic alterations, and different prognoses. This study assessed the potential predictive impact of primary tumor site with regard to anti-epidermal growth factor receptor (EGFR) monoclonal antibody treatment in patients with RAS and BRAF wild-type metastatic colorectal cancer. The results demonstrated the lack of activity of anti-EGFRs in RAS and BRAF wild-type, right-sided tumors, thus suggesting a potential role for primary tumor location in driving treatment choices

Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts

Science.gov (United States)

Tang, Ning; Zhang, Qianqian; Fang, Shu; Han, Xiao; Wang, Zhehai

2017-01-01

Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and recorded, and at the end of experiments, tumor xenografts were removed for Western blot and immunohistochemical analyses. Compared to control groups (negative control, regular-dose icotinib [IcoR], high-dose icotinib [IcoH], and docetaxel [DTX]) and regular icotinib dose (60 mg/kg) with docetaxel, treatment of mice with a high-dose (1200 mg/kg) of icotinib plus sequential docetaxel for 3 weeks (IcoH-DTX) had an additive effect on suppression of tumor xenograft size and volume (P Icotinib-containing treatments markedly reduced phosphorylation of EGFR, mitogen activated protein kinase (MAPK), and protein kinase B (Akt), but only the high-dose icotinib-containing treatments showed an additive effect on CD34 inhibition (P icotinib plus docetaxel had a similar effect on mouse weight loss (a common way to measure adverse reactions in mice), compared to the other treatment combinations. The study indicate that the high dose of icotinib plus sequential docetaxel (IcoH-DTX) have an additive effect on suppressing the growth of wild-type EGFR NSCLC cell nude mouse xenografts, possibly through microvessel density reduction. Future clinical trials are needed to confirm the findings of this study. PMID:27852073

Molecular characterization and phylogenetic relationship of wild type 1 poliovirus strains circulating across Pakistan and Afghanistan bordering areas during 2010-2012.

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Shahzad Shaukat

Full Text Available Pakistan and Afghanistan share a long uncontrolled border with extensive population movement on both sides. Wild poliovirus transmission has never been interrupted in this block due to war against terrorism, poor public health infrastructure, misconceptions about polio vaccines and inadequate immunization activities. All these issues complicate the eradication operations and reinforce the complexity of wiping out poliomyelitis from this region. This study illustrates the origins and routes of cross-border wild poliovirus type 1 (WPV1 transmission during 2010-2012 between Pakistan and Afghanistan. Sequence analyses were conducted based on complete VP1 capsid protein sequences for WPV1 study strains to determine the origin of poliovirus genetic lineages and their evolutionary relationships. Phylogenetic tree was constructed from VP1 gene sequences applying Maximum Likelihood method using Kimura 2- parameter model in MEGA program v 5.0. A total of 72 (14.3% out of 502 wild-type 1 polioviruses were found circulating in border areas of both countries during 2010-2012. Molecular phylogenetic analysis classified these strains in to two sub-genotypes with four clusters and 18 lineages. Genetic data confirmed that the most of WPV1 lineages (12; 66.6% were transmitted from Pakistan to Afghanistan. However, the genetic diversity was significantly reduced during 2012 as most of the lineages were completely eliminated. In conclusion, Pakistan-Afghanistan block has emerged as a single poliovirus reservoir sharing the multiple poliovirus lineages due to uncontrolled movement of people across the borders between two countries. If it is neglected, it can jeopardize the extensive global efforts done so-far to eradicate the poliovirus infection. Our data will be helpful to devise the preventive strategies for effective control of wild poliovirus transmission in this region.

A comparative study of age-related hearing loss in wild type and insulin-like growth factor I deficient mice

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Raquel Riquelme

2010-06-01

Full Text Available Insulin-like growth factor-I (IGF-I belongs to the family of insulin-related peptides that fulfils a key role during the late development of the nervous system. Human IGF1 mutations cause profound deafness, poor growth and mental retardation. Accordingly, Igf1−/− null mice are dwarfs that have low survival rates, cochlear alterations and severe sensorineural deafness. Presbycusis (age-related hearing loss is a common disorder associated with aging that causes social and cognitive problems. Aging is also associated with a decrease in circulating IGF-I levels and this reduction has been related to cognitive and brain alterations, although there is no information as yet regarding the relationship between presbycusis and IGF-I biodisponibility. Here we present a longitudinal study of wild type Igf1+/+ and null Igf1−/− mice from 2 to 12 months of age comparing the temporal progression of several parameters: hearing, brain morphology, cochlear cytoarchitecture, insulin-related factors and IGF gene expression and IGF-I serum levels. Complementary invasive and non-invasive techniques were used, including auditory brainstem-evoked response (ABR recordings and in vivo MRI brain imaging. Igf1−/− null mice presented profound deafness at all the ages studied, without any obvious worsening of hearing parameters with aging. Igf1+/+ wild type mice suffered significant age-related hearing loss, their auditory thresholds and peak I latencies augmenting as they aged, in parallel with a decrease in the circulating levels of IGF-I. Accordingly, there was an age-related spiral ganglion degeneration in wild type mice that was not evident in the Igf1 null mice. However, the Igf1−/− null mice in turn developed a prematurely aged stria vascularis reminiscent of the diabetic strial phenotype. Our data indicate that IGF-I is required for the correct development and maintenance of hearing, supporting the idea that IGF-I-based therapies could contribute to

Rhizosphere bacteria affected by transgenic potatoes with antibacterial activities compared with the effects of soil, wild-type potatoes, vegetation stage and pathogen exposure

NARCIS (Netherlands)

Rasche, F; Hodl, [No Value; Poll, C; Kandeler, E; Gerzabek, MH; van Elsas, JD; Sessitsch, A

A greenhouse experiment was performed to analyze a potential effect of genetically modified potatoes expressing antibacterial compounds (attacin/cecropin, T4 lysozyme) and their nearly isogenic, nontransformed parental wild types on rhizosphere bacterial communities. To compare plant

Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

Science.gov (United States)

Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

2002-01-01

It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM(+/-)) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM(+/-) cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

Primary tumor location predicts poor clinical outcome with cetuximab in RAS wild-type metastatic colorectal cancer.

Science.gov (United States)

Kim, Dalyong; Kim, Sun Young; Lee, Ji Sung; Hong, Yong Sang; Kim, Jeong Eun; Kim, Kyu-Pyo; Kim, Jihun; Jang, Se Jin; Yoon, Young-Kwang; Kim, Tae Won

2017-11-23

In metastatic colorectal cancer, the location of the primary tumor has been suggested to have biological significance. In this study, we investigated whether primary tumor location affects cetuximab efficacy in patients with RAS wild-type metastatic colorectal cancer. Genotyping by the SequenomMassARRAY technology platform (OncoMap) targeting KRAS, NRAS, PIK3CA, and BRAF was performed in tumors from 307 patients who had been given cetuximab as salvage treatment. Tumors with mutated RAS (KRAS or NRAS; n = 127) and those with multiple primary location (n = 10) were excluded. Right colon cancer was defined as a tumor located in the proximal part to splenic flexure. A total of 170 patients were included in the study (right versus left, 23 and 147, respectively). Patients with right colon cancer showed more mutated BRAF (39.1% vs. 5.4%), mutated PIK3CA (13% vs. 1.4%), poorly differentiated tumor (17.4% vs. 3.4%), and peritoneal involvement (26.1% vs. 8.8%) than those with left colon and rectal cancer. Right colon cancer showed poorer progression-free survival (2.0 vs.5.0 months, P = 0.002) and overall survival (4.1 months and 13.0 months, P < 0.001) than the left colon and rectal cancer. By multivariable analysis, BRAF mutation, right colon primary, poorly differentiated histology, and peritoneal involvement were associated with risk of death. In RAS wild-type colon cancer treated with cetuximab as salvage treatment, right colon primary was associated with poorer survival outcomes than left colon and rectal cancer.

Genome re-sequencing of semi-wild soybean reveals a complex Soja population structure and deep introgression.

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Jie Qiu

Full Text Available Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou and a wild line (Lanxi 1 collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1 no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2 besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3 high heterozygous rates (0.19-0.49 were observed in several semi-wild lines; and (4 over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure.

Biosafety of Recombinant and Wild Type Nucleopolyhedroviruses as Bioinsecticides

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Bruce D. Hammock

2007-06-01

Full Text Available The entomopathogenic Autographa californica (Speyer nucleopolyhedrovirus (AcMNPV has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 x 1012 PIBs/feddan, feddan = 4,200 m2 and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field, AcMNPV, and SlNPV using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 μg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%, AcAaIT-field (1.2%, and SlNPV (4.0%. Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal

Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.

Science.gov (United States)

O'Neill, F J; Gao, Y; Xu, X

1993-11-01

The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant

Cross-synergism in enzymatic hydrolysis of lignocellulosics: mathematical correlations according to a hyperbolic model

Energy Technology Data Exchange (ETDEWEB)

Tarantili, P.A.; Koullas, D.P.; Christakopoulos, P.; Kekos, D.; Koukios, E.G.; Macris, B.J. [National Technical Univ. of Athens (Greece). Dept. of Chemical Engineering

1996-10-01

The effect of cross-synergism in enzymatic hydrolysis of ball-milled Avicell, alkali-treated straw cellulose (ATSC), cotton and filter paper was investigated using mixtures of Fusarium oxysporum and Neurospora crassa enzymes. The experimental data were fitted according to an empirical hyperbolic model which utilized two parameters, the maximum conversion ({chi}{sub max}) and the enzymatic hydrolysis time corresponding to 50% of {chi}{sub max} (t{sub 1/2}). The model can predict conversion of polysaccharides as a function of hydrolysis time. Both model parameters were found to be strongly dependent on the crystallinity index as well as on the degree of delignification of the substrate. Up to 60% cellulose hydrolysis can be achieved when the crystallinity index of Avicell is reduced from 94.8% to 63.3%. The percentage increase of {chi}{sub max} due to delignification was higher than the corresponding increase of t{sub 1/2}. The extent of cross-synergism depends strongly on crystallinity index and degree of delignification. This type of synergism has been found to be significant in the case of substrates which are resistant to hydrolysis, such as Avicell (with high crystallinity index) or cotton. Cross-synergism phenomena caused by enzymatic mixtures can double cellulose hydrolysis yield with delignified straw as compared to the hydrolysis yields achieved by single-microorganism cellulases. (author)

Aspergillus oryzae AoSO is a novel component of stress granules upon heat stress in filamentous fungi.

