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Sample records for strains expressing stx2b

  1. Identification of E. coli O157:H7 by Using Specific Primers for rfbE and stx2b Genes

    Directory of Open Access Journals (Sweden)

    Mostafa Bakhshi

    2017-07-01

    Sorbitol-MacConkey agar was used to verification of growth ability of selected colonies during PCR. Results: By appearance of the bonds belong to rfbE and stx2B genes on agarose gel, the ability of designed primers in gene detection in samples of E .coli O157:H7 was verified. Colonies which selected during PCR have growth potency on sorbitol-MacConkey agar medium. Conclusion: It was revealed that we can prepare a fast, precise and relative comfortable method for detection of E. coli O157:H7 strain by using PCR technique and specific primers than other available methods.

  2. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression of Stx2B economically was obtained using 1 mM IPTG for 4 h at ...

  3. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    sunny t

    (SDS-PAGE) and StxB2 yield was 450 µg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using HaloTag technology. Key words: Escherichia coli O157:H7, StxB gene, expression, HaloTag technology, purification. INTRODUCTION. Enterohemorrhagic Escherichia coli ...

  4. Dermatophilus congolensis: strain differences in expression of phospholipase activities.

    Science.gov (United States)

    Masters, A M; Ellis, T M; Grein, S B

    1997-09-01

    Interactions between Dermatophilus congolensis strains and with other bacteria of known haemolytic activities were used to elucidate the complex nature of haemolytic activities present in various D. congolensis strains. This was further analysed by measuring their specific phospholipase activities against defined substrates by thin layer chromatography. D. congolensis strains demonstrated haemolytic interactions (synergistic or antagonistic) with other D. congolensis strains and also other species of bacteria. Most isolates expressed lyso-phospholipase-D activity, while various strains also expressed sphingomyelinase-D activity, phospholipase-A versus phosphatidylcholines and/or cephalins, phospholipase-D versus phosphatidylcholines or all these activities, under the culture conditions used.

  5. A recombinant lactobacillus strain expressing genes coding for ...

    African Journals Online (AJOL)

    SERVER

    2007-08-06

    Aug 6, 2007 ... Using genetically engineered endogenous lactobacillus strains colonizing the vagina mucosa to express heterogenous proteins has of late joined the novel strategies aimed at developing a microbicides against HIV. Using the lactobacillus metabolic genome pathway, we found that these bacteria do not ...

  6. GAL4 enhancer trap strains with reporter gene expression during ...

    Indian Academy of Sciences (India)

    c Indian Academy of Sciences. ONLINE RESOURCES. GAL4 enhancer trap strains with reporter gene expression during the development of adult brain in Drosophila melanogaster. C. R. VENKATESH and B. V. SHYAMALA*. Department of Studies in Zoology, University of Mysore, Manasagangotri, Mysore 570 006, India.

  7. A recombinant lactobacillus strain expressing genes coding for ...

    African Journals Online (AJOL)

    Using genetically engineered endogenous lactobacillus strains colonizing the vagina mucosa to express heterogenous proteins has of late joined the novel ... role of restriction modification systems (RMS), we searched for enzymes that cleave HIV-1, 2 and other SIV genomes using theoretical computational methods.

  8. Comparative gene expression profiling of Neospora caninum strains

    Science.gov (United States)

    To understand the genetic basis of virulence, gene expression profiles of a temperature-sensitive strain (NCts-8) and its wild type (NC-1) of Neospora caninum were characterized and compared using a high-density microarray with approximately 63,000 distinct oligonucleotides. Each sequence is represe...

  9. A comparative study of P450 gene expression in field and laboratory Musca domestica L. strains

    DEFF Research Database (Denmark)

    Højland, Dorte Heidi; Vagn Jensen, Karl-Martin; Kristensen, Michael

    2014-01-01

    unselected strains: a multiresistant strain (791a), a neonicotinoid-resistant strain (766b) and a new field strain (845b). RESULTS CYP4G2 was highly expressed throughout the range of strains and proved to be the one of the most interesting expression profiles of all P450s analysed. CYP6G4 was expressed up...... that CYP6G4 is a major insecticide resistance gene involved in neonicotinoid resistance. © 2013 Society of Chemical Industry...

  10. Comparison of expression vectors in Lactobacillus reuteri strains.

    Science.gov (United States)

    Lizier, Michela; Sarra, Pier G; Cauda, Roberto; Lucchini, Franco

    2010-07-01

    The synthesis of heterologous proteins in lactobacilli is strongly influenced by the promoter selected for the expression. In addition, the activity of the promoters themselves may vary among different bacterial hosts. Three different promoters were investigated for their capability to drive enhanced green fluorescent protein (EGFP) expression in Lactococcus lactis spp. cremoris MG1363, in Lactobacillus reuteri DSM 20016(T) and in five L. reuteri strains isolated from chicken crops. The promoters of the Lactobacillus acidophilus surface layer protein gene (slp), L. acidophilus lactate dehydrogenase gene (ldhL) and enterococcal rRNA adenine N-6-methyltransferase gene (ermB) were fused to the coding sequence of EGFP and inserted into the backbone of the pTRKH3 shuttle vector (pTRKH3-slpGFP, pTRKH3-ldhGFP, pTRKH3-ermGFP). Besides conventional analytical methods, a new quick fluorimetric approach was set up to quantify the EGFP fluorescence in transformed clones using the Qubit() fluorometer. ermB proved to be the most effective promoter in L. reuteri isolates, producing 3.90 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Under the same conditions, the ldhL promoter produced 2.66 x 10(-7) g of fluorescent EGFP (mL OD(stationary culture))(-1). Even though the slp promoter was efficient in L. lactis spp. cremoris MG1363, it was nearly inactive both in L. reuteri DSM 20016(T) and in L. reuteri isolates.

  11. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development.

  12. A mechanism for extremely weak SpaP-expression in Streptococcus mutans strain Z1

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    Yutaka Sato

    2011-04-01

    Full Text Available Background: Streptococcus mutans surface-protein antigen (SpaP, PAc, or antigen I/II has been well known to play an important role in initial attachment to tooth surfaces. However, strains with weak SpaP-expression were recently reported to be found in natural populations of S. mutans. The S. mutans gbpC-negative strain Z1, which we previously isolated from saliva and plaque samples, apparently expresses relatively low levels of SpaP protein compared to S. mutans strains MT8148 or UA159. Objective: To elucidate the mechanism for weak SpaP-expression in this strain, the spaP gene region in strain Z1 was amplified by polymerase chain reaction (PCR and analyzed. Methods: Allelic exchange mutants between strains Z1 and UA159 involving the spaP gene region were constructed. The SpaP protein expressed in the mutants was detected with Coomasie Brilliant Blue (CBB-staining and Western blot analysis following SDS-PAGE. Results: The 4689 bp spaP gene coding sequence for Z1 appeared to be intact. In contrast, a 20 bp nucleotide sequence appeared to be deleted from the region immediately upstream from the Z1 spaP gene when compared to the same region in UA159. The 216 bp and 237 bp intergenic fragments upstream from the spaP gene, respectively, from Z1 and UA159 were isolated, modified, and transformed into the other strain by allelic replacement. The resultant UA159-promoter region-mutant exhibited extremely weak SpaP-expression similar to that of strain Z1 and the Z1 complemented mutant expressed Spa protein levels like that of strain UA159. Conclusion: These results suggest that weak SpaP-expression in strain Z1 resulted from a 20 bp-deletion in the spaP gene promoter region.

  13. EXPRESSION PROFILING OF FIVE RAT STRAINS REVEAL TRANSCRIPTIONAL MODES IN THE ANTIGEN PROCESSING PATHWAY

    Science.gov (United States)

    Comparative gene expression profiling of rat strains with genetic predisposition to diverse cardiovascular diseases can help decode the transcriptional program that governs cellular behavior. We hypothesized that co-transcribed, intra-pathway, functionally coherent genes can be r...

  14. Allelic expression changes in Medaka (Oryzias latipes hybrids between inbred strains derived from genetically distant populations.

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    Yasuhiko Murata

    Full Text Available Variations in allele expressions between genetically distant populations are one of the most important factors which affects their morphological and physiological variations. These variations are caused by natural mutations accumulated in their habitats. It has been reported that allelic expression differences in the hybrids of genetically distant populations are different from parental strains. In that case, there is a possibility that allelic expression changes lead to novel phenotypes in hybrids. Based on genomic information of the genetically distant populations, quantification and comparison of allelic expression changes make importance of regulatory sequences (cis-acting factors or upstream regulatory factors (trans-acting modulators for these changes clearer. In this study, we focused on two Medaka inbred strains, Hd-rR and HNI, derived from genetically distant populations and their hybrids. They are highly polymorphic and we can utilize whole-genome information. To analyze allelic expression changes, we established a method to quantify and compare allele-specific expressions of 11 genes between the parental strains and their reciprocal hybrids. In intestines of reciprocal hybrids, allelic expression was either similar or different in comparison with the parental strains. Total expressions in Hd-rR and HNI were tissue-dependent in the case of HPRT1, with high up-regulation of Hd-rR allele expression in liver. The proportion of genes with differential allelic expression in Medaka hybrids seems to be the same as that in other animals, despite the high SNP rate in the genomes of the two inbred strains. It is suggested that each tissue of the strain difference in trans-acting modulators is more important than polymorphisms in cis-regulatory sequences in producing the allelic expression changes in reciprocal hybrids.

  15. Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

    Energy Technology Data Exchange (ETDEWEB)

    Yousaf, Sohail [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria); Afzal, Muhammad [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria); National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad (Pakistan); Reichenauer, Thomas G. [AIT Austrian Institute of Technology GmbH, Environmental Resources and Technologies Unit, A-2444 Seibersdorf (Austria); Brady, Carrie L. [Forestry and Agricultural Biotechnology Institute, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria (South Africa); Sessitsch, Angela, E-mail: angela.sessitsch@ait.ac.at [AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-2444 Seibersdorf (Austria)

    2011-10-15

    The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants. - Highlights: > E. ludwigii strains efficiently colonized plants in a non-sterile soil environment. > E. ludwigii strains efficiently expressed alkane degradation genes in plants. > E. ludwigii efficiently degraded alkane contaminations and promoted plant growth. > E. ludwigii interacted more effectively with Italian ryegrass than with other plants. > Degradation activity varied with plant and microbial genotype as well as with time. - Enterobacter ludwigii strains belonging to the E. cloacae complex are able to efficiently degrade alkanes when associated with plants and to promote plant growth.

  16. Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

    International Nuclear Information System (INIS)

    Yousaf, Sohail; Afzal, Muhammad; Reichenauer, Thomas G.; Brady, Carrie L.; Sessitsch, Angela

    2011-01-01

    The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants. - Highlights: → E. ludwigii strains efficiently colonized plants in a non-sterile soil environment. → E. ludwigii strains efficiently expressed alkane degradation genes in plants. → E. ludwigii efficiently degraded alkane contaminations and promoted plant growth. → E. ludwigii interacted more effectively with Italian ryegrass than with other plants. → Degradation activity varied with plant and microbial genotype as well as with time. - Enterobacter ludwigii strains belonging to the E. cloacae complex are able to efficiently degrade alkanes when associated with plants and to promote plant growth.

  17. Genetic Diversity of Heat-Labile Toxin Expressed by Enterotoxigenic Escherichia coli Strains Isolated from Humans▿

    Science.gov (United States)

    Lasaro, M. A.; Rodrigues, J. F.; Mathias-Santos, C.; Guth, B. E. C.; Balan, A.; Sbrogio-Almeida, M. E.; Ferreira, L. C. S.

    2008-01-01

    The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease. PMID:18223074

  18. Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17.

    Science.gov (United States)

    Yamanaka, Takeshi; Furukawa, Tomoyo; Matsumoto-Mashimo, Chiho; Yamane, Kazuyoshi; Sugimori, Chieko; Nambu, Takayuki; Mori, Naoki; Nishikawa, Hiroyuki; Walker, Clay B; Leung, Kai-Poon; Fukushima, Hisanori

    2009-01-16

    Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays. Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (sigmaE; putative) and polysialic acid transport protein (KpsD), as compared to those of

  19. Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17

    Directory of Open Access Journals (Sweden)

    Mori Naoki

    2009-01-01

    Full Text Available Abstract Background Prevotella intermedia (P. intermedia, a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2 in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays. Results Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (σE; putative and polysialic acid transport

  20. Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

    NARCIS (Netherlands)

    Martinez Linares, Daniel; Geertsma, Eric R.; Poolman, Bert

    2010-01-01

    The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant

  1. Strain and sex based characterization of behavioral expressions in non-induced compulsive-like mice

    Science.gov (United States)

    Mitra, Swarup; Bastos, Cristiane P.; Chesworth, Savanna; Frye, Cheryl; Bult-Ito, Abel

    2016-01-01

    There is currently a lack of understanding how genetic background and sex differences attribute to the heterogeneity of obsessive-compulsive disorder (OCD). An animal model of compulsive-like behaviors has been developed through bidirectional selection of house mice (Mus musculus) for high (big cotton nests; BIG mice) and low levels (small nests; SMALL mice) of nest-building behavior. The BIG male strains have predictive and face validity as a spontaneous animal model of OCD. Here, we evaluated compulsive-, anxiety-, cognitive-, and depression-like behaviors among male and proestrus female replicate strains each of BIG (BIG1, BIG2) and SMALL (SML1, SML2) nest-builders, and randomly-bred Controls (C1, C2). BIG1 and BIG2 males and females had higher nesting scores when compared to SMALL and Control strains. Male BIG1 and BIG2 strains showed more compulsive-like nesting than BIG1 and BIG2 proestrus females, which was not observed among the other strains. Nesting scores were also different between BIG replicate male strains. A similar pattern was observed in the compulsive-like marble burying behavior with BIG strains burying more marbles than SMALL and Control strains. Significant replicate and sex differences were also observed in marble burying among the BIG strains. The open field test revealed replicate effects while the BIG strains showed less anxiety-like behavior in the elevated plus maze test compared to the SMALL strains. For novel object recognition only the Control strains showed replicate and sex differences. In the depression-like forced swim test proestrus females demonstrated less depression-like behavior than males. BIG and SMALL nest-building strains had a higher corticosterone stress response than the Control strains. Together these results indicate a strong interplay of genetic background and sex in influencing expression of behaviors in our compulsive-like mouse model. These results are in congruence with the clinical heterogeneity of OCD. PMID

  2. Expression of a mineral phosphate solubilizing gene from Erwinia herbicola in two rhizobacterial strains.

    Science.gov (United States)

    Rodríguez, H; Gonzalez, T; Selman, G

    2001-11-30

    A genetic construction was carried out using the broad host range vector pKT230 and plasmid pMCG898, which encodes the Erwinia herbicola pyrroloquinoline quinone (PQQ) synthase, a gene involved in mineral phosphate solubilization (mps). The final construction was transformed and expressed in Escherichia coli MC1061, and the recombinant plasmids were transferred to Burkholderia cepacia IS-16 and Pseudomonas sp. PSS recipient cells by conjugation. Clones containing recombinant plasmids produced higher clearing halos in plates with insoluble phosphate as the unique (P) source, in comparison with those of strains without plasmids, demonstrating the heterologous expression of the E. herbicola gene in the recipient strains. This genetic manipulation allowed the increase in mps ability of both strains, enhancing their potentialities as growth promoters of agricultural crops. These results represent the first report on the application of the recombinant DNA methodology for the obtaining of improved phosphate solubilizing ability from rhizobacterial strains for biofertilization purposes.

  3. Heterologous Expression of the Lactococcus lactis Bacteriocin, Nisin, in a Dairy Enterococcus Strain

    Science.gov (United States)

    Li, Haiping; O'Sullivan, Daniel J.

    2002-01-01

    The bacteriocin nisin is produced only by some strains of Lactococcus lactis, and to date production in other lactic acid bacteria has not been achieved. Enterococcus sp. strain N12β is a nisin-immune transconjugant obtained from a nisin-producing donor (L. lactis ATCC 11454) and a dairy recipient (Enterococcus sp. strain S12β), but it does not produce nisin. In this study, using PCR amplification, we confirmed that the whole nisin operon is likely present in Enterococcus sp. strain N12β. Northern hybridization of total RNA from strain N12β with a nisA probe and the results of reverse transcriptase PCR showed the lack of nisA transcription in this strain. However, nisA transcription was partially restored in strain N12β upon growth in the presence of exogenous nisin, and the nisA transcription signal was intensified after an increase in the external nisin level. Furthermore, bioassays showed that active nisin was produced in a dose-dependent fashion by strain N12β following induction by exogenous nisin. These results indicated that expression of the nisin genes in Enterococcus sp. strain N12β depended on autoinduction via signal transduction. However, the amount of external inducing signal required was significantly greater than the amount needed for autoinduction in L. lactis. PMID:12089020

  4. Controlled Autolysis and Enzyme Release in a Recombinant Lactococcal Strain Expressing the Metalloendopeptidase Enterolysin A

    Science.gov (United States)

    Hickey, Rita M.; Ross, R. Paul; Hill, Colin

    2004-01-01

    This study concerns the exploitation of the lytic enzyme enterolysin A (EntL), produced by Enterococcus faecalis strain DPC5280, to elicit the controlled autolysis of starter lactococci. EntL, a cell wall metalloendopeptidase secreted by some E. faecalis strains, can kill a wide range of gram-positive bacteria, including lactococci. The controlled expression of entL, which encodes EntL, was achieved using a nisin-inducible expression system in a lactococcal host. Zymographic analysis of EntL activity demonstrated that active enzyme is produced by the recombinant lactococcal host. Indeed, expression of EntL resulted in almost complete autolysis of the host strain 2 h after induction with nisin. Model cheese experiments using a starter strain in addition to the inducible enterolysin-producing strain showed a 27-fold increase in activity with respect to the release of lactate dehydrogenase in the strain overexpressing EntL, demonstrating the potential of EntL production in large-scale cheese production systems. Indeed, the observation that a wide range of lactic bacteria are sensitive to EntL suggests that EntL-induced autolysis has potential applications with a variety of lactic acid bacteria and could be a basis for probiotic delivery systems. PMID:15006800

  5. Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Growth yields and morphological characterization

    DEFF Research Database (Denmark)

    Robin, Jarno Jacky Christian; Lettier, G.; Mcintyre, Mhairi

    2003-01-01

    The growth stoichiometry of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus was determined in glucose-limited chemostat cultivations using a chemically defined medium. This strain produces adipoyl-7-aminocleacetoxycephalosporanic acid (ad-7-ADCA) when...

  6. Temporal Profile of Biofilm Formation, Gene Expression and Virulence Analysis in Candida albicans Strains.

    Science.gov (United States)

    de Barros, Patrícia Pimentel; Rossoni, Rodnei Dennis; De Camargo Ribeiro, Felipe; Junqueira, Juliana Campos; Jorge, Antonio Olavo Cardoso

    2017-04-01

    The characterization of Candida albicans strains with different degrees of virulence became very useful to understand the mechanisms of fungal virulence. Then, the objective of this study was to assess and compare the temporal profiles of biofilms formation, gene expression of ALS1, ALS3, HWP1, BCR1, EFG1, TEC1, SAP5, PLB2 and LIP9 and virulence in Galleria mellonella of C. albicans ATCC18804 and a clinical sample isolated from an HIV-positive patient (CA60). Although the CFU/mL counting was higher in biofilms formed in vitro by ATCC strain, the temporal profile of the analysis of the transcripts of the C. albicans strains was elevated to Ca60 compared to strain ATCC, especially in the genes HWP1, ALS3, SAP5, PLB2 and LIP9 (up regulation). Ca60 was more pathogenic for G. mellonella in the survival assay (p = 0.0394) and hemocytes density (p = 0.0349), agreeing with upregulated genes that encode the expression of hyphae and hydrolase genes of Ca60. In conclusion, the C. albicans strains used in this study differ in the amount of biofilm formation, virulence in vivo and transcriptional profiles of genes analyzed that can change factors associated with colonization, proliferation and survival of C. albicans at different niches. SAP5 and HWP1 were the genes more expressed in the formation of biofilm in vitro.

  7. A New Strain Collection for Improved Expression of Outer Membrane Proteins

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    Ina Meuskens

    2017-11-01

    Full Text Available Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3 designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB, both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

  8. Expression of gamma-hemolysin regulated by sae in Staphylococcus aureus strain Smith 5R.

    Science.gov (United States)

    Yamazaki, Kazuko; Kato, Fuminori; Kamio, Yoshiyuki; Kaneko, Jun

    2006-06-01

    Staphylococcus aureus strain Smith 5R produces a two-component pore-forming toxin and forms a rough-surfaced colony with hemolytic haloes on human red blood cell plates (R[+]). Serial subcultures of the strain in broth caused the appearance of gamma-hemolysin negative variants with a smooth colony shape (S[-]), and the S[-] valiant became predominant in culture. The R[+] strain, in which agrA is naturally disrupted by an insertion of IS1181, produced high levels of gamma-hemolysin. In the S[-] variant, expression of both hlg and lukS-F mRNAs was strongly reduced. Nucleotide sequencing of the sae locus revealed that all isolated S[-] variants had spontaneous mutations in the sae locus. Recovery of gamma-hemolysin productivity in S[-] by transformation of the wild-type sae allele strongly suggested that the expression of gamma-hemolysin is positively regulated by sae in an agr-independent manner.

  9. Alveolar macrophages infected with Ames or Sterne strain of Bacillus anthracis elicit differential molecular expression patterns.

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    Felicia D Langel

    Full Text Available Alveolar macrophages (AMs phagocytose Bacillus anthracis following inhalation and induce the production of pro-inflammatory cytokines and chemokines to mediate the activation of innate immunity. Ames, the virulent strain of B. anthracis, contains two plasmids that encode the antiphagocytic poly-γ-d-glutamic acid capsule and the lethal toxin. The attenuated Sterne strain of B. anthracis, which lacks the plasmid encoding capsule, is widely adapted as a vaccine strain. Although differences in the outcome of infection with the two strains may have originated from the presence or absence of an anti-phagocytic capsule, the disease pathogenesis following infection will be manifested via the host responses, which is not well understood. To gain understanding of the host responses at cellular level, a microarray analysis was performed using primary rhesus macaque AMs infected with either Ames or Sterne spores. Notably, 528 human orthologs were identified to be differentially expressed in AMs infected with either strain of the B. anthracis. Meta-analyses revealed genes differentially expressed in response to B. anthracis infection were also induced upon infections with multiple pathogens such as Francisella Novicida or Staphylococcus aureus. This suggests the existence of a common molecular signature in response to pathogen infections. Importantly, the microarray and protein expression data for certain cytokines, chemokines and host factors provide further insights on how cellular processes such as innate immune sensing pathways, anti-apoptosis versus apoptosis may be differentially modulated in response to the virulent or vaccine strain of B. anthracis. The reported differences may account for the marked difference in pathogenicity between these two strains.

  10. Comparative gene expression profiling in two congenic mouse strains following Bordetella pertussis infection

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    Demant Peter

    2007-10-01

    Full Text Available Abstract Background Susceptibility to Bordetella pertussis infection varies widely. These differences can partly be explained by genetic host factors. HcB-28 mice are more resistant to B. pertussis infection than C3H mice, which could partially be ascribed to the B. pertussis susceptibility locus-1 (Bps1 on chromosome 12. The presence of C57BL/10 genome on this locus instead of C3H genome resulted in a decreased number of bacteria in the lung. To further elucidate the role of host genetic factors, in particular in the Bps1 locus, in B. pertussis infection, and to identify candidate genes within in this region, we compared expression profiles in the lungs of the C3H and HcB-28 mouse strains following B. pertussis inoculation. Twelve and a half percent of the genomes of these mice are from a different genetic background. Results Upon B. pertussis inoculation 2,353 genes were differentially expressed in the lungs of both mouse strains. Two hundred and six genes were differentially expressed between the two mouse strains, but, remarkably, none of these were up- or down-regulated upon B. pertussis infection. Of these 206 genes, 17 were located in the Bps1 region. Eight of these genes, which showed a strong difference in gene expression between the two mouse strains, map to the immunoglobulin heavy chain complex (Igh. Conclusion Gene expression changes upon B. pertussis infection are highly identical between the two mouse strains despite the differences in the course of B. pertussis infection. Because the genes that were differentially regulated between the mouse strains only showed differences in expression before infection, it appears likely that such intrinsic differences in gene regulation are involved in determining differences in susceptibility to B. pertussis infection. Alternatively, such genetic differences in susceptibility may be explained by genes that are not differentially regulated between these two mouse strains. Genes in the Igh

  11. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

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    Lobstein Julie

    2012-05-01

    Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

  12. Endometrial gene expression during early pregnancy differs between fertile and subfertile dairy cow strains.

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    Walker, Caroline G; Littlejohn, Mathew D; Mitchell, Murray D; Roche, John R; Meier, Susanne

    2012-01-18

    A receptive uterine environment is a key component in determining a successful reproductive outcome. We tested the hypothesis that endometrial gene expression patterns differ in fertile and subfertile dairy cow strains. Twelve lactating dairy cattle of strains characterized as having fertile (n = 6) and subfertile (n = 6) phenotypes underwent embryo transfer on day 7 of the reproductive cycle. Caruncular and intercaruncular endometrial tissue was obtained at day 17 of pregnancy, and microarrays used to characterize transcriptional profiles. Statistical analysis of microarray data at day 17 of pregnancy revealed 482 and 1,021 differentially expressed transcripts (P value cow strains in intercaruncular and caruncular tissue, respectively. Functional analysis revealed enrichment for several pathways involved in key reproductive processes, including the immune response to pregnancy, luteolysis, and support of embryo growth and development, and in particular, regulation of histotroph composition. Genes implicated in the process of immune tolerance to the embryo were downregulated in subfertile cows, as were genes involved in preventing luteolysis and genes that promote embryo growth and development. This study provides strong evidence that the endometrial gene expression profile may contribute to the inferior reproductive performance of the subfertile dairy cow strain.

  13. Control of apple blue mold by Pichia pastoris recombinant strains expressing cecropin A.

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    Ren, Xueyan; Kong, Qingjun; Wang, Huili; Yu, Ting; Tang, Ya-Jie; Zhou, Wen-Wen; Zheng, Xiaodong

    2012-06-01

    Recombinant Pichia pastoris yeasts expressing cecropin A (GS115/CEC), was evaluated for the control of the blue mold of apple caused by Penicillium expansum due to cecropin A peptide's effective antimicrobial effects on P. expansum spores by the thiazolyl blue (MTT) assay. Then, the protein concentration was determined and it was expressed at high levels up to 14.2 mg/L in the culture medium. Meanwhile, the population growth was assayed in vivo. The population growth of recombinant strain GS115/CEC was higher than that of non-transformed strain GS115 in red Fuji apples wounds. Recombinant yeast strains GS115/CEC significantly inhibited growth of germinated P. expansum spores in vitro and inhibited decay development caused by P. expansum in apple fruits in vivo when compared with apple fruits inoculated with sterile water or the yeast strain GS115/pPIC (plasmid pPIC9k transformed in GS115). This study demonstrated the potential of expression of the antifungal peptide in yeast for the control of postharvest blue mold infections on pome fruits.

  14. Construction of two Listeria ivanovii attenuated strains expressing Mycobacterium tuberculosis antigens for TB vaccine purposes.

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    Lin, Qingqing; Zhou, Mengying; Xu, Zongkai; Khanniche, Asma; Shen, Hao; Wang, Chuan

    2015-02-20

    Bacillus Calmette-Guerin (BCG) has failed in complete control of tuberculosis (TB), thus, novel tuberculosis vaccines are urgently needed. We have constructed several TB vaccine candidates, which are characterized by the use of Listeria ivanovii (LI) strain as an antigen delivery vector. Two L. ivanovii attenuated recombinant strains L. ivanovii△actAplcB-Rv0129c and L. ivanovii△actAplcB-Rv3875 were successfully screened. Results from genome PCR and sequencing showed that the Mycobacterium tuberculosis antigen gene cassette coding for Ag85C or ESAT-6 protein respectively had been integrated into LI genome downstream of mpl gene. Western blot confirmed the secretion of Ag85C or ESAT-6 protein from the recombinant LI strains. These two recombinant strains showed similar growth curves as wide type strain in vitro. In vivo, they transiently propagated in mice spleen and liver, and induced specific CD8(+) IFN-γ secretion. Therefore, in this paper, two novel LI attenuated strains expressing specific TB antigens were successfully constructed. The promising growth characteristics in mice immune system and the capability of induction of IFN-γ secretion make them of potential interest for development of TB vaccines. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Comparative gene expression in toxic versus non-toxic strains of the marine dinoflagellate Alexandrium minutum

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    Glöckner Gernot

    2010-04-01

    Full Text Available Abstract Background The dinoflagellate Alexandrium minutum typically produces paralytic shellfish poisoning (PSP toxins, which are known only from cyanobacteria and dinoflagellates. While a PSP toxin gene cluster has recently been characterized in cyanobacteria, the genetic background of PSP toxin production in dinoflagellates remains elusive. Results We constructed and analysed an expressed sequence tag (EST library of A. minutum, which contained 15,703 read sequences yielding a total of 4,320 unique expressed clusters. Of these clusters, 72% combined the forward-and reverse reads of at least one bacterial clone. This sequence resource was then used to construct an oligonucleotide microarray. We analysed the expression of all clusters in three different strains. While the cyanobacterial PSP toxin genes were not found among the A. minutum sequences, 192 genes were differentially expressed between toxic and non-toxic strains. Conclusions Based on this study and on the lack of identified PSP synthesis genes in the two existent Alexandrium tamarense EST libraries, we propose that the PSP toxin genes in dinoflagellates might be more different from their cyanobacterial counterparts than would be expected in the case of a recent gene transfer. As a starting point to identify possible PSP toxin-associated genes in dinoflagellates without relying on a priori sequence information, the sequences only present in mRNA pools of the toxic strain can be seen as putative candidates involved in toxin synthesis and regulation, or acclimation to intracellular PSP toxins.

  16. Expression of curli by Escherichia coli O157:H7 strains isolated from patients during outbreaks is different from similar strains isolated from leafy green production environments

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    Subbarao Venkata Ravva

    2016-12-01

    Full Text Available We previously reported that the strains of Escherichia coli O157:H7 (EcO157 that survived longer in austere soil environment lacked expression of curli, a fitness trait linked with intestinal colonization. In addition, the proportion of curli-positive variants of EcO157 decreased with repeated soil exposure. Here we evaluated 84 and 176 clinical strains from outbreaks and sporadic infections in the US, plus 211 animal fecal and environmental strains for curli expression. These shiga-toxigenic strains were from 328 different genotypes, as characterized by multi-locus variable-number tandem-repeat analysis (MLVA. More than half of the fecal strains (human and animal and a significant proportion of environmental isolates (82% were found to lack curli expression. EcO157 strains from several outbreaks linked with the consumption of contaminated apple juice, produce, hamburgers, steak and beef were also found to lack curli expression. Phylogenetic analysis of fecal strains indicates curli expression is distributed throughout the population. However, a significant proportion of animal fecal isolates (84% gave no curli expression compared to human fecal isolates (58%. In addition, analysis of environmental isolates indicated nearly exclusive clustering of curli expression to a single branch of the minimal spanning tree. This indicates that curli expression depends primarily upon the type of environmental exposure and the isolation source, although genotypic differences also contribute to clonal variation in curli. Furthermore, curli-deficient phenotype appears to be a selective trait for survival of EcO157 in agricultural environments.

  17. Biological characterization of a b-galactosidase expressing clone of Trypanosoma cruzi CL strain

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    Le-Senne Ana

    2002-01-01

    Full Text Available Clone CL B5 of Trypanosoma cruzi is a beta-galactosidase expressing organism that was genetically transfected to be used for in vitro pharmacological screening. Biological parameters were determined, evaluating growth kinetics of epimastigotes, metacyclogenesis, infectivity to mammalian cell lines, parasitemia kinetics in mice and sensibility to nifurtimox and benznidazole. Differences in relation to other strains and CL parental strain were found, the most important being the incapability to produce death to mice in spite of the high inoculum used. However, it possesses the required features to be used for in vitro drug screening. Data obtained demonstrate that heterogeneity of T. cruzi appears even among clones of the same strain, and that these differences found do not prevent the use of clone CL B5 for the purpose that was engineered.

  18. Relationship between oviposition, virulence gene expression and parasitism success in Cotesia typhae nov. sp. parasitoid strains.

    Science.gov (United States)

    Benoist, R; Chantre, C; Capdevielle-Dulac, C; Bodet, M; Mougel, F; Calatayud, P A; Dupas, S; Huguet, E; Jeannette, R; Obonyo, J; Odorico, C; Silvain, J F; Le Ru, B; Kaiser, L

    2017-12-01

    Studying mechanisms that drive host adaptation in parasitoids is crucial for the efficient use of parasitoids in biocontrol programs. Cotesia typhae nov. sp. (Fernández-Triana) (Hymenoptera: Braconidae) is a newly described parasitoid of the Mediterranean corn borer Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae). Braconidae are known for their domesticated bracovirus, which is injected with eggs in the host larva to overcome its resistance. In this context, we compared reproductive success traits of four Kenyan strains of C. typhae on a French and a Kenyan populations of its host. Differences were found between the four strains and the two most contrasted ones were studied more thoroughly on the French host population. Parasitoid offspring size was correlated with parasitism success and the expression of bracovirus virulence genes (CrV1 and Cystatin) in the host larva after parasitism. Hybrids between these two parasitoid strains showed phenotype and gene expression profiles similar to the most successful parental strain, suggesting the involvement of dominant alleles in the reproductive traits. Ovary dissections revealed that the most successful strain injected more eggs in a single host larva than the less successful one, despite an equal initial ovocyte number in ovaries. It can be expected that the amount of viral particles increase with the number of eggs injected. The ability to bypass the resistance of the allopatric host may in consequence be related to the oviposition behaviour (eggs allocation). The influence of the number of injected eggs on parasitism success and on virulence gene expression was evaluated by oviposition interruption experiments.