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Hsiang-Ting Huang

Full Text Available Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi. The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.

Electrotransformation and expression of cellulase genes in wild-type Lactobacillus reuteri.

Science.gov (United States)

Li, Wang; Yang, Ming-Ming; Zhang, Guang-Qin; He, Wan-Ling; Li, Yuan-Xiao; Chen, Yu-Lin

2012-01-01

Two cellulase genes, Cel15 and Cel73, were amplified from Bacillus subtilis genome DNA in a previous study. Two integrative vectors, pLEM4153 and pLEM4154, containing the genes Cel15 and Cel73, respectively, were constructed and successfully electroporated into the wild-type Lactobacillus reuteri which was isolated from chick guts through an optimized procedure. Two recombinant L. reuteri were selected from a Man, Rogosa, and Sharp (MRS) plate with 10 µg/ml erythromycin, and named L. reuteri XNY-Cel15 and L. reuteri XNY-Cel73, respectively. To verify the transcription and expression of the two cellulase genes in the recombinant L. reuteri strains, the mRNA relative quantity (RQ) and the cellulase activity were determined. The mRNA RQ of Cel15 in L. reuteri XNY-Cel15 is 1,8849.5, and that of Cel73 in L. reuteri XNY-Cel73 is 1,388, and the cellulase activity of the modified MRS broth cultured with L. reuteri XNY-Cel15 was 0.158 U/ml, whereas that with L. reuteri XNY-Cel73 was 0.15 U/ml. Copyright © 2012 S. Karger AG, Basel.

Comparative Study of Nonautolytic Mutant and Wild-Type Strains of Coprinopsis cinerea Supports an Important Role of Glucanases in Fruiting Body Autolysis.

Science.gov (United States)

Liu, Zhonghua; Niu, Xin; Wang, Jun; Zhang, Wenming; Yang, Mingmei; Liu, Cuicui; Xiong, Yuanjing; Zhao, Yan; Pei, Siyu; Qin, Qin; Zhang, Yu; Yu, Yuan; Yuan, Sheng

2015-11-04

Autolysis of Coprinopsis cinerea fruiting bodies affects its commercial value. In this study, a mutant of C. cinerea that exhibits pileus expansion without pileus autolysis was obtained using ultraviolet mutagenesis. This suggests that pileus expansion and pileus autolysis involve different enzymes or proteins. Among the detected hydrolytic enzymes, only β-1,3-glucanase activity increased with expansion and autolysis of pilei in the wild-type strain, but the increase was abolished in the mutant. This suggests that β-1,3-glucanases plays a major role in the autolysis. Although there are 43 possible β-1,3-glucoside hydrolases genes, only 4 known genes, which have products that are thought to act synergistically to degrade the β-1,3-glucan backbone of cell walls during fruiting body autolysis, and an unreported gene were upregulated during pileus expansion and autolysis in the wild-type stain but were suppressed in the mutant. This suggests that expression of these β-1,3-glucanases is potentially controlled by a single regulatory mechanism.

A novel mitochondrial protein of Neurospora crassa immunoprecipitates with known enzyme subunits but is not antigenic

International Nuclear Information System (INIS)

Nixon, E.

1989-01-01

14 C labeled 4'-phosphopantetheine (PAN) is detectable as 2 bands after SDS-PAGE of mitochondrial proteins. The bands comigrate with subunit 6 of cytochrome oxidase (COX) and a small ATPase subunit in tube gel slices of immunoprecipitates. However, other work demonstrated these bands to be due to modification of a novel protein, related to acyl carrier protein (ACP) of spinach and E. coli, that exists in two forms. To resolve this discrepancy, 1-dimensional (1D) slab and 2-dimensional (2D) SDS-PAGE was used for increased resolution over tube gels. Total mitochondrial protein gels from PAN labeled cells were western blotted, probed for COX, and autoradiographed. In 1D there is exact migration of PAN with COX6. In 2D PAN overlaps a protein distinct from and not antigenically related to COX subunits. These data suggest it is the ACP-like protein that in PAN-modified. Its possible association with COX during assembly will be discussed

Identification of concomitant infection with Chlamydia trachomatis IncA-negative mutant and wild-type strains by genomic, transcriptional, and biological characterizations.

Science.gov (United States)

Suchland, Robert J; Jeffrey, Brendan M; Xia, Minsheng; Bhatia, Ajay; Chu, Hencelyn G; Rockey, Daniel D; Stamm, Walter E

2008-12-01

Clinical isolates of Chlamydia trachomatis that lack IncA on their inclusion membrane form nonfusogenic inclusions and have been associated with milder, subclinical infections in patients. The molecular events associated with the generation of IncA-negative strains and their roles in chlamydial sexually transmitted infections are not clear. We explored the biology of the IncA-negative strains by analyzing their genomic structure, transcription, and growth characteristics in vitro and in vivo in comparison with IncA-positive C. trachomatis strains. Three clinical samples were identified that contained a mixture of IncA-positive and -negative same-serovar C. trachomatis populations, and two more such pairs were found in serial isolates from persistently infected individuals. Genomic sequence analysis of individual strains from each of two serovar-matched pairs showed that these pairs were very similar genetically. In contrast, the genome sequence of an unmatched IncA-negative strain contained over 5,000 nucleotide polymorphisms relative to the genome sequence of a serovar-matched but otherwise unlinked strain. Transcriptional analysis, in vitro culture kinetics, and animal modeling demonstrated that IncA-negative strains isolated in the presence of a serovar-matched wild-type strain are phenotypically more similar to the wild-type strain than are IncA-negative strains isolated in the absence of a serovar-matched wild-type strain. These studies support a model suggesting that a change from an IncA-positive strain to the previously described IncA-negative phenotype may involve multiple steps, the first of which involves a translational inactivation of incA, associated with subsequent unidentified steps that lead to the observed decrease in transcript level, differences in growth rate, and differences in mouse infectivity.

Differential effects of silver nanoparticles on DNA damage and DNA repair gene expression in Ogg1-deficient and wild type mice.

Science.gov (United States)

Nallanthighal, Sameera; Chan, Cadia; Murray, Thomas M; Mosier, Aaron P; Cady, Nathaniel C; Reliene, Ramune

2017-10-01

Due to extensive use in consumer goods, it is important to understand the genotoxicity of silver nanoparticles (AgNPs) and identify susceptible populations. 8-Oxoguanine DNA glycosylase 1 (OGG1) excises 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), a pro-mutagenic lesion induced by oxidative stress. To understand whether defects in OGG1 is a possible genetic factor increasing an individual's susceptibly to AgNPs, we determined DNA damage, genome rearrangements, and expression of DNA repair genes in Ogg1-deficient and wild type mice exposed orally to 4 mg/kg of citrate-coated AgNPs over a period of 7 d. DNA damage was examined at 3 and 7 d of exposure and 7 and 14 d post-exposure. AgNPs induced 8-oxoG, double strand breaks (DSBs), chromosomal damage, and DNA deletions in both genotypes. However, 8-oxoG was induced earlier in Ogg1-deficient mice and 8-oxoG levels were higher after 7-d treatment and persisted longer after exposure termination. AgNPs downregulated DNA glycosylases Ogg1, Neil1, and Neil2 in wild type mice, but upregulated Myh, Neil1, and Neil2 glycosylases in Ogg1-deficient mice. Neil1 and Neil2 can repair 8-oxoG. Thus, AgNP-mediated downregulation of DNA glycosylases in wild type mice may contribute to genotoxicity, while upregulation thereof in Ogg1-deficient mice could serve as an adaptive response to AgNP-induced DNA damage. However, our data show that Ogg1 is indispensable for the efficient repair of AgNP-induced damage. In summary, citrate-coated AgNPs are genotoxic in both genotypes and Ogg1 deficiency exacerbates the effect. These data suggest that humans with genetic polymorphisms and mutations in OGG1 may have increased susceptibility to AgNP-mediated DNA damage.

Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography-mass spectrometry.

Science.gov (United States)

Guan, Wenna; Zhao, Hui; Lu, Xuefeng; Wang, Cong; Yang, Menglong; Bai, Fali

2011-11-11

Simple and rapid quantitative determination of fatty-acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae. Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty-acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes. This study founded a gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of seven free fatty acids, nine fatty acid methyl esters, five fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type Synechocystis PCC 6803 and its genetically engineered strain. Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons. The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated. The results demonstrated that fatty-acid-based biofuels can be directly determined by GC-MS without derivation. Therefore, rapid and reliable quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria can be achieved using the GC-MS method founded in this work. Copyright © 2011 Elsevier B.V. All rights reserved.

A comparison of the immune responses of dogs exposed to canine distemper virus (CDV) - Differences between vaccinated and wild-type virus exposed dogs.

Science.gov (United States)

Perrone, Danielle; Bender, Scott; Niewiesk, Stefan

2010-07-01

Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals.

Compositional and Functional Differences in the Human Gut Microbiome Correlate with Clinical Outcome following Infection with Wild-Type Salmonella enterica Serovar Typhi.