  19. Gene expression profiling in the striatum of inbred mouse strains with distinct opioid-related phenotypes

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    Piechota Marcin

    2006-06-01

    Full Text Available Abstract Background Mouse strains with a contrasting response to morphine provide a unique model for studying the genetically determined diversity of sensitivity to opioid reward, tolerance and dependence. Four inbred strains selected for this study exhibit the most distinct opioid-related phenotypes. C57BL/6J and DBA/2J mice show remarkable differences in morphine-induced antinociception, self-administration and locomotor activity. 129P3/J mice display low morphine tolerance and dependence in contrast to high sensitivity to precipitated withdrawal observed in SWR/J and C57BL/6J strains. In this study, we attempted to investigate the relationships between genetic background and basal gene expression profile in the striatum, a brain region involved in the mechanism of opioid action. Results Gene expression was studied by Affymetrix Mouse Genome 430v2.0 arrays with probes for over 39.000 transcripts. Analysis of variance with the control for false discovery rate (q Khdrbs1 and ATPase Na+/K+ alpha2 subunit (Atp1a2 with morphine self-administration and analgesic effects, respectively. Finally, the examination of transcript structure demonstrated a possible inter-strain variability of expressed mRNA forms as for example the catechol-O-methyltransferase (Comt gene. Conclusion The presented study led to the recognition of differences in the gene expression that may account for distinct phenotypes. Moreover, results indicate strong contribution of genetic background to differences in gene transcription in the mouse striatum. The genes identified in this work constitute promising candidates for further animal studies and for translational genetic studies in the field of addictive and analgesic properties of opioids.

  20. Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

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    Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

    1998-01-01

    A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

  1. In planta gene expression analysis of Xanthomonas oryzae pathovar oryzae, African strain MAI1

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    Verdier Valérie

    2010-06-01

    Full Text Available Abstract Background Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo, induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian X. oryzae pv. oryzicola (Xoc. Results Changes in gene expression of the African Xoo strain MAI1 in the susceptible rice cultivar Nipponbare were profiled, using an SSH Xoo DNA microarray. Microarray hybridization was performed comparing bacteria recovered from plant tissues at 1, 3, and 6 days after inoculation (dai with bacteria grown in vitro. A total of 710 bacterial genes were found to be differentially expressed, with 407 up-regulated and 303 down-regulated. Expression profiling indicated that less than 20% of the 710 bacterial transcripts were induced in the first 24 h after inoculation, whereas 63% were differentially expressed at 6 dai. The 710 differentially expressed genes were one-end sequenced. 535 sequences were obtained from which 147 non-redundant sequences were identified. Differentially expressed genes were related to metabolism, secretion and transport, pathogen adherence to plant tissues, plant cell-wall degradation, IS elements, and virulence. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. The Xoo MAI1 non-redundant set of sequences was compared against several X. oryzae genomes, revealing a specific group of genes that was present only in MAI1. Numerous IS elements were also found to be differentially expressed. Quantitative real-time PCR confirmed 86% of the identified profile on a set of 14 genes selected according to the microarray analysis. Conclusions This is the first report to compare the expression of Xoo genes in planta across different time points during infection. This work shows that

  2. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.

    Science.gov (United States)

    Krainer, Florian W; Dietzsch, Christian; Hajek, Tanja; Herwig, Christoph; Spadiut, Oliver; Glieder, Anton

    2012-02-13

    ΒACKGROUND: The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Co-overexpressing enzymes of the

  3. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

    Science.gov (United States)

    2012-01-01

    Βackground The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. Results A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Conclusions Co

  4. Two novel EHEC/EAEC hybrid strains isolated from human infections.

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    Rita Prager

    Full Text Available The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC. Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC. After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H-, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF. The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H-, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.

  5. Laccase Gene Expression and Vinasse Biodegradation by Trametes hirsuta Strain Bm-2

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    Raúl Tapia-Tussell

    2015-08-01

    Full Text Available Vinasse is the dark-colored wastewater that is generated by bioethanol distilleries from feedstock molasses. The vinasse that is generated from molasses contains high amounts of pollutants, including phenolic compounds and melanoindin. The goal of this work was to study the expression of laccase genes in the Trametes hirsuta strain Bm-2, isolated in Yucatan, Mexico, in the presence of phenolic compounds, as well as its effectiveness in removing colorants from vinasse. In the presence of all phenolic compounds tested (guaiacol, ferulic acid, and vanillic acid, increased levels of laccase-encoding mRNA were observed. Transcript levels in the presence of guaiacol were 40 times higher than those in the control. The lcc1 and lcc2 genes of T. hirsuta were differentially expressed; guaiacol and vanillin induced the expression of both genes, whereas ferulic acid only induced the expression of lcc2. The discoloration of vinasse was concomitant with the increase in laccase activity. The highest value of enzyme activity (2543.7 U/mL was obtained in 10% (v/v vinasse, which corresponded to a 69.2% increase in discoloration. This study demonstrates the potential of the Bm-2 strain of T. hirsuta for the biodegradation of vinasse.

  6. The temperature--dependent expression of GST of Schistosoma japonicum (Philippine strain).

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    Cai, Z H; Song, G C; Xu, Y X; Liu, S X

    1993-03-01

    Obtained from pSj5, the cDNA gene encoding GST antigen of Schistosoma japonicum (Philippine strain) was ligated with efficient temperature-dependent PBV220 vector which was constructed in CAPM, and then introduced into host bacterium-DH5 alpha (E. coli) by transformation. Transformants were selected by ampicillin and recombinant clones were identified by restriction mapping. The result showed that recombinant clone 43 was the one carrying recombinant plasmid PBV 220 with the correct insertion of the gene fragment. The GST expression ability of clone 43 was investigated by GST enzymic activity assay and SDS-PAGE. A relatively high level of GST enzymic activity was expressed by this clone under the temperature-dependent condition, that is, cultured at 30 degrees C and expressed at 42 degrees C. A more strongly stained 26 kDa protein band was identified by SDS-PAGE. The result indicated that GST of S. japonicum (Philippine strain) could be expressed not only by IPTG induction, but also by the temperature-dependent method.

  7. Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

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    Horiuchi, Rie [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Akimoto, Takayuki, E-mail: akimoto@m.u-tokyo.ac.jp [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Hong, Zhang [Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Ushida, Takashi [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan)

    2012-08-15

    Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: Black-Right-Pointing-Pointer The expression of Nanog, which is an essential regulator of 'stemness' was reduced during embryonic stem (ES) cell differentiation. Black-Right-Pointing-Pointer Cyclic mechanical strain attenuated the reduction of Nanog expression. Black-Right-Pointing-Pointer Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.

  8. Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus

    Science.gov (United States)

    Ocampo, Clara B.; Caicedo, Paola A.; Jaramillo, Gloria; Ursic Bedoya, Raul; Baron, Olga; Serrato, Idalba M.; Cooper, Dawn M.; Lowenberger, Carl

    2013-01-01

    Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain. PMID:23593426

  9. Differential expression of apoptosis related genes in selected strains of Aedes aegypti with different susceptibilities to dengue virus.

    Directory of Open Access Journals (Sweden)

    Clara B Ocampo

    Full Text Available Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2 and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1 and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain.

  10. Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

    Science.gov (United States)

    Kuyper, Marko; Hartog, Miranda M P; Toirkens, Maurice J; Almering, Marinka J H; Winkler, Aaron A; van Dijken, Johannes P; Pronk, Jack T

    2005-02-01

    After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

  11. [Construction and Fluorescence Analysis of the RecombinantListeria ivanoviiStrain Expressing Green Fluorescent Protein].

    Science.gov (United States)

    Zhang, Xiang; Su, Lin; Liu, Si-Jing; Li, Yong-Yu; Jiang, Ming-Juan; Huang, Huan; Wang, Chuan

    2017-11-01

    Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. The promoter of Listeria monocytogenes Listeriolysin O ( phly ) and the green fluorescent protein (GFP) gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW- phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii ,and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii ,bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW- phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. The exactness of recombinant plasmid pCW- phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW- phly-GFP. The recombinant plasmid pCW- phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. The prokaryotic expression plasmid pCW- phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.

  12. Strain-dependent variations in visceral sensitivity: relationship to stress, anxiety and spinal glutamate transporter expression.

    Science.gov (United States)

    Moloney, R D; Dinan, T G; Cryan, J F

    2015-04-01

    Responses to painful stimuli differ between populations, ethnic groups, sexes and even among individuals of a family. However, data regarding visceral pain are still lacking. Thus, we investigated differences in visceral nociception across inbred and outbred mouse strains using colorectal distension. Anxiety and depression-like behaviour were assessed using the open field and forced swim test as well as the corticosterone stress response. Possible mechanistic targets [excitatory amino acid transporter (EAAT-1), brain-derived neurotrophic factor (BDNF) and 5HT1A receptor] were also assessed using quantitative real-time polymerase chain reaction. Adult, male, inbred and outbred mouse strains were used in all assays (inbred strains; CBA/J Hsd, C3H/HeNHsd, BALB/c OlaHsd, C57 BL/6JOlaHsd, DBA/2J RccHsd, CAST/EiJ, SM/J, A/J OlaHsd, 129P2/OlaHsd, FVB/NHan Hsd and outbred strains: Swiss Webster, CD-1). mRNA expression levels of EAAT-1, BDNF and 5HT1A receptor (HTR1A) were quantified in the lumbosacral spinal cord, amygdala and hippocampus. A significant effect of strain was found in visceral sensitivity, anxiety and depressive-like behaviours. Strain differences were also seen in both baseline and stress-induced corticosterone levels. CBA/J mice consistently exhibited heightened visceral sensitivity, anxiety behaviour and depression-like behaviour which were associated with decreased spinal EAAT-1 and hippocampal BDNF and HTR1A. Our results show the CBA/J mouse strain as a novel mouse model to unravel the complex mechanisms of brain-gut axis disorders such as irritable bowel syndrome, in particular the underlying mechanisms of visceral hypersensitivity, for which there is great need. Furthermore, this study highlights the importance of genotype and the consequences for future development of transgenic strains in pain research. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  13. Genome-wide analysis of peptidase content and expression in a virulent and attenuated Babesia bovis strain pair.

    Science.gov (United States)

    Mesplet, Maria; Palmer, Guy H; Pedroni, Monica J; Echaide, Ignacio; Florin-Christensen, Monica; Schnittger, Leonhard; Lau, Audrey O T

    2011-10-01

    Identifying virulence determinants in Apicomplexan parasites remains a major gap in knowledge for members within this phylum. We hypothesized that peptidases would segregate with virulence between a virulent parent Babesia bovis strain and an attenuated daughter strain derived by rapid in vivo passage. Using the complete genome sequence of the virulent T2Bo strain, 66 peptidases were identified and active sites confirmed. The presence, sequence identity and expression levels were tested for each of the 66 peptidases in the virulent parent and attenuated daughter T2Bo strains using whole genome, targeted sequencing approaches and microarrays analyses. Quantitative PCR revealed that there was no significant difference in peptidase expression between the virulent and attenuated strains. We conclude that while peptidases may well play a required role in B. bovis pathogenesis, neither loss of peptidase gene content nor reduced gene expression underlies the loss of virulence associated with in vivo passage and attenuation. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Analysis of differentially expressed genes related to resistance in spinosad- and neonicotinoid-resistant Musca domestica L. (Diptera: Muscidae) strains

    DEFF Research Database (Denmark)

    Castberg, Dorte Heidi Højland; Kristensen, Michael

    2017-01-01

    interesting in terms of neonicotinoid resistance, while cyp4d9 was overexpressed in 791spin compared to spinosad-susceptible strains. GSTs, ESTs and UGTs were mostly overexpressed, but not to the same degree as P450s. We present a comprehensive and comparative picture of gene expression in three housefly......Background The housefly is a global pest that has developed resistance to most insecticides applied against it. Resistance of the spinosad-resistant strain 791spin and the neonicotinoid-resistant 766b strain is believed to be due to metabolism. We investigate differentially expressed genes...... strains differing significantly in their response to insecticides. High differential expression of P450s and genes coding for cuticle protein indicates a combination of factors involved in metabolic neonicotinoid and spinosad resistance. Conclusion Resistance in these strains is apparently not linked...

  15. Differential protein expression in Corbicula fluminea upon exposure to a Microcystis aeruginosa toxic strain.

    Science.gov (United States)

    Martins, José C; Leão, Pedro N; Vasconcelos, Vítor

    2009-03-15

    Changes in protein expression induced by a Microcystis aeruginosa toxic strain in the freshwater clam Corbicula fluminea were studied using a proteomic approach in an effort to identify new molecular biomarkers. Clams were fed with 1 x 10(6) cells mL(-1) of a M. aeruginosa toxic strain (IZANCYA 2), during 24 b. Cytosolic fractions of gills and digestive tract were analyzed by two-dimensional (2D) electrophoresis in 7 cm IPG strips (pH 4-7). On average, about 400 spots were resolved using Coomassie staining. Altered protein expression was quantitatively detected in 16-13 spots in gills and digestive tract, respectively. In 2D electrophoresis gel protein maps from gills, 10 of 16 spots were downregulated. In the digestive tract, the general tendency was an increase in the protein expression level after the exposure. The altered protein spots were excised and analyzed by MALDI-TOF-MS, with identification of 8 proteins in gills and 5 in the digestive tract. Most of the identified proteins are involved in cytoskeleton assembly. Metabolic proteins were also detected. These results are in agreement with predicted effects of PP1 and PP2A phosphatase inhibition as major effect of microcystins-related toxicity.

  16. Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity.

    Science.gov (United States)

    Crawford, L; Stepan, A M; McAda, P C; Rambosek, J A; Conder, M J; Vinci, V A; Reeves, C D

    1995-01-01

    We demonstrate a novel and efficient bioprocess for production of the cephalosporin intermediates, 7-aminocephalosporanic acid (7-ACA) or 7-amino deacetoxycephalosporanic acid (7-ADCA). The Streptomyces clavuligerus expandase gene or the Cephalosporium acremonium expandase-hydroxylase gene, with and without the acetyltransferase gene, were expressed in a penicillin production strain of Penicillium chrysogenum. Growth of these transformants in media containing adipic acid as the side chain precursor resulted in efficient production of cephalosporins having an adipyl side chain, proving that adipyl-6-APA is a substrate for either enzyme in vivo. Strains expressing expandase produced adipyl-7-ADCA, whereas strains expressing expandase-hydroxylase produced both adipyl-7-ADCA and adipyl-7-ADAC (aminodeacetylcephalosporanic acid). Strains expressing expandase-hydroxylase and acetyltransferase produced adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. The adipyl side chain of these cephalosporins was easily removed with a Pseudomonas-derived amidase to yield the cephalosporin intermediates.

  17. Expression information data table (Strain List) of Drosophila GAL4 enhancer trap lines - GETDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us GETDB Expression information data table (Strain List) of Drosophila GAL4 enhancer trap lines... Data detail Data name Expression information data table (Strain List) of Drosophila GAL4 enhancer trap line...his Database Site Policy | Contact Us Expression information data table (Strain List) of Drosophila GAL4 enhancer trap lines - GETDB | LSDB Archive ...

  18. Cloning and Expression of the Benzoate Dioxygenase Genes from Rhodococcus sp. Strain 19070

    Science.gov (United States)

    Haddad, Sandra; Eby, D. Matthew; Neidle, Ellen L.

    2001-01-01

    The bopXYZ genes from the gram-positive bacterium Rhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic cis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in Rhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both meta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl and ben genes, respectively. Open reading frames downstream of bopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences. PMID:11375157

  19. Avoidance Expression in Rats as a Function of Signal-Shock Interval: Strain and Sex Differences

    Directory of Open Access Journals (Sweden)

    Richard J Servatius

    2015-07-01

    Full Text Available Inbred Wistar Kyoto (WKY rats express inhibited temperament, increased sensitivity to stress, and exaggerated expressions of avoidance. A long-standing observation for lever press escape/avoidance learning in rats is the duration of the warning signal (WS determines whether avoidance is expressed over escape. Outbred female Sprague-Dawley (SD rats trained with a 10-s WS efficiently escaped, but failed to exhibit avoidance; avoidance was exhibited to a high degree with WSs longer than 20-s. We examined this longstanding WS duration function and extended it to male SD and male and female WKY rats. A cross-over design with two WS durations (10 s or 60 s was employed. Rats were trained (20 trials/session in four phases: acquisition (10 sessions, extinction (10 sessions, re-acquisition (8 sessions and re-extinction (8 sessions. Consistent with the literature, female and male SD rats failed to express avoidance to an appreciable degree with a 10-s WS. When these rats were switched to a 60-s WS, performance levels in the initial session of training resembled the peak performance of rats trained with a 60-s WS. Therefore, the avoidance relationship was acquired, but not expressed at 10-s WS. Further, poor avoidance at 10-s does not adversely affect expression at 60-s. Failure to express avoidance with a 10-s WS likely reflects contrasting reinforcement value of avoidance, not a reduction in the amount of time available to respond or competing responses. In contrast, WKY rats exhibited robust avoidance with a 10-s WS, which was most apparent in female WKY rats. Exaggerated expression of avoidances by WKY rats, especially female rats, further confirms this inbred strain as a model of anxiety vulnerability.

  20. Cloning and Expression of Recombinant Plasmid Containing P36/LACK Gene of Leishmania infantum Iranian Strain.

    Directory of Open Access Journals (Sweden)

    Saloomeh Shirali

    2015-06-01

    Full Text Available There are several methods, such as vaccination, to control visceral leishmaniasis. Although there is no efficient vaccine, it seem DNA vaccination with stimulates both cellular and humoral immunity apparently is the best way. The aim of this study was cloning and expression of LACK gene, a 36kD protein, as a candidate protein for vaccination against Iranian L. infantum.Iranian strain of L. infantum [MCAN/IR/07/Moheb-gh] was used as a template for PCR to amplify LACK gene. The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1 and cloned in pcDNA3.1 expression vector. Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction. The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α and its expression was analyzed by SDS-PAGE and Western blot.The results of sequencing, restriction digestion analysis and PCR reaction revealed that LACK gene was cloned correctly in pcDNA3.1 vector and the results of SDS PAGE and Western blot emphasized that LACK protein of Iranian L. infantum is a well-expressed protein.We amplified, cloned and expressed Iranian L. infantum LACK gene successfully.

  1. Cloning and Expression of Recombinant Plasmid Containing P36/LACK Gene of Leishmania infantum Iranian Strain.

    Science.gov (United States)

    Shirali, Saloomeh; Haddadzadeh, Hamidreza; Mohebali, Mehdi; Kazemi, Bahram; Amini, Narges

    2015-01-01

    There are several methods, such as vaccination, to control visceral leishmaniasis. Although there is no efficient vaccine, it seem DNA vaccination with stimulates both cellular and humoral immunity apparently is the best way. The aim of this study was cloning and expression of LACK gene, a 36kD protein, as a candidate protein for vaccination against Iranian L. infantum. Iranian strain of L. infantum [MCAN/IR/07/Moheb-gh] was used as a template for PCR to amplify LACK gene. The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector. Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction. The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot. The results of sequencing, restriction digestion analysis and PCR reaction revealed that LACK gene was cloned correctly in pcDNA3.1 vector and the results of SDS PAGE and Western blot emphasized that LACK protein of Iranian L. infantum is a well-expressed protein. We amplified, cloned and expressed Iranian L. infantum LACK gene successfully.

  2. Synthetic biology toolbox for controlling gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002.

    Science.gov (United States)

    Markley, Andrew L; Begemann, Matthew B; Clarke, Ryan E; Gordon, Gina C; Pfleger, Brian F

    2015-05-15

    The application of synthetic biology requires characterized tools to precisely control gene expression. This toolbox of genetic parts previously did not exist for the industrially promising cyanobacterium, Synechococcus sp. strain PCC 7002. To address this gap, two orthogonal constitutive promoter libraries, one based on a cyanobacterial promoter and the other ported from Escherichia coli, were built and tested in PCC 7002. The libraries demonstrated 3 and 2.5 log dynamic ranges, respectively, but correlated poorly with E. coli expression levels. These promoter libraries were then combined to create and optimize a series of IPTG inducible cassettes. The resultant induction system had a 48-fold dynamic range and was shown to out-perform Ptrc constructs. Finally, a RBS library was designed and tested in PCC 7002. The presented synthetic biology toolbox will enable accelerated engineering of PCC 7002.

  3. Global gene expression profiling of the asymptomatic bacteriuria Escherichia coli strain 83972 in the human urinary tract

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) are an important health problem worldwide, with many million cases each year. Escherichia coli is the most common organism causing UTIs in humans. The asymptomatic bacteriuria E. coli strain 83972 is an excellent colonizer of the human urinary tract, where it causes...... long-term bladder colonization. The strain has been used for prophylactic purposes in patients prone to more severe and recurrent UTIs. For this study, we used DNA microarrays to monitor the expression profile of strain 83972 in the human urinary tract. Significant differences in expression levels were...

  4. In situ Ia expression on brain cells in the rat: autoimmune encephalomyelitis-resistant strain (BN) and susceptible strain (Lewis) compared.

    Science.gov (United States)

    Matsumoto, Y; Kawai, K; Fujiwara, M

    1989-01-01

    In order to examine in situ Ia expression on brain cells of various strains of rat, experimental autoimmune encephalomyelitis (EAE) was induced in both EAE-susceptible (LEW) and EAE-resistant (BN) strains. For induction of EAE in the resistant strain, two methods were applied: one was injection of guinea-pig myelin basic protein (GPBP) in complete Freund's adjuvant into LBNF1----BN chimeras; the other was transfer of GPBP-reactive T-line cells from BN rats into syngeneic rats. LBNF----BN chimeras developed clinical EAE, whereas BN rats that received T-line cells did not. However, histological EAE was apparent in both groups. Immunohistochemical examination using two different monoclonal antibodies (OX3 and OX6) against rat Ia antigens revealed that microglia of LEW, BN and chimera rats expressed Ia antigens in the central nervous system (CNS) with EAE. On the other hand, astrocytes were negative for Ia antigens in all the strains. Furthermore, quantitative analysis was undertaken in order to compare the density of Ia-positive microglia in the BN CNS with that in the LEW CNS. It was revealed that the density of Ia-positive microglia in the vicinity of perivascular inflammatory cell aggregates was essentially the same in both strains regardless of the difference in methods of EAE induction or histological severity of the disease. Ia-positive microglia remote from inflammatory cell aggregates were somewhat fewer in rats with mild histological EAE. However, no strain difference was noted in this analysis. Therefore, we concluded that in situ Ia-inducibility on the brain cells of EAE-resistant rats is not different from that of EAE-susceptible rats. Although Ia-positive microglia in both strains may be involved in the immune responses in the CNS, it is unlikely that the difference in Ia-inducibility on brain cells would contribute to strain-specific susceptibility to EAE. Images Figure 1 Figure 2 PMID:2785488

  5. Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A-oryzae alpha-amylase

    DEFF Research Database (Denmark)

    Agger, Teit; Petersen, J.B.; O'Connor, S.M.

    2002-01-01

    The physiology of three strains of Aspergillus nidulans was examined-a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biom......The physiology of three strains of Aspergillus nidulans was examined-a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations...... and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted...... in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon...

  6. Integrative Gene Cloning and Expression System for Streptomyces sp. US 24 and Streptomyces sp. TN 58 Bioactive Molecule Producing Strains

    Directory of Open Access Journals (Sweden)

    Samiha Sioud

    2009-01-01

    Full Text Available Streptomyces sp. US 24 and Streptomyces sp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed using E. coli ET12567 (pUZ8002, as a conjugal donor, carrying the integrative plasmid pSET152. For the Streptomyces sp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas for Streptomyces sp. TN 58, the integration was in single copy at the attB site. Plasmid pSET152 was inherited every time for all analysed Streptomyces sp. US 24 and Streptomyces sp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA from Streptomyces sp. SK was expressed in strain Streptomyces sp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of the Streptomyces sp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch.

  7. A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.

    Directory of Open Access Journals (Sweden)

    Arthur M Talman

    2010-02-01

    Full Text Available The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

  8. In vivo pathogenicity and resistance to phagocytosis of Streptococcus equi strains with different levels of capsule expression.

    Science.gov (United States)

    Anzai, T; Timoney, J F; Kuwamoto, Y; Fujita, Y; Wada, R; Inoue, T

    1999-07-01

    The glossy non-encapsulated strain of Steptococcus equi, NCTC 9682, was compared with the matt strain Hidaka/95/2 which expresses a medium sized capsule and with the mucoid CF32 which expresses a large sized capsule in phagocytosis assays and for virulence in inoculated horses. The three strains, NCTC 9682, Hidaka /95/2 and CF32 produced 2.0, 3.1, and 5.3 mg/g wet cells respectively after 3 h incubation, but similar amounts of M-like proteins, cytotoxin and mitogen. NCTC 9682 showed no resistance to phagocytosis by equine neutrophils regardless of the presence of opsonin while strains Hidaka /95/2 and CF32 showed almost complete resistance to phagocytosis. Furthermore, NCTC 9682 produced no clinical disease although it infected the guttural pouch and caused seroconversion. Typical strangles with guttural pouch invasion was observed in all horses infected with encapsulated strains.

  9. Induction of feline immunodeficiency virus specific antibodies in cats with an attenuated Salmonella strain expressing the Gag protein.

    NARCIS (Netherlands)

    E.J. Tijhaar (Edwin); C.H.J. Siebelink (Kees); J.A. Karlas (Jos); M.C. Burger; F.R. Mooi (Frits); A.D.M.E. Osterhaus (Albert)

    1997-01-01

    textabstractSalmonella typhimurium aroA strains (SL3261), expressing high levels of the Gag protein of feline immunodeficiency virus (FIV) fused with maltose binding protein (SL3261-MFG), were constructed using an invertible promoter system that allows the stable expression of heterologous antigens

  10. Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger.

    Science.gov (United States)

    Tegtmeyer, Nicole; Rivas Traverso, Francisco; Rohde, Manfred; Oyarzabal, Omar A; Lehn, Norbert; Schneider-Brachert, Wulf; Ferrero, Richard L; Fox, James G; Berg, Douglas E; Backert, Steffen

    2013-01-01

    Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5-6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.

  11. Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger.

    Directory of Open Access Journals (Sweden)

    Nicole Tegtmeyer

    Full Text Available Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5-6 µm and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.

  12. Electron Microscopic, Genetic and Protein Expression Analyses of Helicobacter acinonychis Strains from a Bengal Tiger

    Science.gov (United States)

    Tegtmeyer, Nicole; Rivas Traverso, Francisco; Rohde, Manfred; Oyarzabal, Omar A.; Lehn, Norbert; Schneider-Brachert, Wulf; Ferrero, Richard L.; Fox, James G.; Berg, Douglas E.; Backert, Steffen

    2013-01-01

    Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5–6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections. PMID:23940723

  13. Inactivation and Gene Expression of a Virulent WastewaterEscherichia coliStrain and the Nonvirulent CommensalEscherichia coliDSM1103 Strain upon Solar Irradiation

    KAUST Repository

    Aljassim, Nada I.

    2017-03-06

    This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in <24 h, comparative genomics and transcriptomics revealed differences in the genomes and overall regulation of genes between the two E. coli strains. A wider arsenal of genes related to oxidative stress, cellular repair and protective mechanisms were upregulated in E. coli PI-7. Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation during the late decay phase, which may have accounted for its prolonged persistence. Upon prolonged solar irradiation, both E. coli strains displayed upregulation of genes related to horizontal gene transfer and antibiotic resistance. Virulence functions unique to E. coli PI-7 were also upregulated. Our findings collectively indicated that, whereas solar irradiation is able to reduce total cell numbers, viable E. coli remained and expressed genes that enable survival despite solar treatment. There remains a need for heightened levels of concern regarding risks arising from the dissemination of E. coli that may remain viable in wastewater after solar irradiation.

  14. The pathogenic and vaccine strains of equine infectious anemia virus differentially induce cytokine and chemokine expression and apoptosis in macrophages.

    Science.gov (United States)

    Lin, Yue-Zhi; Cao, Xue-Zhi; Li, Liang; Li, Li; Jiang, Cheng-Gang; Wang, Xue-Feng; Ma, Jian; Zhou, Jian-Hua

    2011-09-01

    The attenuated equine infectious anemia virus (EIAV) vaccine was the first attenuated lentivirus vaccine to be used in a large-scale application and has been used to successfully control the spread of equine infectious anemia (EIA) in China. To better understand the potential role of cytokines in the pathogenesis of EIAV infection and resulting immune response, we used branched DNA technology to compare the mRNA expression levels of 12 cytokines and chemokines, including IL-1α, IL-1β, IL-4, IL-10, TNF-α, IFN-γ, IP-10, IL-8, MIP-1α, MIP-1β, MCP-1, and MCP-2, in equine monocyte-derived macrophages (eMDMs) infected with the EIAV(DLV121) vaccine strain or the parental EIAV(DLV34) pathogenic strain. Infection with EIAV(DLV34) and EIAV(DLV121) both caused changes in the mRNA levels of various cytokines and chemokines in eMDMs. In the early stage of infection with EIAV(DLV34) (0-24h), the expression of the pro-inflammatory cytokines TNF-α and IL-1β were significantly up-regulated, while with EIAV(DLV121), expression of the anti-inflammatory cytokine IL-4 was markedly up-regulated. The effects on the expression of other cytokines and chemokines were similar between these two strains of virus. During the first 4 days after infection, the expression level of IL-4 in cells infected with the pathogenic strain were significantly higher than that in cells infected with the vaccine strain, but the expression of IL-1α and IL-1β induced by the vaccine strain was significantly higher than that observed with the pathogenic strain. In addition, after 4 days of infection with the pathogenic strain, the expression levels of 5 chemokines, but not IP-10, were markedly increased in eMDMs. In contrast, the vaccine strain did not up-regulate these chemokines to this level. Contrary to our expectation, induced apoptosis in eMDMs infected with the vaccine strain was significantly higher than that infected with the pathogenic strain 4 days and 6 days after infection. Together, these

  15. Expression and immunological analysis of the plasmid-borne mlp genes of Borrelia burgdorferi strain B31.

    Science.gov (United States)

    Porcella, S F; Fitzpatrick, C A; Bono, J L

    2000-09-01

    A lipoprotein gene family first identified in Borrelia burgdorferi strain 297, designated 2.9 LP and recently renamed mlp, was found on circular and linear plasmids in the genome sequence of B. burgdorferi strain B31-M1. Sequence analyses of the B31 mlp genes and physically linked variant gene families indicated that mlp gene heterogeneity is unique and unrelated to location or linkage to divergent sequences. Evidence of recombination between B31 mlp alleles was also detected. Northern blot analysis of cultured strain B31 indicated that the mlp genes were not expressed at a temperature (23 degrees C) characteristic of that of ticks in the environment. In striking contrast, expression of many mlp genes increased substantially when strain B31 was shifted to 35 degrees C, a temperature change mimicking that occurring in the natural transmission cycle of the spirochete from tick to mammal. Primer extension analysis of the mlp mRNA transcripts suggested that sigma 70-like promoters are involved in mlp expression during temperature shift conditions. Antibodies were made against strain B31 Mlp proteins within the first 4 weeks after experimental mouse infection. Importantly, Lyme disease patients also had serum antibodies reactive with purified recombinant Mlp proteins from strain B31, a result indicating that humans are exposed to Mlp proteins during infection. Taken together, the data indicate that strain B31 mlp genes encode a diverse array of lipoproteins which may participate in early infection processes in the mammalian host.

  16. Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA gene, encoding an -L-Rhamnosidase of enological interest

    NARCIS (Netherlands)

    Manzanares, P.; Orejas, M.; Vicente Gil, J.; Graaff, de L.H.; Visser, J.; Ramon, D.

    2003-01-01

    The Aspergillus aculeatus rhaA gene encoding an alpha-L-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain

  17. Kinetics of mercury reduction by Serratia marcescens mercuric reductase expressed by pseudomonas putida strains

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.; Deckwer, W.D. [GBF-Gesellschaft fuer Biotechnologische Forschung mbH, Abteilung TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig (Germany)

    2005-10-01

    Mercury (Hg) resistance is widespread among microorganisms and is based on the intracellular transformation of Hg(II) to less toxic elemental Hg(0). The use of microbial consortia to demercurize polluted wastewater streams and environments has been demonstrated. To develop efficient and versatile microbial cleanup strategies requires detailed knowledge of transport and reaction rates. This study focuses on the kinetics of the key enzyme of the microbial transformation, e.g., the mercuric reductase (MerA) under conditions closely resembling the cell interior. To this end, previously constructed and characterized Pseudomonas putida strains expressing MerA from Serratia marcescens were applied. Of the P. putida strains considered in this study P. putida KT2442::mer73 constitutively expressing broad spectrum mercury resistance (merTPAB) yielded the highest mercuric reductase (MerA) activity directly after cell disruption. MerA in the raw extract was further purified (about 100 fold). Reduction rates were measured for various substrates (HgCl{sub 2}, Hg{sub 2}SO{sub 4}, Hg(NO{sub 3}){sub 2} and phenyl mercury acetate) up to high concentrations dependent on the purification grade. In all cases, a pronounced substrate inhibition was found. The kinetic constants determined for the cell raw extract are in agreement with those measured for intact cells. However, the rate data exhibit reduced affinity and inhibition with rising purification grade (specific activity). Therefore, the findings seemingly point to reactions preceding the catalytic reduction. Based on simplified assumptions, a kinetic model is suggested which reasonably describes the experimental findings and can advantageously be applied to the bioreactor design. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

  18. Lipase production by recombinant strains of Aspergillus niger expressing a lipase-encoding gene from Thermomyces lanuginosus

    DEFF Research Database (Denmark)

    Prathumpai, Wai; Flitter, S.J.; Mcintyre, Mhairi

    2004-01-01

    Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on differe...... shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall....

  19. Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus

    OpenAIRE

    Ocampo, Clara B.; Caicedo, Paola A.; Jaramillo, Gloria; Bedoya, Raul Ursic; Baron, Olga; Serrato, Idalba M.; Cooper, Dawn M.; Lowenberger, Carl

    2013-01-01

    Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential ex...

  20. Construction of a heterologous gene expression system in the banana rhizobacterium strain GW-3 and its colonization ability.

    Science.gov (United States)

    Wang, Yuguang; Xia, Qiyu; Zhang, He; Lu, Xuehua; Sun, Jianbo; Zhang, Xin

    2014-03-01

    Rhizobacteria inhabiting the rhizosphere are beneficial to their host plants, and can potentially serve as biocontrol agents to control plant diseases. We isolated the rhizobacterium strain GW-3, which was the dominant bacterium in the rhizosphere soils of healthy banana plants. Then, we constructed an expression system with a kanamycin resistance gene to express a heterologous protein in GW-3. Using the green fluorescent protein gene as the reporter, we monitored expression of the heterologous protein by detecting fluorescence intensity and conducting western blot analyses. The standard fluorescence intensity of the recombinant strain reached 1,482 ± 3.49 RFU. To study the colonization ability of GW-3, we inoculated this bacterium into sterilized and unsterilized rhizosphere soils and monitored the bacterial population over 25 days. The populations of GW-3 in rhizosphere soils first increased, then decreased, and finally reached a balance. Laser scanning confocal microscope analyses of fluorescence in banana roots after inoculation with GW-3 confirmed that the recombinant GW-3 strain stably colonized banana root surfaces. Analyses of the bacterial population in unsterilized rhizosphere soils showed that the recombinant GW-3 strain was still the dominant bacterium in banana rhizosphere soils at 25 days after inoculation. Together, these results showed that this expression system can be used to express a heterologous protein at high levels in a dominant rhizobacterium. By incorporating relevant resistance genes into the expression system, this method could be used to genetically engineer GW-3 to control banana wilt disease.