Science.gov (United States)

Zhang, Yan; Brady, Arthur; Jones, Cheron; Song, Yang; Darton, Thomas C; Jones, Claire; Blohmke, Christoph J; Pollard, Andrew J; Magder, Laurence S; Fasano, Alessio; Sztein, Marcelo B; Fraser, Claire M

2018-05-08

Insights into disease susceptibility as well as the efficacy of vaccines against typhoid and other enteric pathogens may be informed by better understanding the relationship between the effector immune response and the gut microbiota. In the present study, we characterized the composition (16S rRNA gene profiling) and function (RNA sequencing [RNA-seq]) of the gut microbiota following immunization and subsequent exposure to wild-type Salmonella enterica serovar Typhi in a human challenge model to further investigate the central hypothesis that clinical outcomes may be linked to the gut microbiota. Metatranscriptome analysis of longitudinal stool samples collected from study subjects revealed two stable patterns of gene expression for the human gut microbiota, dominated by transcripts from either Methanobrevibacter or a diverse representation of genera in the Firmicutes phylum. Immunization with one of two live oral attenuated vaccines against S.  Typhi had minimal effects on the composition or function of the gut microbiota. It was observed that subjects harboring the methanogen-dominated transcriptome community at baseline displayed a lower risk of developing symptoms of typhoid following challenge with wild-type S.  Typhi. Furthermore, genes encoding antioxidant proteins, metal homeostasis and transport proteins, and heat shock proteins were expressed at a higher level at baseline or after challenge with S.  Typhi in subjects who did not develop symptoms of typhoid. These data suggest that functional differences relating to redox potential and ion homeostasis in the gut microbiota may impact clinical outcomes following exposure to wild-type S.  Typhi. IMPORTANCE S.  Typhi is a significant cause of systemic febrile morbidity in settings with poor sanitation and limited access to clean water. It has been demonstrated that the human gut microbiota can influence mucosal immune responses, but there is little information available on the impact of the human gut

Comparison of the Tastes of L-Alanine and Monosodium Glutamate in C57BL/6J Wild Type and T1r3 Knockout Mice.

Science.gov (United States)

Eddy, Meghan C; Eschle, Benjamin K; Delay, Eugene R

2017-09-01

Previous research showed that L-alanine and monosodium L-glutamate elicit similar taste sensations in rats. This study reports the results of behavioral experiments designed to compare the taste capacity of C57BL/6J wild type and T1r3- mice for these 2 amino acids. In conditioned taste aversion (CTA) experiments, wild-type mice exhibited greater sensitivity than knockout mice for both L-amino acids, although knockout mice were clearly able to detect both amino acids at 50 mM and higher concentrations. Generalization of CTA between L-alanine and L-glutamate was bidirectionally equivalent for both mouse genotypes, indicating that both substances elicited similar tastes in both genotypes. This was verified by the discrimination experiments in which both mouse genotypes performed at or near chance levels at 75 and 150 mM. Above 150 mM, discrimination performance improved, suggesting the taste qualities of the 2 L-amino acids are not identical. No differences between knockout and wild-type mice in discrimination ability were detected. These results indicate that while the T1r3 receptor is important for tasting L-alanine and L-glutamate, other receptors are also important for tasting these amino acids. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification and comparative profiling of miRNAs in an early flowering mutant of trifoliate orange and its wild type by genome-wide deep sequencing.

Directory of Open Access Journals (Sweden)

Lei-Ming Sun

Full Text Available MicroRNAs (miRNAs are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf. and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes.

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

DEFF Research Database (Denmark)

Röpke, M; Hald, J; Guldberg, Per

1996-01-01

p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules...

Hunting with lead: association between blood lead levels and wild game consumption.

Science.gov (United States)

Iqbal, Shahed; Blumenthal, Wendy; Kennedy, Chinaro; Yip, Fuyuen Y; Pickard, Stephen; Flanders, W Dana; Loringer, Kelly; Kruger, Kirby; Caldwell, Kathleen L; Jean Brown, Mary

2009-11-01

Wild game hunting is a popular activity in many regions of the United States. Recently, the presence of lead fragments in wild game meat, presumably from the bullets or shot used for hunting, has raised concerns about health risks from meat consumption. This study examined the association between blood lead levels (PbB) and wild game consumption. We recruited 742 participants, aged 2-92 years, from six North Dakota cities. Blood lead samples were collected from 736 persons. Information on socio-demographic background, housing, lead exposure source, and types of wild game consumption (i.e., venison, other game such as moose, birds) was also collected. Generalized estimating equations (GEE) were used to determine the association between PbB and wild game consumption. Most participants reported consuming wild game (80.8%) obtained from hunting (98.8%). The geometric mean PbB were 1.27 and 0.84 microg/dl among persons who did and did not consume wild game, respectively. After adjusting for potential confounders, persons who consumed wild game had 0.30 microg/dl (95% confidence interval: 0.16-0.44 microg/dl) higher PbB than persons who did not. For all game types, recent (game consumption was associated with higher PbB. PbB was also higher among those who consumed a larger serving size (> or = 2 oz vs. game' consumption only. Participants who consumed wild game had higher PbB than those who did not consume wild game. Careful review of butchering practices and monitoring of meat-packing processes may decrease lead exposure from wild game consumption.

[An overview of surveillance of avian influenza viruses in wild birds].

Science.gov (United States)

Zhu, Yun; Shi, Jing-Hong; Shu, Yue-Long

2014-05-01

Wild birds (mainly Anseriformes and Charadriiformes) are recognized as the natural reservoir of avian influenza viruses (AIVs). The long-term surveillance of AIVs in wild birds has been conducted in North America and Europe since 1970s. More and more surveillance data revealed that all the HA and NA subtypes of AIVs were identified in the wild ducks, shorebirds, and gulls, and the AIVs circulating in wild birds were implicated in the outbreaks of AIVs in poultry and humans. Therefore, the AIVs in wild birds pose huge threat to poultry industry and human health. To gain a better understanding of the ecology and epidemiology of AIVs in wild birds, we summarize the transmission of AIVs between wild birds, poultry, and humans, the main results of surveillance of AIVs in wild birds worldwide and methods for surveillance, and the types of samples and detection methods for AIVs in wild birds, which would be vital for the effective control of avian influenza and response to possible influenza pandemic.

Wild carnivores (Mammalia) as hosts for ticks (Ixodida) in Panama

NARCIS (Netherlands)

Bermudez, S.E.; Esser, H.J.; Miranda, R.; Moreno, R.S.

2015-01-01

This study reports ticks collected from wild carnivores from different habitat types in Panama. We examined 94 individual wild carnivores and we found 87 parasitized by ticks: seven coyotes, six crab-eating foxes, 54 coatis, four raccoons, five ocelots, two pumas, two gray foxes, two skunks, and one

The Phenotypic Effects of Royal Jelly on Wild-Type D. melanogaster Are Strain-Specific.

Directory of Open Access Journals (Sweden)

Stefanie L Morgan

Full Text Available The role for royal jelly (RJ in promoting caste differentiation of honeybee larvae into queens rather than workers is well characterized. A recent study demonstrated that this poorly understood complex nutrition drives strikingly similar phenotypic effects in Drosophila melanogaster, such as increased body size and reduced developmental time, making possible the use of D. melanogaster as a model system for the genetic analysis of the cellular mechanisms underlying RJ and caste differentiation. We demonstrate here that RJ increases the body size of some wild-type strains of D. melanogaster but not others, and report significant delays in developmental time in all flies reared on RJ. These findings suggest that cryptic genetic variation may be a factor in the D. melanogaster response to RJ, and should be considered when attempting to elucidate response mechanisms to environmental changes in non-honeybee species.

Effects of ketoconazole on the biodistribution and metabolism of [{sup 11}C]loperamide and [{sup 11}C]N-desmethyl-loperamide in wild-type and P-gp knockout mice

Energy Technology Data Exchange (ETDEWEB)

Seneca, Nicholas; Zoghbi, Sami S.; Shetty, H. Umesha; Tuan, Edward; Kannan, Pavitra; Taku, Andrew; Innis, Robert B. [Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 (United States); Pike, Victor W. [Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 (United States)], E-mail: pikev@mail.nih.gov

2010-04-15

Introduction: [{sup 11}C]Loperamide and [{sup 11}C]N-desmethyl-loperamide ([{sup 11}C]dLop) have been proposed as radiotracers for imaging brain P-glycoprotein (P-gp) function. A major route of [{sup 11}C]loperamide metabolism is N-demethylation to [{sup 11}C]dLop. We aimed to test whether inhibition of CYP3A4 with ketoconazole might reduce the metabolism of [{sup 11}C]loperamide and [{sup 11}C]dLop in mice, and thereby improve the quality of these radiotracers. Methods: Studies were performed in wild-type and P-gp knockout (mdr-1a/b -/-) mice. During each of seven study sessions, one pair of mice, comprising one wild-type and one knockout mouse, was pretreated with ketoconazole (50 mg/kg, ip), while another such pair was left untreated. Mice were sacrificed at 30 min after injection of [{sup 11}C]loperamide or [{sup 11}C]dLop. Whole brain and plasma samples were measured for radioactivity and analyzed with radio-high-performance liquid chromatography. Results: Ketoconazole increased the plasma concentrations of [{sup 11}C]loperamide and its main radiometabolite, [{sup 11}C]dLop, by about twofold in both wild-type and knockout mice, whereas the most polar radiometabolite was decreased threefold. Furthermore, ketoconazole increased the brain concentrations of [{sup 11}C]loperamide and the radiometabolite [{sup 11}C]dLop by about twofold in knockout mice, and decreased the brain concentrations of the major and most polar radiometabolite in wild-type and knockout mice by 82% and 49%, respectively. In contrast, ketoconazole had no effect on plasma and brain distribution of administered [{sup 11}C]dLop and its radiometabolites in either wild-type or knockout mice, except to increase the low plasma [{sup 11}C]dLop concentration. The least polar radiometabolite of [{sup 11}C]dLop was identified with LC-MS{sup n} as the N-hydroxymethyl analog of [{sup 11}C]dLop and this also behaved as a P-gp substrate. Conclusion: In this study, ketoconazole (50 mg/kg, ip) proved

Humoral and cell-mediated immune responses in DNA immunized mink challenged with wild-type canine distemper virus.