  1. [Immunogenicity of attenuated Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae in mice].

    Science.gov (United States)

    Ma, Fengying; Zou, Haoyong; He, Qigai

    2011-09-01

    The study was carried out to construct and characterize Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae and to test its immunogenicity in mice. We made p36, p46, p65 and p97R1-Nrdf, the main immunogenic genes of Mycoplasma hyopneumoniae, to insert into the prokaryotic expression plasmid pYA3493. Then these recombinant plasmids and pYA3493 were electroporated into C500 asd-mutant, resulting in the recombinant Salmonella choleraesuis vaccine strains C36 (pYA-36), C46 (pYA-46), C65 (pYA-65), C97R1-Nrdf(pYA-97R1-Nrdf) and CpYA(pYA3493). We characterized these recombinant Salmonella choleraesuis vaccine strains and tested the immunogenicity in mice by intramuscular injection or orally immunized. The results of the immunogenicity in mice indicated that the group orally immunized with C36, C46, C65, C97R1-Nrdf showed significantly higher Mycoplasma pneumoniae antibody than both the group orally immunized with C36, C46, C65 and the group intramuscular injected with the Mycoplasma hyopneumoniae bacterin (M + PAC) (P Mycoplasma hyopneumoniae bacterin (M + PAC) (P 0.05). The highest level of IL-4 was found in the group orally immunized with C36, C46, C65; higher levels of IL-4 was observed in the group orally immunized with C36, C46, C65, C97R1-Nrdf than the group injected with the Mycoplasma hyopneumoniae bacterin (M + PAC); and the lowest IL-4 level was found in the group injected with C36, C46, C65. There were no significant differences among them (P > 0.05). The Mycoplasma pneumoniae antibody, IFN-gamma or IL-4 production of the each group was obviously higher than the control group (P Mycoplasma hyopneumoniae which has immunogenicity in mice especially by intramuscular injection could probably serve as a vaccine against mycoplasmal pneumonia of swine.

  2. Sex specific gene regulation and expression QTLs in mouse macrophages from a strain intercross.

    Directory of Open Access Journals (Sweden)

    Jeffrey M Bhasin

    2008-01-01

    Full Text Available A powerful way to identify genes for complex traits it to combine genetic and genomic methods. Many trait quantitative trait loci (QTLs for complex traits are sex specific, but the reason for this is not well understood.RNA was prepared from bone marrow derived macrophages of 93 female and 114 male F(2 mice derived from a strain intercross between apoE-deficient mice on the AKR and DBA/2 genetic backgrounds, and was subjected to transcriptome profiling using microarrays. A high density genome scan was performed using a mouse SNP chip, and expression QTLs (eQTLs were located for expressed transcripts. Using suggestive and significant LOD score cutoffs of 3.0 and 4.3, respectively, thousands of eQTLs in the female and male cohorts were identified. At the suggestive LOD threshold the majority of the eQTLs were trans eQTLs, mapping unlinked to the position of the gene. Cis eQTLs, which mapped to the location of the gene, had much higher LOD scores than trans eQTLs, indicating their more direct effect on gene expression. The majority of cis eQTLs were common to both males and females, but only approximately 1% of the trans eQTLs were shared by both sexes. At the significant LOD threshold, the majority of eQTLs were cis eQTLs, which were mostly sex-shared, while the trans eQTLs were overwhelmingly sex-specific. Pooling the male and female data, 31% of expressed transcripts were expressed at different levels in males vs. females after correction for multiple testing.These studies demonstrate a large sex effect on gene expression and trans regulation, under conditions where male and female derived cells were cultured ex vivo and thus without the influence of endogenous sex steroids. These data suggest that eQTL data from male and female mice should be analyzed separately, as many effects, such as trans regulation are sex specific.

  3. Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Kinetics of adipoyl-7-aminodeacetoxycephalosporanic acid and byproduct formations

    DEFF Research Database (Denmark)

    Robin, Jarno Jacky Christian; Bruheim, P.; Nielsen, M.L.

    2003-01-01

    The production kinetics of a transformed strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was investigated in chemostat cultivations. The recombinant strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) as the major product; howeve...

  4. Role of Morphological Growth State and Gene Expression in Desulfovibrio africanus strain Walvis Bay Mercury Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Moberly, James G [ORNL; Miller, Carrie L [ORNL; Brown, Steven D [ORNL; Biswas, Abir [ORNL; Brandt, Craig C [ORNL; Palumbo, Anthony Vito [ORNL; Elias, Dwayne A [ORNL

    2012-01-01

    The biogeochemical transformations of mercury are a complex process, with the production of methylmercury, a potent human neurotoxin, repeatedly demonstrated in sulfate- and Fe(III)- reducing as well as methanogenic bacteria. However, little is known regarding the morphology, genes or proteins involved in methylmercury generation. Desulfovibrio africanus strain Walvis Bay is a Hg-methylating -proteobacterium with a sequenced genome and has unusual pleomorphic forms. In this study, a relationship between the pleomorphism and Hg methylation was investigated. Proportional increases in the sigmoidal (regular) cell form corresponded with increased net MeHg production, but decreased when the pinched cocci (persister) form became the major morphotype. D. africanus microarrays indicated that the ferrous iron transport genes (feoAB), as well as ribosomal genes and several genes whose products are predicted to have metal binding domains (CxxC), were up-regulated during exposure to Hg in the exponential phase. While no specific methylation pathways were identified, the finding that Hg may interfere with iron transport and the correlation of growth-phase dependent morphology with MeHg production are notable. The identification of these relationships between differential gene expression, morphology, and the growth phase dependence of Hg transformations suggests that actively growing cells are primarily responsible for methylation, and so areas with ample carbon and electron-acceptor concentrations may also generate a higher proportion of methylmercury than more oligotrophic environments. The observation of increased iron transporter expression also suggests that Hg methylation may interfere with iron biogeochemical cycles.

  5. Expression of Genes by Aflatoxigenic and Nonaflatoxigenic Strains of Aspergillus flavus Isolated from Brazil Nuts.

    Science.gov (United States)

    Baquião, Arianne Costa; Rodriges, Aline Guedes; Lopes, Evandro Luiz; Tralamazza, Sabina Moser; Zorzete, Patricia; Correa, Benedito

    2016-08-01

    The aims of the present study were to monitor the production of aflatoxin B1 (AFB1) and mycelial growth, and to evaluate the expression of genes directly and indirectly involved in the biosynthesis of aflatoxins by Aspergillus flavus isolated from Brazil nuts. Six previously identified A. flavus strains were grown on coconut agar at 25°C for up to 10 days. Mycotoxins were separated by high-performance liquid chromatography and fungal growth was measured daily using the diametric mycelial growth rate. Transcriptional analysis was performed by real-time polymerase chain reaction (PCR) after 2 and 7 d of incubation using specific primers (aflR, aflD, aflP, lipase, metalloprotease, and LaeA). Three (50%) of the six A. flavus isolates produced AFB1 (ICB-1, ICB-12, and ICB-54) and three (50%) were not aflatoxigenic (ICB-141, ICB-161, and ICB-198). Aflatoxin production was observed from d 2 of incubation (1.5 ng/g for ICB-54) and increased gradually with time of incubation until d 10 (15,803.6 ng/g for ICB-54). Almost all A. flavus isolates exhibited a similar gene expression pattern after 2 d of incubation (p > 0.10). After 7 d of incubation, the LaeA (p aflatoxin production in A. flavus and that overexpression of aflR could affect the transcriptional and aflatoxigenic pattern (ICB-54). Elucidation of the molecular mechanisms that regulate the secondary metabolism of toxigenic fungi may permit the rational silencing of the genes involved and consequently the programmed inhibition of aflatoxin production. Knowledge of the conditions, under which aflatoxin genes are expressed, should contribute to the development of innovative and more cost-effective strategies to reduce and prevent aflatoxin contamination in Brazil nuts.

  6. Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239

    Directory of Open Access Journals (Sweden)

    Stougaard Peter

    2010-01-01

    Full Text Available Abstract Background Chitosanases (EC 3.2.1.132 hydrolyze the polysaccharide chitosan, which is composed of partially acetylated β-(1,4-linked glucosamine residues. In nature, chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. Chitosanases may also be utilized in eukaryotic cell manipulation for intracellular delivery of molecules formulated with chitosan as well as for transformation of filamentous fungi by temporal modification of the cell wall structures. However, the chitosanases used so far in transformation and transfection experiments show optimal activity at high temperature, which is incompatible with most transfection and transformation protocols. Thus, there is a need for chitosanases, which display activity at lower temperatures. Results This paper describes the isolation of a chitosanase-producing, cold-active bacterium affiliated to the genus Janthinobacterium. The 876 bp chitosanase gene from the Janthinobacterium strain was isolated and characterized. The chitosanase was related to the Glycosyl Hydrolase family 46 chitosanases with Streptomyces chitosanase as the closest related (64% amino acid sequence identity. The chitosanase was expressed recombinantly as a periplasmic enzyme in Escherichia coli in amounts about 500 fold greater than in the native Janthinobacterium strain. Determination of temperature and pH optimum showed that the native and the recombinant chitosanase have maximal activity at pH 5-7 and at 45°C, but with 30-70% of the maximum activity at 10°C and 30°C, respectively. Conclusions A novel chitosanase enzyme and its corresponding gene was isolated from Janthinobacterium and produced recombinantly in E. coli as a periplasmic enzyme. The Janthinobacterium chitosanase displayed reasonable activity at 10°C to 30°C, temperatures that are preferred in

  7. Prion Strain Differences in Accumulation of PrPSc on Neurons and Glia Are Associated with Similar Expression Profiles of Neuroinflammatory Genes: Comparison of Three Prion Strains.

    Science.gov (United States)

    Carroll, James A; Striebel, James F; Rangel, Alejandra; Woods, Tyson; Phillips, Katie; Peterson, Karin E; Race, Brent; Chesebro, Bruce

    2016-04-01

    Misfolding and aggregation of host proteins are important features of the pathogenesis of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, frontotemporal dementia and prion diseases. In all these diseases, the misfolded protein increases in amount by a mechanism involving seeded polymerization. In prion diseases, host prion protein is misfolded to form a pathogenic protease-resistant form, PrPSc, which accumulates in neurons, astroglia and microglia in the CNS. Here using dual-staining immunohistochemistry, we compared the cell specificity of PrPSc accumulation at early preclinical times post-infection using three mouse scrapie strains that differ in brain regional pathology. PrPSc from each strain had a different pattern of cell specificity. Strain 22L was mainly associated with astroglia, whereas strain ME7 was mainly associated with neurons and neuropil. In thalamus and cortex, strain RML was similar to 22L, but in substantia nigra, RML was similar to ME7. Expression of 90 genes involved in neuroinflammation was studied quantitatively using mRNA from thalamus at preclinical times. Surprisingly, despite the cellular differences in PrPSc accumulation, the pattern of upregulated genes was similar for all three strains, and the small differences observed correlated with variations in the early disease tempo. Gene upregulation correlated with activation of both astroglia and microglia detected in early disease prior to vacuolar pathology or clinical signs. Interestingly, the profile of upregulated genes in scrapie differed markedly from that seen in two acute viral CNS diseases (LaCrosse virus and BE polytropic Friend retrovirus) that had reactive gliosis at levels similar to our prion-infected mice.

  8. Significant and systematic expression differentiation in long-lived yeast strains.

    Directory of Open Access Journals (Sweden)

    Chao Cheng

    2007-10-01

    Full Text Available Recent studies suggest that the regulation of longevity may be partially conserved in many eukaryotes ranging from yeast to mammals. The three yeast mutants sch9Delta, ras2Delta, tor1Delta show extended chronological life span up to three folds. Our aim is to dissect the mechanisms that lead to the yeast life span extension.We obtain gene expression profiles of sch9Delta, ras2Delta, tor1Delta as well as that for a wild type at day 2.5 in SDC medium using Affymetrix Yeast2.0 arrays. To accurately estimate the expression differentiation between the wild type and the long-lived mutants, we use sub-array normalization followed by a variant of the median-polishing summarization. The results are validated by the probe sets of S. pombe on the same chips. To translate the differentiation into changes of biological activities, we make statistical inference by integrating the expression profiles with biological gene subsets defined by Gene Ontology, KEGG pathways, and cellular localization of proteins. Other than subset-versus-other comparisons, we also make local comparisons between two directly-related gene subsets such as cytosolic and mitochondrial ribosomes. Our consensus is obtained by cross-examination of these inferences. The significant and systematic differentiation in the three long-lived strains includes: lower transcriptional activities; down-regulation of TCA cycle and oxidative phosphorylation versus up-regulation of the KEGG pathway Glycolysis/Gluconeogenesis; the overall reduction of mitochondrial activities. We also report some different expression patterns such as reduction of the activities relating to mitosis in ras2Delta.The modification of energy pathways and modification of compartment activities such as down-regulation of mitochondrial ribosome proteins versus up-regulation of cytosolic ribosome proteins are directly associated with the life span extension in yeast. The results provide a new and systematic S. cerevisiae version of

  9. Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme

    NARCIS (Netherlands)

    Venkataraman, Harini; Te Poele, Evelien M; Rosłoniec, Kamila Z; Vermeulen, Nico; Commandeur, Jan N M; van der Geize, Robert; Dijkhuizen, Lubbert

    In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the

  10. Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Ying Kong

    Full Text Available The slow growth of Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis (TB, hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

  11. The Inhibitory Effects of 2 Commercial Probiotic Strains on the Growth of Staphylococcus aureus and Gene Expression of Enterotoxin A

    Directory of Open Access Journals (Sweden)

    Mahnoosh Parsaeimehr

    2017-08-01

    Full Text Available Background: Food-borne intoxications are current problems in human society and most of them are caused by the enterotoxins of Staphylococcus aureus. Staphylococcal enterotoxin A (SEA is the most frequently responsible for staphylococcal food poisoning outbreaks. From a food safety and human health point of view, lactic acid bacteria (LAB may provide a promising strategy to combat the pathogenic bacteria, particularly S. aureus. Objective: The objective of this study was to evaluate the inhibitory activity of two commercial lactobacillus strains on growth and enterotoxin A production by S. aureus. Moreover, the inhibitory effect of these strains on gene expression of enterotoxin type A was assessed using real-time Polymerase chain reaction (PCR. Materials and Methods: In this study the inhibitory effect of two commercial probiotic strains, Lactobacillus acidophilus (LA5 and Lactobacillus casei 01 on the growth and enterotoxin production of S. aureus was evaluated at 25 and 35°C. The gene expression of SEA of S. aureus was also evaluated by real time (RT PCR technique. Results: The lactobacillus strains decreased the bacterial count at both temperatures compared with the control group. This reduced effect was greater at 25°C (3 log/CFU than 35°C (2 log/CFU. The production of SEA, SEC and SEE was inhibited by the lactobacillus strains. Furthermore, the gene expression of SEA was significantly suppressed in S. aureus co cultured with studied lactobacillus strains and the greatest down-regulation of sea (10.31 fold was observed in co-incubation of S. aureus with LC01 at 25°C. Conclusion: This research raises important implications for the potential use of LAB as a natural preservative in foodstuffs by correct microbial ecology of the environment and a new approach for biocontrol of S. aureus.

  12. Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield.

    Science.gov (United States)

    Papapetridis, Ioannis; Goudriaan, Maaike; Vázquez Vitali, María; de Keijzer, Nikita A; van den Broek, Marcel; van Maris, Antonius J A; Pronk, Jack T

    2018-01-01

    Reduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae . Anaerobic cultures of wild-type S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD + balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO 2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield. Multi-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2 , which encodes an isoenzyme of NAD + -dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO 2 -reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield. The metabolic engineering

  13. A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis

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    Krijger Jorrit-Jan

    2012-08-01

    Full Text Available Abstract Background The Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining. Results Selection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the β-galactosidase gene indicated the desired integration event of the expression cassette, and β-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox and a viral envelope protein (BVDV-E2, respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained. Conclusions A novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of

  14. Heterologous expression of Ralp3 in Streptococcus pyogenes M2 and M6 strains affects the virulence characteristics.

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    Nikolai Siemens

    Full Text Available BACKGROUND: Ralp3 is a transcriptional regulator present in a serotype specific fashion on the chromosome of the human pathogen Streptococcus pyogenes (group A streptococci, GAS. In serotypes harbouring the ralp3 gene either positive or negative effects on important metabolic and virulence genes involved in colonization and immune evasion in the human host were observed. A previous study revealed that deletion of ralp3 in a GAS M49 serotype significantly attenuated many virulence traits and caused metabolic disadvantages. This leads to two questions: (i which kind of consequences could Ralp3 expression have in GAS serotypes naturally lacking this gene, and (ii is Ralp3 actively lost during evolution in these serotypes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of Ralp3 in GAS M2 and M6 pathogenesis. Both serotypes lack ralp3 on their chromosome. The heterologous expression of ralp3 in both serotypes resulted in reduced attachment to and internalization into the majority of tested epithelial cells. Both ralp3 expression strains showed a decreased ability to survive in human blood and exclusively M2::ralp3 showed decreased survival in human serum. Both mutants secreted more active SpeB in the supernatant, resulting in a higher activity compared to wild type strains. The respective M2 and M6 wild type strains outcompeted the ralp3 expression strains in direct metabolic competition assays. The phenotypic changes observed in the M2:ralp3 and M6:ralp3 were verified on the transcriptional level. Consistent with the virulence data, tested genes showed transcript level changes in the same direction. CONCLUSIONS/SIGNIFICANCE: Together these data suggest that Ralp3 can take over transcriptional control of virulence genes in serotypes lacking the ralp3 gene. Those serotypes most likely lost Ralp3 during evolution since obviously expression of this gene is disadvantageous for metabolism and pathogenesis.

  15. Gene expression profiles among murine strains segregate with distinct differences in the progression of radiation-induced lung disease

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    Isabel L. Jackson

    2017-04-01

    Full Text Available Molecular mechanisms underlying development of acute pneumonitis and/or late fibrosis following thoracic irradiation remain poorly understood. Here, we hypothesize that heterogeneity in disease progression and phenotypic expression of radiation-induced lung disease (RILD across murine strains presents an opportunity to better elucidate mechanisms driving tissue response toward pneumonitis and/or fibrosis. Distinct differences in disease progression were observed in age- and sex-matched CBA/J, C57L/J and C57BL/6J mice over 1 year after graded doses of whole-thorax lung irradiation (WTLI. Separately, comparison of gene expression profiles in lung tissue 24 h post-exposure demonstrated >5000 genes to be differentially expressed (Ptwofold change between strains with early versus late onset of disease. An immediate divergence in early tissue response between radiation-sensitive and -resistant strains was observed. In pneumonitis-prone C57L/J mice, differentially expressed genes were enriched in proinflammatory pathways, whereas in fibrosis-prone C57BL/6J mice, genes were enriched in pathways involved in purine and pyrimidine synthesis, DNA replication and cell division. At 24 h post-WTLI, different patterns of cellular damage were observed at the ultrastructural level among strains but microscopic damage was not yet evident under light microscopy. These data point toward a fundamental difference in patterns of early pulmonary tissue response to WTLI, consistent with the macroscopic expression of injury manifesting weeks to months after exposure. Understanding the mechanisms underlying development of RILD might lead to more rational selection of therapeutic interventions to mitigate healthy tissue damage.

  16. Gene expression profiles among murine strains segregate with distinct differences in the progression of radiation-induced lung disease.

    Science.gov (United States)

    Jackson, Isabel L; Baye, Fitsum; Goswami, Chirayu P; Katz, Barry P; Zodda, Andrew; Pavlovic, Radmila; Gurung, Ganga; Winans, Don; Vujaskovic, Zeljko

    2017-04-01

    Molecular mechanisms underlying development of acute pneumonitis and/or late fibrosis following thoracic irradiation remain poorly understood. Here, we hypothesize that heterogeneity in disease progression and phenotypic expression of radiation-induced lung disease (RILD) across murine strains presents an opportunity to better elucidate mechanisms driving tissue response toward pneumonitis and/or fibrosis. Distinct differences in disease progression were observed in age- and sex-matched CBA/J, C57L/J and C57BL/6J mice over 1 year after graded doses of whole-thorax lung irradiation (WTLI). Separately, comparison of gene expression profiles in lung tissue 24 h post-exposure demonstrated >5000 genes to be differentially expressed ( P twofold change) between strains with early versus late onset of disease. An immediate divergence in early tissue response between radiation-sensitive and -resistant strains was observed. In pneumonitis-prone C57L/J mice, differentially expressed genes were enriched in proinflammatory pathways, whereas in fibrosis-prone C57BL/6J mice, genes were enriched in pathways involved in purine and pyrimidine synthesis, DNA replication and cell division. At 24 h post-WTLI, different patterns of cellular damage were observed at the ultrastructural level among strains but microscopic damage was not yet evident under light microscopy. These data point toward a fundamental difference in patterns of early pulmonary tissue response to WTLI, consistent with the macroscopic expression of injury manifesting weeks to months after exposure. Understanding the mechanisms underlying development of RILD might lead to more rational selection of therapeutic interventions to mitigate healthy tissue damage. © 2017. Published by The Company of Biologists Ltd.

  17. Construction of new synthetic biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002.

    Science.gov (United States)

    Zess, Erin K; Begemann, Matthew B; Pfleger, Brian F

    2016-02-01

    Predictive control of gene expression is an essential tool for developing synthetic biological systems. The current toolbox for controlling gene expression in cyanobacteria is a barrier to more in-depth genetic analysis and manipulation. Towards relieving this bottleneck, this work describes the use of synthetic biology to construct an anhydrotetracycline-based induction system and adapt a trans-acting small RNA (sRNA) system for use in the cyanobacterium Synechococcus sp. strain PCC 7002. An anhydrotetracycline-inducible promoter was developed to maximize intrinsic strength and dynamic range. The resulting construct, PEZtet , exhibited tight repression and a maximum 32-fold induction upon addition of anhydrotetracycline. Additionally, a sRNA system based on the Escherichia coli IS10 RNA-IN/OUT regulator was adapted for use in Synechococcus sp. strain PCC 7002. This system exhibited 70% attenuation of target gene expression, providing a demonstration of the use of sRNAs for differential gene expression in cyanobacteria. These systems were combined to produce an inducible sRNA system, which demonstrated 59% attenuation of target gene expression. Lastly, the role of Hfq, a critical component of sRNA systems in E. coli, was investigated. Genetic studies showed that the Hfq homolog in Synechococcus sp. strain PCC 7002 did not impact repression by the engineered sRNA system. In summary, this work describes new synthetic biology tools that can be applied to physiological studies, metabolic engineering, or sRNA platforms in Synechococcus sp. strain PCC 7002. © 2015 Wiley Periodicals, Inc.

  18. Impact of GR24 on gene expression of solopathogenic strain of ...

    African Journals Online (AJOL)

    MAHTA

    2011-11-09

    Nov 9, 2011 ... Quantitative polymerase chain reaction (qPCR) analysis showed that transcription levels of genes involved in cell respiration ... the pathogen can become solopathogenic, whereby strains are able to switch from the ... annotated genome has been used to identify the genes in a solopathogenic strain of S.

  19. An engineered Lactococcus lactis strain exerts significant immune responses through efficient expression and delivery of Helicobacter pylori Lpp20 antigen.

    Science.gov (United States)

    Zhang, Rongguang; Peng, Xiaoyan; Duan, Guangcai; Shi, Qingfeng; Chen, Shuaiyin; Wang, Chen; Fan, Qingtang; Xi, Yuanlin

    2016-12-01

    To produce and deliver Helicobacter pylori lipoprotein Lpp20 via using Lactococcus lactis with aim of developing an efficient way to use this protective antigen in vaccine formulation. An engineered L. lactis strain carrying the lpp20 gene from H. pylori was constructed. The inducible expression of Lpp20 in L. lactis was detected as a 20 kDa intracellular protein by SDS-PAGE. Lpp20 constituted 10 % of the L. lactis cellular proteins. The expression product was highly immunoreactive, as demonstrated by western blot assays using mouse anti-H. pylori sera. Animal experimentation showed that oral vaccination with the engineered strain excited significantly elevated levels of serum Lpp20-specific IgG antibodies in BALB/c mice (P lactis, demonstrating an efficient utilization mode of Lpp20 in anti-H. pylori vaccination.

  20. Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer's yeast strain.

    Science.gov (United States)

    Wang, Jin-Jing; Wang, Zhao-Yue; He, Xiu-Ping; Zhang, Bo-Run

    2012-01-01

    Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase's shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21's better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.

  1. Polyphosphate metabolic gene expression analyses reveal mechanisms of phosphorus accumulation and release in Microlunatus phosphovorus strain JN459.

    Science.gov (United States)

    Zhong, Chuanqing; Fu, Jiafang; Jiang, Tianyi; Zhang, Chunming; Cao, Guangxiang

    2018-03-01

    The ability of Microlunatus phosphovorus to accumulate large amounts of polyphosphate (Poly-P) plays an important role in removing soluble phosphorus from wastewater. Strain JN459, isolated from a sewage system, was previously demonstrated to be Microlunatus phosphovorus. In this study, we analyzed the phosphorus-accumulating and phosphorus-releasing characteristics of strain JN459. Our analyses indicate that strain JN459 accumulates Poly-P under aerobic conditions but releases phosphorus under anaerobic conditions. To determine the mechanisms underlying Poly-P metabolism in strain JN459, we compared transcriptional profiles under aerobic and anaerobic conditions. Significant differences were detected in the expression levels of genes associated with Poly-P metabolism between aerobic and anaerobic conditions, including ppk (MLP_47700, MLP_50300 and MLP_05750), ppgk (MLP_05430 and MLP_26610), ppx (MLP_44770), pap (MLP_23310) and ppnk (MLP_17420). The high expression of polyphosphate glucokinase (MLP_05430) and polyphosphate/ATP-dependent NAD kinase (MLP_17420) indicated that both of them might be responsible for utilizing Poly-P as the energy resource for growth under anaerobic conditions. These findings enhance our understanding of phosphate metabolism in a major bacterial species involved in wastewater phosphorus reduction.

  2. Expression profile of genes during resistance reversal in a temephos selected strain of the dengue vector, Aedes aegypti.

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    Clare Strode

    Full Text Available BACKGROUND: The mosquito Aedes aegypti is one of the most important disease vectors because it transmits two major arboviruses, dengue and yellow fever, which cause significant global morbidity and mortality. Chemical insecticides form the cornerstone of vector control. The organophosphate temephos a larvicide recommended by WHO for controlling Ae. aegypti, however, resistance to this compound has been reported in many countries, including Brazil. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to identify genes implicated in metabolic resistance in an Ae. aegypti temephos resistant strain, named RecR, through microarray analysis. We utilized a custom 'Ae. aegypti detox chip' and validated microarray data through RT-PCR comparing susceptible and resistant individuals. In addition, we analyzed gene expression in 4(th instar larvae from a reversed susceptible strain (RecRev, exposed and unexposed to temephos. The results obtained revealed a set of 13 and 6 genes significantly over expressed in resistant adult mosquitoes and larvae, respectively. One of these genes, the cytochrome P450 CYP6N12, was up-regulated in both stages. RT-PCR confirmed the microarray results and, additionally, showed no difference in gene expression between temephos exposed and unexposed RecRev mosquitoes. This suggested that the differences in the transcript profiles among the strains are heritable due to a selection process and are not caused by immediate insecticide exposure. Reversal of temephos resistance was demonstrated and, importantly, there was a positive correlation between a decrease in the resistance ratio and an accompanying decrease in the expression levels of previously over expressed genes. Some of the genes identified here have also been implicated in metabolic resistance in other mosquito species and insecticide resistant populations of Ae. aegypti. CONCLUSIONS/SIGNIFICANCE: The identification of gene expression signatures associated to

  3. A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

    Science.gov (United States)

    Vaughan, Ashley M; Mikolajczak, Sebastian A; Camargo, Nelly; Lakshmanan, Viswanathan; Kennedy, Mark; Lindner, Scott E; Miller, Jessica L; Hume, Jen C C; Kappe, Stefan H I

    2012-12-01

    Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Different cytokine production and Toll-like receptor expression induced by heat-killed invasive and carrier strains of Neisseria meningitidis.

    Science.gov (United States)

    Potmesil, Roman; Beran, Ondrej; Musilek, Martin; Kriz, Pavla; Holub, Michal

    2014-01-01

    Neisseria meningitidis may cause severe invasive disease. The carriage state of the pathogen is common, and the reasons underlying why the infection becomes invasive are not fully understood. The aim of this study was to compare the differences between invasive and carrier strains in the activation of innate immunity. The monocyte expression of TLR2, TLR4, CD14, and HLA-DR, cytokine production, and the granulocyte oxidative burst were analyzed after in vitro stimulation by heat-killed invasive (n = 14) and carrier (n = 9) strains of N. meningitidis. The expression of the cell surface markers in monocytes, the oxidative burst, and cytokine concentrations were measured using flow cytometry. Carrier strains stimulated a higher production of inflammatory cytokines and oxidative burst in granulocytes than invasive strains (all p < 0.001), whereas invasive strains significantly up-regulated TLR2, TLR4 (p < 0.001), and CD14 (p < 0.01) expression on monocytes. Conversely, the monocyte expression of HLA-DR was higher after the stimulation by carrier strains (p < 0.05) in comparison to invasive strains. The LPS inhibitor polymyxin B abolished the differences between the strains. Our findings indicate different immunostimulatory potencies of invasive strains of N. meningitidis compared with carrier strains. © 2013 APMIS Published by Blackwell Publishing Ltd.

  5. Integrative Expression of Glucoamylase Gene in a Brewer’s Yeast Saccharomyces pastorianus Strain

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    Guangyi Zhang

    2008-01-01

    Full Text Available The recombinant brewer’s yeast Saccharomyces pastorianus strain was constructed byintroducing the ilv2:GLA fragment released from pMGI6, carrying glucoamylase gene (GLA and using the yeast α-acetolactate synthase gene (ILV2 as the recombination sequence. The strain was able to utilise starch as the sole carbon source, its glucoamylase activity was 6.3 U/mL and its α-acetolactate synthase activity was lowered by 33.3 %. The introduced GLA gene was integrated at the recipient genomic ILV2 gene, one copy of ILV2 gene was disrupted and the other copy remained intact. Primary wort fermentation test confirmed that the diacetyl and residual sugar concentration in the wort fermented by the recombinant strain were reduced by 65.6 and 34.2 % respectively, compared to that of the recipient strain. Under industrial operating conditions, the maturation time of beer fermented by the recombinant strain was reduced from 7 to 4 days, there were no significant differences in the appearance and mouthfeel, and the beer satisfied the high quality demands. That is why the strain could be used in beer production safely.

  6. Expression of class 5 antigens by meningococcal strains obtained from patients in Brazil and evaluation of two new monoclonal antibodies

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    Elizabeth N. De Gaspari

    Full Text Available Determining the profile of antigen expression among meningococci is important for epidemiologic surveillance and vaccine development. To this end, two new mouse monoclonal antibodies (MAbs have been derived against Neisseria meningitidis proteins (class 5. The MAbs were reactive against outer membrane antigens and were bactericidal. Selected anti-class 5 MAbs [(5.1-3E6-2; (5.3-3BH4-C7; (5.4-1BG11-C7; (5.5-3DH-F5G9 also 5F1F4-T3(5.c], and the two new monoclonal antibodies C14F10Br2 (5.8 and 7F11B5Br3 (5.9, were then tested against different meningococcal strains, (63 strains of serogroup A, 60 strains of serogroup C (from 1972 to 1974; and 136 strains of serogroup B (from 1992 meningococci. Our results demonstrated that the expression of class 5 proteins in the N. meningitidis B Brazilian strains studied is highly heterogeneous. The serotypes and subtypes of B:4:P1.15, B:4:P1.9, B:4:P1.7, B:4:P1.3, B:4:P1.14, B:4:P1.16, B:4:NT, and B:NT:NT were detected in N. meningitidis B serogroups.The strains C:2a:P1.2 and A:4.21:P1.9 were dominant in the C and A serogroups, respectively. Serogroup B organisms expressed the class 5 epitopes 5.4 (18%, 5.5 (22%, 5.8 (3.6%, 5.9 (8.0% and 5c (38%. Serogroup C expressed class 5 epitopes 5.1 (81%, 5.4 (35%, 5.5 (33% and 5.9 (5.0%; and serogroup A showed reactivity directed at the class 5 protein 5c (47%; and reactivity was present with the new monoclonal antibody, 5.9 (5.5%. We conclude that the two new MAbs are useful in detecting important group B, class 5 antigens, and that a broad selection of serogroup B, class 5 proteins would be required for an effective vaccine based on the class 5 proteins.

  7. Viability, biofilm formation, and MazEF expression in drug-sensitive and drug-resistant Mycobacterium tuberculosis strains circulating in Xinjiang, China.

    Science.gov (United States)

    Zhao, Ji-Li; Liu, Wei; Xie, Wan-Ying; Cao, Xu-Dong; Yuan, Li

    2018-01-01

    Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is one of the most common chronic infectious amphixenotic diseases worldwide. Prevention and control of TB are greatly difficult, due to the increase in drug-resistant TB, particularly multidrug-resistant TB. We speculated that there were some differences between drug-sensitive and drug-resistant MTB strains and that mazEF 3,6,9 toxin-antitoxin systems (TASs) were involved in MTB viability. This study aimed to investigate differences in viability, biofilm formation, and MazEF expression between drug-sensitive and drug-resistant MTB strains circulating in Xinjiang, China, and whether mazEF 3,6,9 TASs contribute to MTB viability under stress conditions. Growth profiles and biofilm-formation abilities of drug-sensitive, drug-resistant MTB strains and the control strain H37Rv were monitored. Using molecular biology experiments, the mRNA expression of the mazF 3, 6, and 9 toxin genes, the mazE 3, 6, and 9 antitoxin genes, and expression of the MazF9 protein were detected in the different MTB strains, H37RvΔ mazEF 3,6,9 mutants from the H37Rv parent strain were generated, and mutant viability was tested. Ex vivo culture analyses demonstrated that drug-resistant MTB strains exhibit higher survival rates than drug-sensitive strains and the control strain H37Rv. However, there was no statistical difference in biofilm-formation ability in the drug-sensitive, drug-resistant, and H37Rv strains. mazE 3,6 mRNA-expression levels were relatively reduced in the drug-sensitive and drug-resistant strains compared to H37Rv. Conversely, mazE 3,9 expression was increased in drug-sensitive strains compared to drug-resistant strains. Furthermore, compared with the H37Rv strain, mazF 3,6 expression was increased in drug-resistant strains, mazF 9 expression was increased in drug-sensitive strains, and mazF 9 exhibited reduced expression in drug-resistant strains compared with drug-sensitive strains. Protein expression of mazF9

  8. Systematic analysis of gene expression differences between left and right atria in different mouse strains and in human atrial tissue.