Science.gov (United States)

Nielsen, Line; Søgaard, Mette; Karlskov-Mortensen, Peter; Jensen, Trine Hammer; Jensen, Tove Dannemann; Aasted, Bent; Blixenkrone-Møller, Merete

2009-07-30

The aim of the study was to investigate the different phases of the immune response after DNA immunization with the hemagglutinin and nucleoprotein genes from canine distemper virus (CDV). Although attenuated live CDV vaccines have effectively reduced the incidence of disease, canine distemper is still a problem worldwide. The broad host range of CDV creates a constant viral reservoir among wildlife animals. Our results demonstrated early humoral and cell-mediated immune responses (IFN-gamma) in DNA vaccinated mink compared to mock-vaccinated mink after challenge with a Danish wild-type CDV. The DNA vaccine-induced immunity protected the natural host against disease development.

Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor.

Science.gov (United States)

Yamaura, Takumi; Ezaki, Junji; Okabe, Naoyuki; Takagi, Hironori; Ozaki, Yuki; Inoue, Takuya; Watanabe, Yuzuru; Fukuhara, Mitsuro; Muto, Satoshi; Matsumura, Yuki; Hasegawa, Takeo; Hoshino, Mika; Osugi, Jun; Shio, Yutaka; Waguri, Satoshi; Tamura, Hirosumi; Imai, Jun-Ichi; Ito, Emi; Yanagisawa, Yuka; Honma, Reiko; Watanabe, Shinya; Suzuki, Hiroyuki

2018-02-01

Lung adenocarcinoma (ADC) patients with tumors that harbor no targetable driver gene mutation, such as epidermal growth factor receptor ( EGFR ) gene mutations, have unfavorable prognosis, and thus, novel therapeutic targets are required. Family with sequence similarity 83, member B ( FAM83B ) is a biomarker for squamous cell lung cancer. FAM83B has also recently been shown to serve an important role in the EGFR signaling pathway. In the present study, the molecular and clinical impact of FAM83B in lung ADC was investigated. Matched tumor and adjacent normal tissue samples were obtained from 216 patients who underwent complete lung resection for primary lung ADC and were examined for FAM83B expression using cDNA microarray analysis. The associations between FAM83B expression and clinicopathological parameters, including patient survival, were examined. FAM83B was highly expressed in tumors from males, smokers and in tumors with wild-type EGFR . Multivariate analyses further confirmed that wild-type EGFR tumors were significantly positively associated with FAM83B expression. In survival analysis, FAM83B expression was associated with poor outcomes in disease-free survival and overall survival, particularly when stratified against tumors with wild-type EGFR . Furthermore, FAM83B knockdown was performed to investigate its phenotypic effect on lung ADC cell lines. Gene silencing by FAM83B RNA interference induced growth suppression in the HLC-1 and H1975 lung ADC cell lines. FAM83B may be involved in lung ADC tumor proliferation and can be a predictor of poor survival. FAM83B is also a potential novel therapeutic target for ADC with wild-type EGFR .

Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4+ T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host.

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Jodi L McGill

Full Text Available Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4+ T cell proliferation and IFNγ production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection.

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

Science.gov (United States)

Brent, G A; Williams, G R; Harney, J W; Forman, B M; Samuels, H H; Moore, D D; Larsen, P R

1992-04-01

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common

BACHD rats expressing full-length mutant huntingtin exhibit differences in social behavior compared to wild-type littermates.

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Giuseppe Manfré

Full Text Available Huntington disease (HD is a devastating inherited neurodegenerative disorder characterized by progressive motor, cognitive, and psychiatric symptoms without any cure to slow down or stop the progress of the disease. The BACHD rat model for HD carrying the human full-length mutant huntingtin protein (mHTT with 97 polyQ repeats has been recently established as a promising model which reproduces several HD-like features. While motor and cognitive functions have been characterized in BACHD rats, little is known about their social phenotype.This study focuses especially on social behavior since evidence for social disturbances exists in human patients. Our objective was to compare social behavior in BACHD and wild-type (WT rats at different ages, using two different measures of sociability.Animals were tested longitudinally at the age of 2, 4 and 8 months in the social interaction test to examine different parameters of sociability. A separate cohort of 7 month old rats was tested in the three chamber social test to measure both sociability and social novelty. Gene expression analyses in 8 months old animals were performed by real time qRT-PCR to evaluate a potential involvement of D1 and D2 dopaminergic receptors and the contribution of Brain-derived neurotrophic factor (BDNF to the observed behavioral alterations.In the social interaction test, BACHD rats showed age-dependent changes in behaviour when they were-re introduced to their cagemate after a 24 hours-period of individual housing. The time spent on nape attacks increased with aging. Furthermore, a significant higher level of pinning at 2 months of age was shown in the BACHD rats compared to wild-types, followed by a reduction at 4 and 8 months. On the other hand, BACHD rats exhibited a decreased active social behaviour compared to wild-types, reflected by genotype-effects on approaching, following and social nose contact. In the three chamber social test, BACHD rats seemed to show a mild

BACHD rats expressing full-length mutant huntingtin exhibit differences in social behavior compared to wild-type littermates.

Science.gov (United States)

Manfré, Giuseppe; Novati, Arianna; Faccini, Ilaria; Rossetti, Andrea C; Bosch, Kari; Molteni, Raffaella; Riva, Marco A; Van der Harst, Johanneke E; Nguyen, Huu Phuc; Homberg, Judith R

2018-01-01

Huntington disease (HD) is a devastating inherited neurodegenerative disorder characterized by progressive motor, cognitive, and psychiatric symptoms without any cure to slow down or stop the progress of the disease. The BACHD rat model for HD carrying the human full-length mutant huntingtin protein (mHTT) with 97 polyQ repeats has been recently established as a promising model which reproduces several HD-like features. While motor and cognitive functions have been characterized in BACHD rats, little is known about their social phenotype. This study focuses especially on social behavior since evidence for social disturbances exists in human patients. Our objective was to compare social behavior in BACHD and wild-type (WT) rats at different ages, using two different measures of sociability. Animals were tested longitudinally at the age of 2, 4 and 8 months in the social interaction test to examine different parameters of sociability. A separate cohort of 7 month old rats was tested in the three chamber social test to measure both sociability and social novelty. Gene expression analyses in 8 months old animals were performed by real time qRT-PCR to evaluate a potential involvement of D1 and D2 dopaminergic receptors and the contribution of Brain-derived neurotrophic factor (BDNF) to the observed behavioral alterations. In the social interaction test, BACHD rats showed age-dependent changes in behaviour when they were-re introduced to their cagemate after a 24 hours-period of individual housing. The time spent on nape attacks increased with aging. Furthermore, a significant higher level of pinning at 2 months of age was shown in the BACHD rats compared to wild-types, followed by a reduction at 4 and 8 months. On the other hand, BACHD rats exhibited a decreased active social behaviour compared to wild-types, reflected by genotype-effects on approaching, following and social nose contact. In the three chamber social test, BACHD rats seemed to show a mild deficit in

BACHD rats expressing full-length mutant huntingtin exhibit differences in social behavior compared to wild-type littermates

Science.gov (United States)

Manfré, Giuseppe; Novati, Arianna; Faccini, Ilaria; Rossetti, Andrea C.; Bosch, Kari; Molteni, Raffaella; Riva, Marco A.; Van der Harst, Johanneke E.; Homberg, Judith R.

2018-01-01

Background Huntington disease (HD) is a devastating inherited neurodegenerative disorder characterized by progressive motor, cognitive, and psychiatric symptoms without any cure to slow down or stop the progress of the disease. The BACHD rat model for HD carrying the human full-length mutant huntingtin protein (mHTT) with 97 polyQ repeats has been recently established as a promising model which reproduces several HD-like features. While motor and cognitive functions have been characterized in BACHD rats, little is known about their social phenotype. Objective This study focuses especially on social behavior since evidence for social disturbances exists in human patients. Our objective was to compare social behavior in BACHD and wild-type (WT) rats at different ages, using two different measures of sociability. Methods Animals were tested longitudinally at the age of 2, 4 and 8 months in the social interaction test to examine different parameters of sociability. A separate cohort of 7 month old rats was tested in the three chamber social test to measure both sociability and social novelty. Gene expression analyses in 8 months old animals were performed by real time qRT-PCR to evaluate a potential involvement of D1 and D2 dopaminergic receptors and the contribution of Brain-derived neurotrophic factor (BDNF) to the observed behavioral alterations. Results In the social interaction test, BACHD rats showed age-dependent changes in behaviour when they were-re introduced to their cagemate after a 24 hours-period of individual housing. The time spent on nape attacks increased with aging. Furthermore, a significant higher level of pinning at 2 months of age was shown in the BACHD rats compared to wild-types, followed by a reduction at 4 and 8 months. On the other hand, BACHD rats exhibited a decreased active social behaviour compared to wild-types, reflected by genotype-effects on approaching, following and social nose contact. In the three chamber social test, BACHD rats

Histone H3 lysine 9 methyltransferase FvDim5 regulates fungal development, pathogenicity and osmotic stress responses in Fusarium verticillioides.