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    Peter C Kahr

    Full Text Available BACKGROUND: Normal development of the atria requires left-right differentiation during embryonic development. Reduced expression of Pitx2c (paired-like homeodomain transcription factor 2, isoform c, a key regulator of left-right asymmetry, has recently been linked to atrial fibrillation. We therefore systematically studied the molecular composition of left and right atrial tissue in adult murine and human atria. METHODS: We compared left and right atrial gene expression in healthy, adult mice of different strains and ages by employing whole genome array analyses on freshly frozen atrial tissue. Selected genes with enriched expression in either atrium were validated by RT-qPCR and Western blot in further animals and in shock-frozen left and right atrial appendages of patients undergoing open heart surgery. RESULTS: We identified 77 genes with preferential expression in one atrium that were common in all strains and age groups analysed. Independent of strain and age, Pitx2c was the gene with the highest enrichment in left atrium, while Bmp10, a member of the TGFβ family, showed highest enrichment in right atrium. These differences were validated by RT-qPCR in murine and human tissue. Western blot showed a 2-fold left-right concentration gradient in PITX2 protein in adult human atria. Several of the genes and gene groups enriched in left atria have a known biological role for maintenance of healthy physiology, specifically the prevention of atrial pathologies involved in atrial fibrillation, including membrane electrophysiology, metabolic cellular function, and regulation of inflammatory processes. Comparison of the array datasets with published array analyses in heterozygous Pitx2c(+/- atria suggested that approximately half of the genes with left-sided enrichment are regulated by Pitx2c. CONCLUSIONS: Our study reveals systematic differences between left and right atrial gene expression and supports the hypothesis that Pitx2c has a functional

  9. Optimized expression of full-length IgG1 antibody in a common E. coli strain.

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    Conrad En Zuo Chan

    Full Text Available Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc portion of the immunoglobulin are not required.

  10. Suppression of different classes of somatic mutations in Arabidopsis by vir gene-expressing Agrobacterium strains.

    Science.gov (United States)

    Shah, Jasmine M; Ramakrishnan, Anantha Maharasi; Singh, Amit Kumar; Ramachandran, Subalakshmi; Unniyampurath, Unnikrishnan; Jayshankar, Ajitha; Balasundaram, Nithya; Dhanapal, Shanmuhapreya; Hyde, Geoff; Baskar, Ramamurthy

    2015-08-26

    Agrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of blue spots visible in plants grown for a further two weeks, we estimated the mutation frequencies. For plants co-cultivated for 48 h with Agrobacterium, if the strain contained vir genes, then the rates of transversions, SHRs and FSMs (measured 2 weeks later) were lower than those of uninfected controls. In contrast, co-cultivation for 48 h with any of the Agrobacterium strains raised the transposition rates above control levels. The multiple time-point study showed that in seedlings co-cultivated with wild type Ach5, the reduced rates of transversions and SHRs after 48 h co-cultivation represent an apparent suppression of an earlier short-lived increase in mutation rates (peaking for plants co-cultivated for 3 h). An increase after 3 h co-cultivation was also seen for rates of transversions (but not SHR) in seedlings exposed to the strain lacking vir genes, oncogenes and T-DNA. However, the mutation rates in plants co-cultivated for longer times with this strain subsequently

  11. Expression and inducibility of endosulfan metabolizing gene in Rhodococcus strain isolated from earthworm gut microflora for its application in bioremediation.

    Science.gov (United States)

    Verma, Ankit; Ali, Daoud; Farooq, M; Pant, A B; Ray, R S; Hans, R K

    2011-02-01

    The metabolizing potential of a bacterial strain Rhodococcus MTCC 6716, isolated from the gut of an Indian earthworm (Metaphire posthuma) was studied for endosulfan bioremediation. In the present work, the optimum conditions for the maximum growth, kinetic of endosulfan degradation, regression equation, half life and correlation coefficient were studied. Endosulfan induced alterations in the expression of mRNA and protein of specific endosulfan metabolizing marker gene (Esd) was studied. Maximum growth of bacteria was observed at pH 7.0, 30°C and 0.085 M sodium chloride concentration in a liquid culture medium. Endosulfan was degraded by Rhodococcus strain up to 97.23% within 15 days without producing toxic metabolite and with strong correlation coefficient (-0.728) and half life 5.99 days. Endosulfan degradation was mediated through gene(s) present in genomic DNA. Expression of marker gene was found endosulfan concentration dependent. The results suggest that this novel strain (Rhodococcus) may be utilized for bioremediation of endosulfan. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Sorbitol synthesis by an engineered Lactobacillus casei strain expressing a sorbitol-6-phosphate dehydrogenase gene within the lactose operon.

    Science.gov (United States)

    Nissen, Lorenzo; Pérez-Martínez, Gaspar; Yebra, María J

    2005-08-01

    Sorbitol is claimed to have important health-promoting effects and Lactobacillus casei is a lactic acid bacterium relevant as probiotic and used as a cheese starter culture. A sorbitol-producing L. casei strain might therefore be of considerable interest in the food industry. A recombinant strain of L. casei was constructed by the integration of a d-sorbitol-6-phosphate dehydrogenase-encoding gene (gutF) in the chromosomal lactose operon (strain BL232). gutF expression in this strain followed the same regulation as that of the lac genes, that is, it was repressed by glucose and induced by lactose. (13)C-nuclear magnetic resonance analysis of supernatants of BL232 resting cells demonstrated that, when pre-grown on lactose, cells were able to synthesize sorbitol from glucose. Inactivation of the l-lactate dehydrogenase gene in BL232 led to an increase in sorbitol production, suggesting that the engineered route provides an alternative pathway for NAD(+) regeneration.

  13. Optimalisation of expression conditions for production of round-leaf sundew chitinase (Drosera rotundifolia L. in three E. coli expression strains

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    Miroslav Rajninec

    2016-12-01

    Full Text Available Round-leaf sundew (Drosera rotundifolia L., family Droseraceae, genus Drosera, is one of a few plant species with a strong antifungal potential. Chitinases of carnivorous plants play an important role in decomposition of chitin-containing cell structures of insect prey. The cell wall of many phytopathogenic fungi also contains chitin, which can be utilized by chitinases, thus round-leaf sundew represents an interesting gene source for plant biotechnology. The purpose of this study was to compare the suitability of 3 different E. coli expression strains (E. coli BL21- CodonPlus® (DE3-RIPL, E. coli ArcticExpress (DE3RIL and E. coli SHuffle® T7 for production and isolation of heterologous round-leaf sundew chitinase (DrChit. Results showed that the recombinant protein was successfully expressed in all three strains, but occurred in insoluble protein fraction. To get the DrChit protein into soluble protein fraction some modifications concerning to induction temperatures and concentration of the IPTG inductor were tested. In addition, composition of lysis buffer has been modified with supplementation of strong non-ionic detergents, Triton® X100 and Tween® 20, respectively. As these modifications didn’t increase the amount of the DrChit protein in soluble fraction, therefore, its isolation under denaturing conditions and subsequent refolding for activity assays is recommended.

  14. Expression of cytokines in chicken peripheral mononuclear blood cells (PMBCs exposed to probiotic strains and Salmonella Enteritidis

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    Eva Husáková

    2015-01-01

    Full Text Available The mRNA expression of interleukin (IL-1β, LITAF, iNOS, macrophage inflammatory protein (MIP1-ß, and K60 were examined in peripheral blood mononuclear cells (PMBCs. The PMBCs were isolated from the chicken blood and in vitro exposed to the probiotic strains E. faecium AL41, E. faecium H31, L. fermentum AD1, and infected with Salmonella enterica serovar Enteritidis (SE147. The PMBCs were evaluated for mRNA expression levels at 24 h and 48 h post infection (p.i. using the reverse transcriptase polymerase chain reaction (RT-PCR. The level of expression of IL-1ß and MIP1-ß was upregulated (P S. Enteritidis + E. faecium AL41 group 48 h p.i. compared to 24 h p.i. Similarly, expression of LITAF was upregulated (P S. Enteritidis (SE group 48 h p.i. In PMBCs treated with E. faecium H31 and S. Enteritidis expression of IL-1ß (P P P E. faecium AL41 demonstrated the highest immunostimulatory effect on expression of selected cytokines by chicken PMBCs after Salmonella infection. It is supposed that the differences in cytokine induction within SE groups are related to lymphocytes isolated from different animals.

  15. Comparison of 432 Pseudomonas strains through integration of genomic, functional, metabolic and expression data

    NARCIS (Netherlands)

    Koehorst, Jasper J.; Dam, van Jesse C.J.; Heck, van Ruben G.A.; Saccenti, Edoardo; Martins dos Santos, Vitor; Suarez-Diez, Maria; Schaap, Peter J.

    2016-01-01

    Pseudomonas is a highly versatile genus containing species that can be harmful to humans and plants while others are widely used for bioengineering and bioremediation. We analysed 432 sequenced Pseudomonas strains by integrating results from a large scale functional comparison using protein

  16. Whole transcriptome expression analysis and comparison of two different strains of Plasmodium falciparum using RNA-Seq

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    Hiasindh Ashmi Antony

    2016-06-01

    Full Text Available The emergence and distribution of drug resistance in malaria are serious public health concerns in tropical and subtropical regions of the world. However, the molecular mechanism of drug resistance remains unclear. In the present study, we performed a high-throughput RNA-Seq to identify and characterize the differentially expressed genes between the chloroquine (CQ sensitive (3D7 and resistant (Dd2 strains of Plasmodium falciparum. The parasite cells were cultured in the presence and absence of CQ by in vitro method. Total RNA was isolated from the harvested parasite cells using TRIzol, and RNA-Seq was conducted using an Illumina HiSeq 2500 sequencing platform with paired-end reads and annotated using Tophat. The transcriptome analysis of P. falciparum revealed the expression of ~5000 genes, in which ~60% of the genes have unknown function. Cuffdiff program was used to identify the differentially expressed genes between the CQ-sensitive and resistant strains. Here, we furnish a detailed description of the experimental design, procedure, and analysis of the transcriptome sequencing data, that have been deposited in the National Center for Biotechnology Information (accession nos. PRJNA308455 and GSE77499.

  17. Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni.

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    Anne E Lockyer

    Full Text Available The outcome of infection in the host snail Biomphalaria glabrata with the digenean parasite Schistosoma mansoni is determined by the initial molecular interplay occurring between them. The mechanisms by which schistosomes evade snail immune recognition to ensure survival are not fully understood, but one possibility is that the snail internal defence system is manipulated by the schistosome enabling the parasite to establish infection. This study provides novel insights into the nature of schistosome resistance and susceptibility in B. glabrata at the transcriptomic level by simultaneously comparing gene expression in haemocytes from parasite-exposed and control groups of both schistosome-resistant and schistosome-susceptible strains, 2 h post exposure to S. mansoni miracidia, using an novel 5K cDNA microarray. Differences in gene expression, including those for immune/stress response, signal transduction and matrix/adhesion genes were identified between the two snail strains and tests for asymmetric distributions of gene function also identified immune-related gene expression in resistant snails, but not in susceptible. Gene set enrichment analysis revealed that genes involved in mitochondrial electron transport, ubiquinone biosynthesis and electron carrier activity were consistently up-regulated in resistant snails but down-regulated in susceptible. This supports the hypothesis that schistosome-resistant snails recognize schistosomes and mount an appropriate defence response, while in schistosome-susceptible snails the parasite suppresses this defence response, early in infection.

  18. Lipase production by recombinant strains of Aspergillus niger expressing a lipase-encoding gene from Thermomyces lanuginosus.

    Science.gov (United States)

    Prathumpai, Wai; Flitter, Simon J; McIntyre, Mhairi; Nielsen, Jens

    2004-11-01

    Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y(xp total)), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7+/-0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3+/-0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r(p total), the sum of extracellular and intracellular lipase productivity) was found to be 1.60+/-0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h(-1), compared with a total specific lipase productivity of 1.10+/-0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.

  19. Expression of the neutral protease gene from a thermophilic Bacillus sp BT1 strain in Bacillus subtilis and its natural host : Identification of a functional promoter

    NARCIS (Netherlands)

    Vecerek, B; Venema, G

    The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp. BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5' to the gene. In

  20. Sphingomonas wittichii Strain RW1 Genome-Wide Gene Expression Shifts in Response to Dioxins and Clay.

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    Benli Chai

    Full Text Available Sphingomonas wittichii strain RW1 (RW1 is one of the few strains that can grow on dibenzo-p-dioxin (DD. We conducted a transcriptomic study of RW1 using RNA-Seq to outline transcriptional responses to DD, dibenzofuran (DF, and the smectite clay mineral saponite with succinate as carbon source. The ability to grow on DD is rare compared to growth on the chemically similar DF even though the same initial dioxygenase may be involved in oxidation of both substrates. Therefore, we hypothesized the reason for this lies beyond catabolic pathways and may concern genes involved in processes for cell-substrate interactions such as substrate recognition, transport, and detoxification. Compared to succinate (SUC as control carbon source, DF caused over 240 protein-coding genes to be differentially expressed, whereas more than 300 were differentially expressed with DD. Stress response genes were up-regulated in response to both DD and DF. This effect was stronger with DD than DF, suggesting a higher toxicity of DD compared to DF. Both DD and DF caused changes in expression of genes involved in active cross-membrane transport such as TonB-dependent receptor proteins, but the patterns of change differed between the two substrates. Multiple transcription factor genes also displayed expression patterns distinct to DD and DF growth. DD and DF induced the catechol ortho- and the salicylate/gentisate pathways, respectively. Both DD and DF induced the shared down-stream aliphatic intermediate compound pathway. Clay caused category-wide down-regulation of genes for cell motility and chemotaxis, particularly those involved in the synthesis, assembly and functioning of flagella. This is an environmentally important finding because clay is a major component of soil microbes' microenvironment influencing local chemistry and may serve as a geosorbent for toxic pollutants. Similar to clay, DD and DF also affected expression of genes involved in motility and chemotaxis.

  1. Sphingomonas wittichii Strain RW1 Genome-Wide Gene Expression Shifts in Response to Dioxins and Clay

    Science.gov (United States)

    Tsoi, Tamara V.; Iwai, Shoko; Liu, Cun; Fish, Jordan A.; Gu, Cheng; Johnson, Timothy A.; Zylstra, Gerben; Teppen, Brian J.; Li, Hui; Hashsham, Syed A.; Boyd, Stephen A.; Cole, James R.; Tiedje, James M.

    2016-01-01

    Sphingomonas wittichii strain RW1 (RW1) is one of the few strains that can grow on dibenzo-p-dioxin (DD). We conducted a transcriptomic study of RW1 using RNA-Seq to outline transcriptional responses to DD, dibenzofuran (DF), and the smectite clay mineral saponite with succinate as carbon source. The ability to grow on DD is rare compared to growth on the chemically similar DF even though the same initial dioxygenase may be involved in oxidation of both substrates. Therefore, we hypothesized the reason for this lies beyond catabolic pathways and may concern genes involved in processes for cell-substrate interactions such as substrate recognition, transport, and detoxification. Compared to succinate (SUC) as control carbon source, DF caused over 240 protein-coding genes to be differentially expressed, whereas more than 300 were differentially expressed with DD. Stress response genes were up-regulated in response to both DD and DF. This effect was stronger with DD than DF, suggesting a higher toxicity of DD compared to DF. Both DD and DF caused changes in expression of genes involved in active cross-membrane transport such as TonB-dependent receptor proteins, but the patterns of change differed between the two substrates. Multiple transcription factor genes also displayed expression patterns distinct to DD and DF growth. DD and DF induced the catechol ortho- and the salicylate/gentisate pathways, respectively. Both DD and DF induced the shared down-stream aliphatic intermediate compound pathway. Clay caused category-wide down-regulation of genes for cell motility and chemotaxis, particularly those involved in the synthesis, assembly and functioning of flagella. This is an environmentally important finding because clay is a major component of soil microbes’ microenvironment influencing local chemistry and may serve as a geosorbent for toxic pollutants. Similar to clay, DD and DF also affected expression of genes involved in motility and chemotaxis. PMID:27309357

  2. Repression of Proteases and Hsp90 Chaperone Expression Induced by an Antiretroviral in Virulent Environmental Strains of Cryptococcus neoformans.