Science.gov (United States)

Gu, Qin; Ji, Tiantian; Sun, Xiao; Huang, Hai; Zhang, Hao; Lu, Xi; Wu, Liming; Huo, Rong; Wu, Huijun; Gao, Xuewen

2017-10-16

Histone methylation plays important biological roles in eukaryotic cells. Methylation of lysine 9 at histone H3 (H3K9me) is critical for regulating chromatin structure and gene transcription. Dim5 is a lysine histone methyltransferase (KHMTase) enzyme, which is responsible for the methylation of H3K9 in eukaryotes. In the current study, we identified a single ortholog of Neurospora crassa Dim5 in Fusarium verticillioides. In this study, we report that FvDim5 regulates the trimethylation of H3K9 (H3K9me3). The FvDIM5 deletion mutant (ΔFvDim5) showed significant defects in conidiation, perithecium production and fungal virulence. Unexpectedly, we found that deletion of FvDIM5 resulted in increased tolerance to osmotic stresses and upregulated FvHog1 phosphorylation. These results indicate the importance of FvDim5 for the regulation of fungal development, pathogenicity and osmotic stress responses in F. verticillioides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

2010 CELL AND MOLECULAR FUNGAL BIOLOGY GORDON RESEARCH CONFERENCE, JUNE 13-18, 2010

Energy Technology Data Exchange (ETDEWEB)

Michelle Momany

2010-06-18

The Cellular and Molecular Fungal Biology Conference provides a forum for presentation of the latest advances in fungal research with an emphasis on filamentous fungi. This open-registration scientific meeting brings together the leading scientists from academia, government and industry to discuss current research results and future directions at Holderness School, an outstanding venue for scientific interaction. A key objective of the conference is to foster interaction among scientists working on model fungi such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa and Aspergillus nidulans and scientists working on a variety of filamentous fungi whose laboratory tractability is often inversely proportional to their medical, industrial or ecological importance. Sessions will be devoted to Systems Biology, Fungi and Cellulosic Biomass, Small RNAs, Population Genomics, Symbioses, Pathogenesis, Membrane Trafficking and Polarity, and Cytoskeleton and Motors. A session will also be devoted to hot topics picked from abstracts. The CMFB conference provides a unique opportunity to examine the breadth of fungal biology in a small meeting format that encourages in-depth discussion among the attendees.

Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells

Directory of Open Access Journals (Sweden)

Bernardas Morkunas

2016-07-01

Full Text Available Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell–cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable.

Pulsed growth of fungal hyphal tips.

Science.gov (United States)

López-Franco, R; Bartnicki-Garcia, S; Bracker, C E

1994-12-06

Somatic fungal hyphae are generally assumed to elongate at steady linear rates when grown under constant environmental conditions with ample nutrients. However, patterns of pulsed hyphal elongation were detected during apparent steady growth of hyphal tips in fungi from several major taxonomic groups (Oomycetes, Pythium aphanidermatum and Saprolegnia ferax; Zygomycetes, Gilbertella persicaria; Deuteromycetes, Trichoderma viride; Ascomycetes, Neurospora crassa and Fusarium culmorum; Basidiomycetes, Rhizoctonia solani). Growing hyphal tips were recorded with video-enhanced phase-contrast microscopy at high magnification, and digital images were measured at very short time intervals (1-5 s). In all fungi tested, the hyphal elongation rate was never perfectly steady but fluctuated continuously with alternating periods of fast and slow growth at more or less regular intervals. Pulsed growth was observed in fungi differing in cell diameter, overall growth rate, taxonomic position, and presence and pattern of Spitzenkörper organization, suggesting that this is a general phenomenon. Frequency and amplitude of the pulses varied among the test organisms. T. viride and N. crassa showed the most frequent pulses (average of 13-14 per min), and F. culmorum the least frequent (2.7 per min). Average pulse amplitude varied from 0.012 microns/s for F. culmorum to 0.068 microns/s for G. persicaria. In F. culmorum and T. viride, the fast phase of the growth pulses was correlated with the merger of satellite Spitzenkörper with the main Spitzenkörper. These findings are consistent with a causal relationship between fluctuations in the overall rate of secretory vesicle delivery/discharge at the hyphal apex and the fluctuations in hyphal elongation rate.

On the mechanistic differences of benzene-induced leukemogenesis between wild type and p53 knockout mice

International Nuclear Information System (INIS)

Hirabayashi, Yoko; Yoon, Byung-Il; Kawasaki, Yasushi; Li, Guang-Xun; Kanno, Jun; Inoue, Tohru

2003-01-01

Leukemia induction by benzene inhalation was first reported by Le Noire in 1887, described multiple cases of leukemia among Parisian cobblers. However, experimental induction of leukemia by benzene exposure was not succeeded for a hundred years, until Snyder et al. and our group reported it nearly 20 years ago. Nevertheless, the mechanistic background of benzene-induced leukemia was still an enigma until recently a benzene-induced peculiar cell kinetics of the stem/progenitor cells has been elucidated by our study, demonstrated a marked repeated oscillatory decrease in peripheral blood and bone marrow (BM) cellularity during and after benzene exposure, which epigenetically preceded and developed the leukemia more than a year later. We utilized the BUUV (bromodeoxyuridine + UV exposure) method to study stem/progenitor cell kinetics during and/or after benzene exposure. Using these methods, we were able to measure the labeling rate, cycling fraction of clonogenic progenitor cells, and other cell cycle parameters. The cycling fraction of stem/progenitor cells was found not to turn into an active hematopoiesis but to remain low during benzene inhalation and further we found evidence that the cycling fraction depression may be mediated in part by a slowing of stem/progenitor cell cycling perse by up-regulation of p21. The benzene induced leukemogenicity between mice carrying wild-type p53 and mice lacking p53 seem to differ from one another. In the case of p53 knockout mouse, DNA damage such as weak mutagenicity and or chromosomal damages are retained, and those damages participated in the induction of a consequent activation of proto-oncogenes and the like, which led cells to further neoplastic changes. In contrast, in the case of wild type mice, a dramatic oscillational change in the cell cycle of the stem cell compartment seems to be an important factor for mice carrying the p53 gene. (author)

Proline metabolism in the wild-type and in a salt-tolerant mutant of nicotiana plumbaginifolia studied by (13)C-nuclear magnetic resonance imaging

Science.gov (United States)

Roosens; Willem; Li; Verbruggen; Biesemans; Jacobs

1999-12-01

To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. (13)C-Nuclear magnetic resonance with [5-(13)C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mM NaCl stress in the presence of [5-(13)C]Glu, a significant enrichment in [5-(13)C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-(13)C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-(13)C]Glu into [5-(13)C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Delta-pyrroline-5-carboxylate synthetase enzyme.

Antibody production of wild-type and enzyme V279F variants of PAF-AH as a risk factor for Cardiovascular disease

Science.gov (United States)

Ramadhani, Anggia N.; Puspitarini, Sapti; Sari, Anissa N.; Widodo

2017-11-01

Coronary artery disease (CAD) has emerged as a leading cause of death in Indonesia nowadays. WHO data in 2012 revealed that 37% of the Indonesian population died from this disease. CAD occurs because of endothelial dysfunction in the arteries. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a phospholipase A2 enzyme, encoded by the PLA2G7 gene. This protein is predicted to be involved in inflammatory phospholipid metabolism so it can be used as a biomarker of CAD in the early phase. Thus, the purpose of this research is to discover the difference in antibody production between wild-type and mutant V279F. The PAF-AH enzyme was isolated from mice lymphocyte cells in order to develop this enzyme as a biomarker of cardiovascular disease. PAF-AH migrates at 55kDa according to SDS-PAGE analysis. Flow cytometry analysis showed that mutant PAF-AH (V279F) is more antigenic than wild-type PAF-AH. The missense mutation of V279F PAF-AH means this enzyme cannot catabolize the acetyl group at the sn-2 position of PAF.

iTRAQ-facilitated proteomic profiling of anthers from a photosensitive male sterile mutant and wild-type cotton (Gossypium hirsutum L.).

Science.gov (United States)

Liu, Ji; Pang, Chaoyou; Wei, Hengling; Song, Meizhen; Meng, Yanyan; Ma, Jianhui; Fan, Shuli; Yu, Shuxun

2015-08-03

Male sterility is a common phenomenon in flowering plants, and it has been successfully developed in several crops by taking advantage of heterosis. Cotton (Gossypium hirsutum L.) is an important economic crop, used mainly for the production of textile fiber. Using a space mutation breeding technique, a novel photosensitive genetic male sterile mutant CCRI9106 was isolated from the wild-type upland cotton cultivar CCRI040029. To use CCRI9106 in cotton hybrid breeding, it is of great importance to study the molecular mechanisms of its male sterility. Here, histological and iTRAQ-facilitated proteomic analyses of anthers were performed to explore male sterility mechanisms of the mutant. Scanning and transmission electron microscopy of the anthers showed that the development of pollen wall in CCRI9106 was severely defective with a lack of exine formation. At the protein level, 6121 high-confidence proteins were identified and 325 of them showed differential expression patterns between mutant and wild-type anthers. The proteins up- or down-regulated in MT anthers were mainly involved in exine formation, protein degradation, calcium ion binding,etc. These findings provide valuable information on the proteins involved in anther and pollen development, and contribute to elucidate the mechanism of male sterility in upland cotton. Copyright © 2015. Published by Elsevier B.V.

Comparative analysis of hyoscine in wild-type and in vitro- grown ...

African Journals Online (AJOL)

regeneration was obtained with 2 mg/L BAP and 1 mg/L kinetin. ... Wild root, stem and leaves exhibited higher amounts (approx. ... Despite several medical benefits offered by .... The initial screening of hyoscine from all samples ... Mobile phase of 0.02 mol/L ..... compounds in commercial herbal drugs and spices from.

Genetic Characterization of the Hemagglutinin Genes of Wild-Type Measles Virus Circulating in China, 1993–2009

Science.gov (United States)

Zhu, Zhen; Liu, Chunyu; Mao, Naiying; Ji, Yixin; Wang, Huiling; Jiang, Xiaohong; Li, Chongshan; Tang, Wei; Feng, Daxing; Wang, Changyin; Zheng, Lei; Lei, Yue; Ling, Hua; Zhao, Chunfang; Ma, Yan; He, Jilan; Wang, Yan; Li, Ping; Guan, Ronghui; Zhou, Shujie; Zhou, Jianhui; Wang, Shuang; Zhang, Hong; Zheng, Huanying; Liu, Leng; Ma, Hemuti; Guan, Jing; Lu, Peishan; Feng, Yan; Zhang, Yanjun; Zhou, Shunde; Xiong, Ying; Ba, Zhuoma; Chen, Hui; Yang, Xiuhui; Bo, Fang; Ma, Yujie; Liang, Yong; Lei, Yake; Gu, Suyi; Liu, Wei; Chen, Meng; Featherstone, David; Jee, Youngmee; Bellini, William J.; Rota, Paul A.; Xu, Wenbo

2013-01-01

Background China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. Principal Findings Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was measles in China. PMID:24073194

The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer.

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Jatin Roper

Full Text Available To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC.PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC(50 = 9.0-14.3 nM. Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01 vs. a 43% decrease (p = 0.008 in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003, no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013.These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.

Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics

Science.gov (United States)

Wong, Min Hao; Giraldo, Juan P.; Kwak, Seon-Yeong; Koman, Volodymyr B.; Sinclair, Rosalie; Lew, Tedrick Thomas Salim; Bisker, Gili; Liu, Pingwei; Strano, Michael S.

2017-02-01

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors--single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal--embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm-1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

Comparison of mating performance of medfly (Diptera: Tephritidae) genetic sexing and wild type strains: field cage and video recording experiments

International Nuclear Information System (INIS)

Calcagno, G.E.; Vilardi, J.C.; Manso, F.

2002-01-01

To improve the efficiency of the sterile insect technique (SIT) efforts are being devoted to obtain genetic sexing strains (GSS). The present work was carried out in order to compare the mating efficiency of flies from the GSS [(Ty34228 y + /X)sw x ] and from a wild type strain (Mendoza). Females of the GSS (T228) exhibit longer embryonic development, while males develop in a normal time period. In a field-cage experiment, mating competitiveness was compared between the T228 and the Mendoza, Argentina mass reared strain. The number and duration of matings and the location of copula in the tree were recorded. The analysis was repeated using irradiated males of T228. The results showed that mating efficiency of the GSS is good in comparison with that of the Mendoza strain. Although copulatory success in T228 is reduced by the radiation treatment, the high numbers of sterilized males released would compensate this effect in the control programs. In a second experiment, under laboratory conditions, video recording techniques were applied. In this case two virgin males, one of the GSS and one emerged from wild collected fruits, competed during 30 min for a virgin wild female. The proportion of successful males did not differ between strains, but some differences were observed between strains in the time spent in different stages of the courtship. Males of the T228 were more aggressive, and they attempted to copulate with the other male more frequently than did wild males. These differences may be due to selection for more aggressive individuals under the overcrowded laboratory breeding conditions for this strain. (author)

Wild Maid, Wild Soul, A Wild Wild Weed: Niki de Saint Phalle’s Fierce Femininities, c. 1960-66

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Amelia Jones

2015-11-01

Full Text Available Self-described as “wild maid,” “wild soul,” “a wild wild weed,” Niki de Saint Phalle narrated herself as artistic subject in a concerted way that stands out in the history of art: as both creatively driven and emotionally renegade and excessive, as both definitively woman and definitively artist. In this essay I take this special case of self-narration, and the particular power of St. Phalle’s work, as an opportunity to explore the relationship between.(auto-biography and artistic practice. The case of St. Phalle, a radical sculptor, performance artist, writer, and filmmaker, allows us to understand the exaggerated way in which women artists were until very recently forced to adopt “fierce femininities” to make a place for themselves as artists. In this way, I suggest that St. Phalle represents a key inspirational force opening the door for second wave feminism and the feminist art movement.

Occurrence of Edwardsiella tarda in wild European eels Anguilla anguilla from Mediterranean Spain.

Science.gov (United States)

Alcaide, Elena; Herraiz, Sonia; Esteve, Consuelo

2006-11-21

Pure cultures of Edwarsiella tarda were isolated from body ulcers and internal organs of wild European eels caught in a Mediterranean freshwater coastal lagoon (Albufera Lake, Valencia, Spain) over a 1 yr period. Overall, the E. tarda isolation rate from wild eels was 9%, but this increased to 22.8% in diseased individuals. All 22 E. tarda isolates belonged to the 'wild-type' biogroup of the species and were virulent for eels (lethal dose that kills 50% of exposed individuals [LD50 dose]: 10(4.85) to 10(6.83) CFU ind.(-1)), and therefore represented the aetiological agent of the haemorrhagic disease observed in wild European eels. The E. tarda isolates and E. tarda CECT 894T type strain were biochemically and serologically related and resistant to macrolides, antifolates, and glycopeptides, but only the isolates from wild eels were resistant to clindamicyn. This study is the first description of edwardsiellosis in a wild European eel population, and alerts us to the presence of E. tarda in natural wetland environments in Mediterranean Europe.

Oxygen effect and influence of the anoxic radiosensitizing agent TAN on the induction of λ-prophage in polA and wild type E.coli strains after gamma irradiation

International Nuclear Information System (INIS)

Bonev, M.N.; Sivriev, I.K.; Kolev, S.D.

1998-01-01

The modification effect of both oxygen and radiosensitizing agent TAN on the λ-prophage induction in polA mutant and wild type E.coli cells after γ-irradiation was studied. The oxygen and TAN enhancement ratio concerning the cell sensitivity is more significant in polA mutant cells as compared to that in the wild type ones. The same behaviour has been observed for the oxygen and TAN enhancement ratio for the λ-prophage induction. The TAN effect on the survival and on the λ-induction was smaller than the oxygen effect. The bigger efficiency of oxygen and DNA-radicals are more difficult to repair than those created by an interaction of TAN and DNA-radicals

A comparison of the immune responses of dogs exposed to canine distemper virus (CDV) — Differences between vaccinated and wild-type virus exposed dogs

Science.gov (United States)

Perrone, Danielle; Bender, Scott; Niewiesk, Stefan

2010-01-01

Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals. PMID:20885846

Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1,000 Genomes Project.

Science.gov (United States)

Santpere, Gabriel; Darre, Fleur; Blanco, Soledad; Alcami, Antonio; Villoslada, Pablo; Mar Albà, M; Navarro, Arcadi

2014-04-01

Most people in the world (∼90%) are infected by the Epstein-Barr virus (EBV), which establishes itself permanently in B cells. Infection by EBV is related to a number of diseases including infectious mononucleosis, multiple sclerosis, and different types of cancer. So far, only seven complete EBV strains have been described, all of them coming from donors presenting EBV-related diseases. To perform a detailed comparative genomic analysis of EBV including, for the first time, EBV strains derived from healthy individuals, we reconstructed EBV sequences infecting lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project. As strain B95-8 was used to transform B cells to obtain LCLs, it is always present, but a specific deletion in its genome sets it apart from natural EBV strains. After studying hundreds of individuals, we determined the presence of natural EBV in at least 10 of them and obtained a set of variants specific to wild-type EBV. By mapping the natural EBV reads into the EBV reference genome (NC007605), we constructed nearly complete wild-type viral genomes from three individuals. Adding them to the five disease-derived EBV genomic sequences available in the literature, we performed an in-depth comparative genomic analysis. We found that latency genes harbor more nucleotide diversity than lytic genes and that six out of nine latency-related genes, as well as other genes involved in viral attachment and entry into host cells, packaging, and the capsid, present the molecular signature of accelerated protein evolution rates, suggesting rapid host-parasite coevolution.

Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data.

Science.gov (United States)

Angeby, K A; Jureen, P; Giske, C G; Chryssanthou, E; Sturegård, E; Nordvall, M; Johansson, A G; Werngren, J; Kahlmeter, G; Hoffner, S E; Schön, T

2010-05-01

To describe wild-type distributions of the MIC of fluoroquinolones for Mycobacterium tuberculosis in relation to current critical concentrations used for drug susceptibility testing and pharmacokinetic/pharmacodynamic (PK/PD) data. A 96-stick replicator on Middlebrook 7H10 medium was used to define the MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin for 90 consecutive clinical strains and 24 drug-resistant strains. The MICs were compared with routine BACTEC 460 susceptibility results and with MIC determinations in the BACTEC MGIT 960 system in a subset of strains using ofloxacin as a class representative. PK/PD data for each drug were reviewed in relation to the wild-type MIC distribution. The wild-type MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin were distributed from 0.125 to 1, 0.25 to 1, 0.032 to 0.5 and 0.125 to 0.5 mg/L, respectively. The MIC data correlated well with the BACTEC 960 MGIT and BACTEC 460 results. PD indices were the most favourable for levofloxacin, followed by moxifloxacin, ofloxacin and ciprofloxacin. We propose S (susceptible)

Oral Challenge with Wild-Type Salmonella Typhi Induces Distinct Changes in B Cell Subsets in Individuals Who Develop Typhoid Disease.

OpenAIRE

Franklin R Toapanta; Paula J Bernal; Stephanie Fresnay; Laurence S Magder; Thomas C Darton; Claire Jones; Claire S Waddington; Christoph J Blohmke; Brian Angus; Myron M Levine; Andrew J Pollard; Marcelo B Sztein

2016-01-01

A novel human oral challenge model with wild-type Salmonella Typhi (S. Typhi) was recently established by the Oxford Vaccine Group. In this model, 104 CFU of Salmonella resulted in 65% of participants developing typhoid fever (referred here as typhoid diagnosis -TD-) 6?9 days post-challenge. TD was diagnosed in participants meeting clinical (oral temperature ?38?C for ?12h) and/or microbiological (S. Typhi bacteremia) endpoints. Changes in B cell subpopulations following S. Typhi challenge re...

Chinese herbal extract Su-duxing had potent inhibitory effects on both wild-type and entecavir-resistant hepatitis B virus (HBV) in vitro and effectively suppressed HBV replication in mouse model.

Science.gov (United States)

Liu, Yan; Yao, Weiming; Si, Lanlan; Hou, Jun; Wang, Jiabo; Xu, Zhihui; Li, Weijie; Chen, Jianhong; Li, Ruisheng; Li, Penggao; Bo, Lvping; Xiao, Xiaohe; Lan, Jinchu; Xu, Dongping

2018-04-24

The present study aimed to investigate anti-HBV effect and major active compounds of Su-duxing, a medicine extracted from Chinese herbs. HBV-replicating cell lines HepG2.2.15 (wild-type) and HepG2. A64 (entecavir-resistant) were used for in vitro test. C57BL/6 mice infected by adeno-associated virus carrying 1.3 mer wild-type HBV genome were used for in vivo test. Inhibitory rates of Su-duxing (10 μg/mL) on HBV replicative intermediate and HBsAg levels were 75.1%, 51.0% in HepG2.2.15 cells and 65.2%, 42.9% in HepG2. A64 cells. The 50% inhibitory concentration of Su-duxing and entecavir on HBV replicative intermediates had 0.2-fold and 712.5-fold increase respectively for entecavir-resistant HBV compared to wild-type HBV. Mice treated with Su-duxing (45.0 mg kg -1  d -1 for 2 weeks) had 1.39 log 10 IU/mL decrease of serum HBV DNA, and 48.9% and 51.7% decrease of serum HBsAg and HBeAg levels. GeneChip and KEGG analysis proposed that anti-HBV mechanisms included relief of HBx stability and viral replication, deregulation of early cell cycle checkpoints, and induction of type I interferon. Six active compounds (Matrine, Oxymatrine, Chlorogenic acid, Sophocarpine, Baicalein, and Wogonin) against HBV were identified in Su-duxing. Greater anti-HBV effects were observed in some compound pairs compared to each single compound. In conclusion, Su-duxing had potent inhibitory effects on both wild-type and entecavir-resistant HBV. Its effects were associated with coordinated roles of active compounds in its composition. Copyright © 2018. Published by Elsevier B.V.

Intraperitoneal Infection of Wild-Type Mice with Synthetically Generated Mammalian Prion.

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Xinhe Wang

2015-07-01

Full Text Available The prion hypothesis postulates that the infectious agent in transmissible spongiform encephalopathies (TSEs is an unorthodox protein conformation based agent. Recent successes in generating mammalian prions in vitro with bacterially expressed recombinant prion protein provide strong support for the hypothesis. However, whether the pathogenic properties of synthetically generated prion (rec-Prion recapitulate those of naturally occurring prions remains unresolved. Using end-point titration assay, we showed that the in vitro prepared rec-Prions have infectious titers of around 104 LD50/μg. In addition, intraperitoneal (i.p. inoculation of wild-type mice with rec-Prion caused prion disease with an average survival time of 210-220 days post inoculation. Detailed pathological analyses revealed that the nature of rec-Prion induced lesions, including spongiform change, disease specific prion protein accumulation (PrP-d and the PrP-d dissemination amongst lymphoid and peripheral nervous system tissues, the route and mechanisms of neuroinvasion were all typical of classical rodent prions. Our results revealed that, similar to naturally occurring prions, the rec-Prion has a titratable infectivity and is capable of causing prion disease via routes other than direct intra-cerebral challenge. More importantly, our results established that the rec-Prion caused disease is pathogenically and pathologically identical to naturally occurring contagious TSEs, supporting the concept that a conformationally altered protein agent is responsible for the infectivity in TSEs.

Nrf2 but not autophagy inhibition is associated with the survival of wild-type epidermal growth factor receptor non-small cell lung cancer cells

International Nuclear Information System (INIS)

Zhou, Yan; Li, Yuan; Ni, Hong-Min; Ding, Wen-Xing; Zhong, Hua

2016-01-01

Non-small cell lung cancer (NSCLC) is one of the most common malignancies in the world. Icotinib and Gefitinib are two epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) that have been used to treat NSCLC. While it is well known that mutations of EGFR can affect the sensitivity of NSCLC to the EGFR-TKI, other mechanisms may also be adopted by lung cancer cells to develop resistance to EGFR-TKI treatment. Cancer cells can use multiple adaptive mechanisms such as activation of autophagy and Nrf2 to protect against various stresses and chemotherapeutic drugs. Whether autophagy or Nrf2 activation contributes to the resistance of NSCLC to EGFR-TKI treatment in wild-type EGFR NSCLC cells remains elusive. In the present study, we confirmed that Icotinib and Gefitinib induced apoptosis in EGFR mutant HCC827 but not in EGFR wild-type A549 NSCLC cells. Icotinib and Gefitinib did not induce autophagic flux or inhibit mTOR in A549 cells. Moreover, suppression of autophagy by chloroquine, a lysosomal inhibitor, did not affect Icotinib- or Gefitinib-induced cell death in A549 cells. In contrast, Brusatol, an Nrf2 inhibitor, significantly suppressed the cell survival of A549 cells. However, Brusatol did not further sensitize A549 cells to EGFR TKI-induced cell death. Results from this study suggest that inhibition of Nrf2 can decrease cell vitality of EGFR wild-type A549 cells independent of autophagy. - Highlights: • Cancer cells use adaptive mechanisms against chemotherapy. • Autophagy is not essential for the drug resistance of lung cancer A549 cells. • Inhibition of Nrf2 decreases cell survival of lung cancer A549 cells.

Proline Metabolism in the Wild-Type and in a Salt-Tolerant Mutant of Nicotiana plumbaginifolia Studied by 13C-Nuclear Magnetic Resonance Imaging1

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Roosens, Nancy H.; Willem, Rudolph; Li, Yan; Verbruggen, Ingrid; Biesemans, Monique; Jacobs, Michel

1999-01-01

To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. 13C-Nuclear magnetic resonance with [5-13C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mm NaCl stress in the presence of [5-13C]Glu, a significant enrichment in [5-13C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-13C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-13C]Glu into [5-13C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Δ-pyrroline-5-carboxylate synthetase enzyme. PMID:10594115

Nrf2 but not autophagy inhibition is associated with the survival of wild-type epidermal growth factor receptor non-small cell lung cancer cells

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Zhou, Yan [Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030 (China); Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160 (United States); Li, Yuan; Ni, Hong-Min; Ding, Wen-Xing [Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160 (United States); Zhong, Hua, E-mail: eddiedong8@hotmail.com [Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030 (China)

2016-11-01

Non-small cell lung cancer (NSCLC) is one of the most common malignancies in the world. Icotinib and Gefitinib are two epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) that have been used to treat NSCLC. While it is well known that mutations of EGFR can affect the sensitivity of NSCLC to the EGFR-TKI, other mechanisms may also be adopted by lung cancer cells to develop resistance to EGFR-TKI treatment. Cancer cells can use multiple adaptive mechanisms such as activation of autophagy and Nrf2 to protect against various stresses and chemotherapeutic drugs. Whether autophagy or Nrf2 activation contributes to the resistance of NSCLC to EGFR-TKI treatment in wild-type EGFR NSCLC cells remains elusive. In the present study, we confirmed that Icotinib and Gefitinib induced apoptosis in EGFR mutant HCC827 but not in EGFR wild-type A549 NSCLC cells. Icotinib and Gefitinib did not induce autophagic flux or inhibit mTOR in A549 cells. Moreover, suppression of autophagy by chloroquine, a lysosomal inhibitor, did not affect Icotinib- or Gefitinib-induced cell death in A549 cells. In contrast, Brusatol, an Nrf2 inhibitor, significantly suppressed the cell survival of A549 cells. However, Brusatol did not further sensitize A549 cells to EGFR TKI-induced cell death. Results from this study suggest that inhibition of Nrf2 can decrease cell vitality of EGFR wild-type A549 cells independent of autophagy. - Highlights: • Cancer cells use adaptive mechanisms against chemotherapy. • Autophagy is not essential for the drug resistance of lung cancer A549 cells. • Inhibition of Nrf2 decreases cell survival of lung cancer A549 cells.

Early Transcriptional Responses of Bovine Chorioallantoic Membrane Explants to Wild Type, ΔvirB2 or ΔbtpB Brucella abortus Infection

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Mol, Juliana P. S.; Costa, Erica A.; Carvalho, Alex F.; Sun, Yao-Hui; Tsolis, Reneé M.; Paixão, Tatiane A.; Santos, Renato L.

2014-01-01

The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; Pabortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion. PMID:25259715

High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A

International Nuclear Information System (INIS)

Casal, J.J.; Clough, R.C.; Vierstra, R.D.

1996-01-01

The occurrence of phytochrome-mediated high irradiance responses (HIR), previously characterised largely in dicotyledonous plants, was investigated in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam. and in both wild-type Oryza sativa L. and in transgenic plants overexpressing oat phytochrome A under the control of a 35S promoter. Coleoptile growth was promoted (maize, ryegrass) or inhibited (wild-type rice) by continuous far-red light (FRc). However, at equal fluences, hourly pulses of far-red light (FRp) were equally effective, indicating that the growth responses to FRc were not true HIR. In contrast, in maize and rice, FRc increased anthocyanin content in the coleoptile in a fluence-rate dependent manner. This response was a true HIR as FRp had reduced effects. In maize, anthocyanin levels were significantly higher under FRc than under continuous red light. In rice, overexpression of phytochrome A increased the inhibition of coleoptile growth and the levels of anthocyanin under FRc but not under FRp or under continuous red light. The effect of FRc was fluence-rate dependent. In light-grown rice, overexpression of phytochrome A reduced leaf-sheath length, impaired the response to supplementary far-red light, but did not affect the response to canopy shade-light. In grasses, typical HIR, i.e. fluence-rate dependent responses showing reciprocity failure, can be induced by FRc. Under FRc, overexpressed phytochrome A operates through this action mode in transgenic rice. (author)

Innate resistance to myxomatosis in wild rabbits in England*

Science.gov (United States)

Ross, J.; Sanders, M. F.

1977-01-01

Wild rabbits (Oryctolagus cuniculus) from one study area in England have been used over a period of 11 years to investigate the possible appearance of innate resistance to myxomatosis. Rabbits of 4-6 weeks old were captured alive, retained in the laboratory until at least 4 months old, and then infected with a type of myxoma virus which kills 90-95% of laboratory rabbits. Observations were made of symptoms, mortality rate and survival times. In the first 4 years of the study (1966-9), mortality rates were not significantly different from those of laboratory rabbits, although survival times of wild rabbits were appreciably longer. In 1970, the mortality rate amongst wild rabbits was 59%, in 1974 it was 17%, and in 1976 it was 20%, thus showing that a considerable degree of inherited resistance to myxomatosis has developed. The types of myxoma virus most commonly isolated from wild rabbits in Great Britain in recent years have been those which cause 70-95% mortality in laboratory rabbits. Therefore, if the degree of innate resistance demonstrated is widespread in Great Britain, there are serious implications regarding the size of the rabbit population, because myxomatosis has been an important factor in holding rabbit numbers at a relatively low level. PMID:270526

Innate resistance to myxomatosis in wild rabbits in England.

Science.gov (United States)

Ross, J; Sanders, M F

1977-12-01

Wild rabbits (Oryctolagus cuniculus) from one study area in England have been used over a period of 11 years to investigate the possible appearance of innate resistance to myxomatosis. Rabbits of 4-6 weeks old were captured alive, retained in the laboratory until at least 4 months old, and then infected with a type of myxoma virus which kills 90-95% of laboratory rabbits. Observations were made of symptoms, mortality rate and survival times.In the first 4 years of the study (1966-9), mortality rates were not significantly different from those of laboratory rabbits, although survival times of wild rabbits were appreciably longer. In 1970, the mortality rate amongst wild rabbits was 59%, in 1974 it was 17%, and in 1976 it was 20%, thus showing that a considerable degree of inherited resistance to myxomatosis has developed.The types of myxoma virus most commonly isolated from wild rabbits in Great Britain in recent years have been those which cause 70-95% mortality in laboratory rabbits. Therefore, if the degree of innate resistance demonstrated is widespread in Great Britain, there are serious implications regarding the size of the rabbit population, because myxomatosis has been an important factor in holding rabbit numbers at a relatively low level.

Staurosporine scaffold-based rational discovery of the wild-type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog-sensitive kinase technology.

Science.gov (United States)

Song, Xiaoyun; Liu, Xingcai; Ding, Xi

2017-04-01

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first-line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small-molecule kinase inhibitors in EGFR-targeted chemotherapy. Previously, the reversible pan-kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR T790M ) over wild-type kinase (EGFR WT ), suggesting that the staurosporine scaffold is potentially to develop the wild-type sparing reversible inhibitors of EGFR T790M . Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold-based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico-in vitro analog-sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild-type sparing inhibitors UCN-01, UCN-02, AFN941, and SB-218078 with high or moderate selectivity of 30-, 45-, 5-, and 8-fold for EGFR T790M over EGFR WT , respectively, which are comparable with or even better than that of the parent compound staurosporine (24-fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN-02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding. A hydroxyl group of UCN-02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

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Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gapsys, Vytautas; Ucurum, Zöhre; de Groot, Bert L; Fotiadis, Dimitrios

2016-09-13

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

Epidermal Growth Factor Receptor (EGFR gene copy number (GCN correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH and chromogenic in situ hybridization (CISH analysis

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Scartozzi Mario

2009-08-01

Full Text Available Abstract Background K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Methods Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Results Forty-four patients were available for analysis. We observed a partial remission in 9 (60% and 2 (9% cases with a FISH EGFR GCN ≥ 2.6 and Conclusion FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

Testing projected wild bee distributions in agricultural habitats: predictive power depends on species traits and habitat type.

Science.gov (United States)

Marshall, Leon; Carvalheiro, Luísa G; Aguirre-Gutiérrez, Jesús; Bos, Merijn; de Groot, G Arjen; Kleijn, David; Potts, Simon G; Reemer, Menno; Roberts, Stuart; Scheper, Jeroen; Biesmeijer, Jacobus C

2015-10-01

Species distribution models (SDM) are increasingly used to understand the factors that regulate variation in biodiversity patterns and to help plan conservation strategies. However, these models are rarely validated with independently collected data and it is unclear whether SDM performance is maintained across distinct habitats and for species with different functional traits. Highly mobile species, such as bees, can be particularly challenging to model. Here, we use independent sets of occurrence data collected systematically in several agricultural habitats to test how the predictive performance of SDMs for wild bee species depends on species traits, habitat type, and sampling technique. We used a species distribution modeling approach parametrized for the Netherlands, with presence records from 1990 to 2010 for 193 Dutch wild bees. For each species, we built a Maxent model based on 13 climate and landscape variables. We tested the predictive performance of the SDMs with independent datasets collected from orchards and arable fields across the Netherlands from 2010 to 2013, using transect surveys or pan traps. Model predictive performance depended on species traits and habitat type. Occurrence of bee species specialized in habitat and diet was better predicted than generalist bees. Predictions of habitat suitability were also more precise for habitats that are temporally more stable (orchards) than for habitats that suffer regular alterations (arable), particularly for small, solitary bees. As a conservation tool, SDMs are best suited to modeling rarer, specialist species than more generalist and will work best in long-term stable habitats. The variability of complex, short-term habitats is difficult to capture in such models and historical land use generally has low thematic resolution. To improve SDMs' usefulness, models require explanatory variables and collection data that include detailed landscape characteristics, for example, variability of crops and

Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers.

OpenAIRE

Cox, D. G.; Simard, J.; Sinnett, D.; Hamdi, Y.; Soucy, P.; Ouimet, M.; Barjhoux, L.; Verny-Pierre, C.; McGuffog, L.; Healey, S.; Szabo, C.; Greene, M. H.; Mai, P. L.; Andrulis, I. L.; Thomassen, M.

2011-01-01

Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA...

Expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of glyceraldehyde-3-phosphate dehydrogenase from Campylobacter jejuni

International Nuclear Information System (INIS)

Tourigny, David S.; Elliott, Paul R.; Edgell, Louise J.; Hudson, Gregg M.; Moody, Peter C. E.

2010-01-01

The cloning, expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of C. jejuni glyceraldehyde-3-phosphate dehydrogenase is reported. The genome of the enteric pathogen Campylobacter jejuni encodes a single glyceraldehyde-3-phosphate dehydrogenase that can utilize either NADP + or NAD + as coenzymes for the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of both the wild type and an active-site mutant of the enzyme are presented. Preliminary X-ray analysis revealed that in both cases the crystals diffracted to beyond 1.9 Å resolution. The space group is shown to be I4 1 22, with unit-cell parameters a = 90.75, b = 90.75, c = 225.48 Å, α = 90.46, β = 90.46, γ = 222.79°; each asymmetric unit contains only one subunit of the tetrameric enzyme

Wild-type bone marrow transplant partially reverses neuroinflammation in progranulin-deficient mice.

Science.gov (United States)

Yang, Yue; Aloi, Macarena S; Cudaback, Eiron; Josephsen, Samuel R; Rice, Samantha J; Jorstad, Nikolas L; Keene, C Dirk; Montine, Thomas J

2014-11-01

Frontotemporal dementia (FTD) is a neurodegenerative disease with devastating changes in behavioral performance and social function. Mutations in the progranulin gene (GRN) are one of the most common causes of inherited FTD due to reduced progranulin expression or activity, including in brain where it is expressed primarily by neurons and microglia. Thus, efforts aimed at enhancing progranulin levels might be a promising therapeutic strategy. Bone marrow (BM)-derived cells are able to engraft in the brain and adopt a microglial phenotype under myeloablative irradiation conditioning. This ability makes BM-derived cells a potential cellular vehicle for transferring therapeutic molecules to the central nervous system. Here, we utilized BM cells from Grn(+/+) (wild type or wt) mice labeled with green fluorescence protein for delivery of progranulin to progranulin-deficient (Grn(-/-)) mice. Our results showed that wt bone marrow transplantation (BMT) partially reconstituted progranulin in the periphery and in cerebral cortex of Grn(-/-) mice. We demonstrated a pro-inflammatory effect in vivo and in ex vivo preparations of cerebral cortex of Grn(-/-) mice that was partially to fully reversed 5 months after BMT. Our findings suggest that BMT can be administered as a stem cell-based approach to prevent or to treat neurodegenerative diseases.

Wild Type Bone Marrow Transplant Partially Reverses Neuroinflammation in Progranulin-Deficient Mice

Science.gov (United States)

Yang, Yue; Aloi, Macarena S.; Cudaback, Eiron; Josephsen, Samuel R.; Rice, Samantha J.; Jorstad, Nikolas L.; Keene, C. Dirk; Montine, Thomas J.

2014-01-01

Frontotemporal dementia (FTD) is a neurodegenerative disease with devastating changes in behavioral performance and social function. Mutations in the progranulin gene (GRN) are one of the most common causes of inherited FTD due to reduced progranulin expression or activity, including in brain where it is expressed primarily by neurons and microglia. Thus, efforts aimed at enhancing progranulin levels might be a promising therapeutic strategy. Bone marrow-derived cells are able to engraft in the brain and adopt a microglial phenotype under myeloablative irradiation conditioning. This ability makes bone marrow (BM)-derived cells a potential cellular vehicle for transferring therapeutic molecules to the central nervous system. Here, we utilized BM cells from Grn+/+ (wild type or wt) mice labeled with green fluorescence protein for delivery of progranulin to progranulin deficient (Grn−/−) mice. Our results showed that wt bone marrow transplantation (BMT) partially reconstituted progranulin in the periphery and in cerebral cortex of Grn−/− mice. We demonstrated a pro-inflammatory effect in vivo and in ex vivo preparations of cerebral cortex of Grn−/− mice that was partially to fully reversed five months after BMT. Our findings suggest that BMT can be administered as a stem cell-based approach to prevent or to treat neurodegenerative diseases. PMID:25199051

Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

Science.gov (United States)

Jakobsen, Oyvind M; Brautaset, Trygve; Degnes, Kristin F; Heggeset, Tonje M B; Balzer, Simone; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

2009-02-01

Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V(max) values (between 47 and 58 micromol/min/mg protein) and K(m) values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC(50)], 0.1 mM) and by l-lysine (IC(50), 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC(50), 4 mM) and by l-lysine (IC(50), 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

Spatiotemporal trends in Canadian domestic wild boar production and habitat predict wild pig distribution