    Science.gov (United States)

    Serafin, Cleber Fernando; Paris, Ana Paula; Paula, Claudete Rodrigues; Simão, Rita Cássia Garcia; Gandra, Rinaldo Ferreira

    2017-04-01

    This study evaluated the effect of the antiretroviral ritonavir on protease secretion in different strains of Cryptococcus neoformans isolated from the environment and investigated the expression of heat shock protein (Hsp90), classically described virulence factors in other yeast in the presence of the same antiretroviral. The presence of the enzyme was detected by the formation of a degradation of the halo around the colonies. The results were classified as follows: level 1 (without proteases), level 2 (positive for proteases), and level 3 (strongly positive for proteases). Total protein extract isolated from the cell walls of the 12 strains incubated in the absence and presence of ritonavir (0.3125 mg mL -1 ) were resolved by SDS-PAGE and analyzed by Western blot assays using an antiserum against Hsp90 from Blastocladiella emersonii. All strains tested showed inhibition of proteinase activity in the presence of ritonavir at 0.3125 to 1.25 mg mL -1 . High levels of Hsp90 were observed in the absence of ritonavir (0.3125 mg mL -1 ), except for the non-virulent control cells. In contrast, in the presence of the antiretroviral, a drastic reduction in the expression of the chaperone was observed. The data suggest that ritonavir, in addition to containing viral replication, could inhibit the expression of virulence factors in opportunistic yeast, as proteases and Hsp90. According to our current knowledge, this is the first time that the inhibition of Hsp90 by an antiretroviral was reported for environmental isolates of C. neoformans.

  3. Exploring suppression subtractive hybridization (SSH) for discriminating Lactococcus lactis ssp. cremoris SK11 and ATCC 19257 in mixed culture based on the expression of strain-specific genes.

    Science.gov (United States)

    Ndoye, B; Lessard, M-H; LaPointe, G; Roy, D

    2011-02-01

    An approach based on quantitative reverse transcriptase PCR (RT-qPCR) was developed for monitoring two strains of lactococci in co-culture in milk by measuring the expression of specific genes identified by suppression subtractive hybridization (SSH). SSH was used to identify strain-specific genes of Lactococcus lactis ssp. cremoris SK11 and ATCC 19257. RT-qPCR was then employed to validate gene specificity and compare the expression of selected specific genes (glycosyltransferase and amidase genes for L. lactis ssp. cremoris ATCC 19257 and a hypothetical protein for SK11) identified by SSH. The time profile of changes in gene expression relative to ldh transcription differed between pure and mixed cultures as well as between media. At the stationary phase, gene expression of mixed cultures in GM17 attained the highest proportion of ldh transcription while mixed cultures in milk peaked at the postexponential phase. Strain ratios expressed as RNA proportion appear to favour SK11 in GM17 medium, while ATCC 19257 dominated in milk co-cultures. This approach was useful to determine the contribution of strain SK11 in relation to strain ATCC 19257 during co-culture in milk compared to rich medium. The ability to track the metabolic contribution of each lactococcal strain during fermentation of milk or cheese ripening will extend our understanding of the impact of process parameters on the production performance of strains. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

  4. Imperatorin inhibits the expression of alpha-hemolysin in Staphylococcus aureus strain BAA-1717 (USA300).

    Science.gov (United States)

    Ouyang, Ping; Chen, Junjie; Sun, Mao; Yin, Zhongqiong; Lin, Juchun; Fu, Hualin; Shu, Gang; He, Changliang; Lv, Cheng; Deng, Xuming; Wang, Kaiyu; Geng, Yi; Yin, Lizi

    2016-07-01

    Both community-associated and hospital-acquired infections with methicillin-resistant Staphylococcus aureus (MRSA) have been increasingly reported around the world in the past 20 years. In 2006, the Centers for Disease Control and Prevention reported that 64 % of MRSA isolates were of the USA300 clonal type in infected patients in USA. The aim of our study was to estimate the in vitro effect of imperatorin on MRSA strain BAA-1717 (USA300). The effects of imperatorin on alpha-hemolysin (Hla) production, when strain BAA-1717 was co-cultured with sub-inhibitory concentrations of imperatorin, were analysed using susceptibility testing, hemolysis assays, western blotting and real-time PCR. Live/Dead analysis and cytotoxicity assays were employed to examine the protective effect of imperatorin against the strain BAA-1717-mediated injury of human alveolar epithelial cells (A549). The results showed that imperatorin has no anti-S. aureus activity at the tested concentrations in vitro. However, imperatorin can observably inhibit the production of Hla in culture supernatants and reduce the transcriptional levels of hla (the gene encoding Hla) and arg (the accessory gene regulator). Imperatorin prevented Hla-mediated A549 epithelial cell injury in a co-culture system. In conclusion, our results suggested that imperatorin has the potential to be developed as a new anti-virulence drug candidate for managing S. aureus infection.

  5. Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice.

    Directory of Open Access Journals (Sweden)

    Dejia Li

    2010-12-01

    Full Text Available Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv mice express a near-full length dystrophin protein at ∼5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.

  6. Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain.

    Science.gov (United States)

    Wilde, Caroline; Gold, Nicholas D; Bawa, Nancy; Tambor, José Humberto M; Mougharbel, Lina; Storms, Reginald; Martin, Vincent J J

    2012-08-01

    Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.

  7. Strain Stimulations with Different Intensities on Fibroblast Viability and Protein Expression

    Directory of Open Access Journals (Sweden)

    Jia Ying

    2017-10-01

    Full Text Available Mechanical stimulation via acupuncture and tuina massage triggers various cell responses. This study aims to understand these cellular bio-physical mechanisms by investigating the effect of different stimulation intensities on cell viability and protein expression.

  8. [Establishment of 5 resistant ovarian cancer cell strains and expression of resistance-related genes].

    Science.gov (United States)

    Luan, Ying-zi; Li, Li; Li, Dang-rong; Zhang, Wei; Tang, Bu-jian

    2004-06-01

    To investigate expression difference of several drug resistance related genes between sensitive and resistant ovarian carcinoma cells. Cell lines resistant to cisplatin, carboplatin and taxol were established from ovarian carcinoma cell lines of SKOV3 and A2780, and their biological features were detected. The expressions of several genes related to drug resistance were measured by RT-PCR method. (1) The values of resistance index (RI) of resistant cells to relevant drugs were elevated 3 times or more, with different degrees of cross-resistance to several other drugs (RI 2 approximately 20). They grew more slowly than primary cells (Td elongated 1.4 approximately 2.4 times, P 0.05). Intracellular concentrations of relevant drugs were reduced 2.0 approximately 8.5 times in resistant cells (P p53, lung resistance protein-1 (LRP-1), multiple drug resistance related protein-1 (MRP-1) genes were expressed at lower levels in resistant cells than in sensitive cells; while protein kinase C (PKC), topoisomerase (topo) I, and topo II beta were expressed higher, no obvious alterations were found concerning glutathione S transferase-pi (GST-pi), and topo II alpha. Expression of multiple drug resistance-1 (MDR-1) gene was either elevated or reduced in different cells. The expressions of resistance related genes were widely different in different kinds of resistant cells, suggesting more than one pathway leading to resistance transformation. This adds more difficulties for clinical management.

  9. Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

    International Nuclear Information System (INIS)

    Joo Lee, Pom; Ahn, Ji-Young; Kim, Yang-Hoon; Wook Kim, Seung; Kim, Ji-Yeon; Park, Jae-Sung; Lee, Jeewon

    2004-01-01

    We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus (AJ308438), Photorhabdus luminescens W14 (AF346499) P. luminescens TTO1 (BX571873), and Yersinia pestis CO92 (NC 0 03143). The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity

  10. Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

    Science.gov (United States)

    Ye, Xudong; Chen, Yurong; Wan, Yuechun; Hong, Yun-Jeong; Ruebelt, Martin C; Gilbertson, Larry A

    2016-03-01

    KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells.

  11. Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

    Directory of Open Access Journals (Sweden)

    Schaudinn Christoph

    2005-01-01

    Full Text Available Abstract Background Serotyping of O-(lipopolysaccharide and H-(flagellar antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies. Results The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4 and P12b (O15:H17 was determined and both were found 99.3% (1043 of 1050 nucleotides identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella. Conclusion The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of

  12. Mechanical strain stimulates vasculogenesis and expression of angiogenesis guidance molecules of embryonic stem cells through elevation of intracellular calcium, reactive oxygen species and nitric oxide generation.

    Science.gov (United States)

    Sharifpanah, Fatemeh; Behr, Sascha; Wartenberg, Maria; Sauer, Heinrich

    2016-12-01

    Differentiation of embryonic stem (ES) cells may be regulated by mechanical strain. Herein, signaling molecules underlying mechanical stimulation of vasculogenesis and expression of angiogenesis guidance cues were investigated in ES cell-derived embryoid bodies. Treatment of embryoid bodies with 10% static mechanical strain using a Flexercell strain system significantly increased CD31-positive vascular structures and the angiogenesis guidance molecules plexinB1, ephrin B2, neuropilin1 (NRP1), semaphorin 4D (sem4D) and robo4 as well as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) as evaluated by Western blot and real time RT-PCR. In contrast ephrin type 4 receptor B (EphB4) expression was down-regulated upon mechanical strain, indicating an arterial-type differentiation. Robo1 protein expression was modestly increased with no change in mRNA expression. Mechanical strain increased intracellular calcium as well as reactive oxygen species (ROS) and nitric oxide (NO). Mechanical strain-induced vasculogenesis was abolished by the NOS inhibitor L-NAME, the NADPH oxidase inhibitor VAS2870, upon chelation of intracellular calcium by BAPTA as well as upon siRNA inactivation of ephrin B2, NRP1 and robo4. BAPTA blunted the strain-induced expression of angiogenic growth factors, the increase in NO and ROS as well as the expression of NRP1, sem4D and plexinB1, whereas ephrin B2, EphB4 as well as robo1 and robo4 expression were not impaired. Mechanical strain stimulates vasculogenesis of ES cells by the intracellular messengers ROS, NO and calcium as well as by upregulation of angiogenesis guidance molecules and the angiogenic growth factors VEGF, FGF-2 and PDGF-BB. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Gene expression variation resolves species and individual strains among coral-associated dinoflagellates within the genus Symbiodinium

    KAUST Repository

    Parkinson, John Everett

    2016-02-11

    Reef-building corals depend on symbiotic mutualisms with photosynthetic dinoflagellates in the genus Symbiodinium. This large microalgal group comprises many highly divergent lineages (“Clades A-I”) and hundreds of undescribed species. Given their ecological importance, efforts have turned to genomic approaches to characterize the functional ecology of Symbiodinium. To date, investigators have only compared gene expression between representatives from separate clades—the equivalent of contrasting genera or families in other dinoflagellate groups—making it impossible to distinguish between clade-level and species-level functional differences. Here, we examined the transcriptomes of four species within one Symbiodinium clade (Clade B) at ~20,000 orthologous genes, as well as multiple isoclonal cell lines within species (i.e. cultured strains). These species span two major adaptive radiations within Clade B, each encompassing both host-specialized and ecologically cryptic taxa. Species-specific expression differences were consistently enriched for photosynthesis-related genes, likely reflecting selection pressures driving niche diversification. Transcriptional variation among strains involved fatty acid metabolism and biosynthesis pathways. Such differences among individuals are potentially a major source of physiological variation, contributing to the functional diversity of coral holobionts composed of unique host-symbiont genotype pairings. Our findings expand the genomic resources available for this important symbiont group and emphasize the power of comparative transcriptomics as a method for studying speciation processes and inter-individual variation in non-model organisms.

  14. Gene Expression Variation Resolves Species and Individual Strains among Coral-Associated Dinoflagellates within the Genus Symbiodinium.

    Science.gov (United States)

    Parkinson, John E; Baumgarten, Sebastian; Michell, Craig T; Baums, Iliana B; LaJeunesse, Todd C; Voolstra, Christian R

    2016-02-11

    Reef-building corals depend on symbiotic mutualisms with photosynthetic dinoflagellates in the genus Symbiodinium. This large microalgal group comprises many highly divergent lineages ("Clades A-I") and hundreds of undescribed species. Given their ecological importance, efforts have turned to genomic approaches to characterize the functional ecology of Symbiodinium. To date, investigators have only compared gene expression between representatives from separate clades-the equivalent of contrasting genera or families in other dinoflagellate groups-making it impossible to distinguish between clade-level and species-level functional differences. Here, we examined the transcriptomes of four species within one Symbiodinium clade (Clade B) at ∼20,000 orthologous genes, as well as multiple isoclonal cell lines within species (i.e., cultured strains). These species span two major adaptive radiations within Clade B, each encompassing both host-specialized and ecologically cryptic taxa. Species-specific expression differences were consistently enriched for photosynthesis-related genes, likely reflecting selection pressures driving niche diversification. Transcriptional variation among strains involved fatty acid metabolism and biosynthesis pathways. Such differences among individuals are potentially a major source of physiological variation, contributing to the functional diversity of coral holobionts composed of unique host-symbiont genotype pairings. Our findings expand the genomic resources available for this important symbiont group and emphasize the power of comparative transcriptomics as a method for studying speciation processes and interindividual variation in nonmodel organisms. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Acetylsalicylic acid differentially limits the activation and expression of cell death markers in human platelets exposed to Staphylococcus aureus strains.

    Science.gov (United States)

    Chabert, Adrien; Damien, Pauline; Verhoeven, Paul O; Grattard, Florence; Berthelot, Philippe; Zeni, Fabrice; Panicot-Dubois, Laurence; Robert, Stéphane; Dignat-George, Françoise; Eyraud, Marie-Ange; Pozzetto, Bruno; Payrastre, Bernard; Cognasse, Fabrice; Garraud, Olivier; Hamzeh-Cognasse, Hind

    2017-07-17

    Beyond their hemostatic functions, platelets alter their inflammatory response according to the bacterial stimulus. Staphylococcus aureus is associated with exacerbated inflammation and thrombocytopenia, which is associated with poor prognosis during sepsis. Acetylsalicylic acid and statins prevent platelet aggregation and decrease the mortality rate during sepsis. Therefore, we assessed whether these two molecules could reduce in vitro platelet activation and the inflammatory response to S. aureus. Platelets were exposed to clinical strains of S. aureus in the presence or absence of acetylsalicylic acid or fluvastatin. Platelet activation, aggregation, and release of soluble sCD62P, sCD40 Ligand, RANTES and GROα were assessed. Platelet cell death was evaluated by analyzing the mitochondrial membrane potential, phosphatidylserine exposure, platelet microparticle release and caspase-3 activation. All S. aureus strains induced platelet activation but not aggregation and decreased the platelet count, the expression of cell death markers and the release of RANTES and GROα. Acetylsalicylic acid but not fluvastatin limited platelet activation and inflammatory factor release and restored the platelet count by protecting platelets from Staphylococcus-induced expression of cell death markers. This study demonstrates that acetylsalicylic acid limits S. aureus-induced effects on platelets by reducing cell death, revealing new strategies to reduce the platelet contribution to bacteremia-associated inflammation.

  16. Mushroom body miscellanea: transgenic Drosophila strains expressing anatomical and physiological sensor proteins in Kenyon cells

    Science.gov (United States)

    Pech, Ulrike; Dipt, Shubham; Barth, Jonas; Singh, Priyanka; Jauch, Mandy; Thum, Andreas S.; Fiala, André; Riemensperger, Thomas

    2013-01-01

    The fruit fly Drosophila melanogaster represents a key model organism for analyzing how neuronal circuits regulate behavior. The mushroom body in the central brain is a particularly prominent brain region that has been intensely studied in several insect species and been implicated in a variety of behaviors, e.g., associative learning, locomotor activity, and sleep. Drosophila melanogaster offers the advantage that transgenes can be easily expressed in neuronal subpopulations, e.g., in intrinsic mushroom body neurons (Kenyon cells). A number of transgenes has been described and engineered to visualize the anatomy of neurons, to monitor physiological parameters of neuronal activity, and to manipulate neuronal function artificially. To target the expression of these transgenes selectively to specific neurons several sophisticated bi- or even multipartite transcription systems have been invented. However, the number of transgenes that can be combined in the genome of an individual fly is limited in practice. To facilitate the analysis of the mushroom body we provide a compilation of transgenic fruit flies that express transgenes under direct control of the Kenyon-cell specific promoter, mb247. The transgenes expressed are fluorescence reporters to analyze neuroanatomical aspects of the mushroom body, proteins to restrict ectopic gene expression to mushroom bodies, or fluorescent sensors to monitor physiological parameters of neuronal activity of Kenyon cells. Some of the transgenic animals compiled here have been published already, whereas others are novel and characterized here for the first time. Overall, the collection of transgenic flies expressing sensor and reporter genes in Kenyon cells facilitates combinations with binary transcription systems and might, ultimately, advance the physiological analysis of mushroom body function. PMID:24065891

  17. High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526

    Directory of Open Access Journals (Sweden)

    Feng Hang

    2016-01-01

    Full Text Available Paenibacillus spp. BD3526, a bacterium exhibiting a protein hydrolysis circle surrounded with an obvious precipitation zone on skim milk agar, was isolated from raw yak (Bos grunniens milk collected in Tibet, China. Phylogenetic analysis based on 16S rRNA and whole genome sequence comparison indicated the isolate belong to the genus Paenibacillus. The strain BD3526 demonstrated strong ability to produce protease with milk clotting activity (MCA in wheat bran broth. The protease with MCA was predominantly accumulated during the late-exponential phase of growth. The proteolytic activity (PA of the BD3526 protease was 1.33-fold higher than that of the commercial R. miehei coagulant. A maximum MCA (6470 ± 281 SU mL−1 of the strain BD3526 was reached under optimal cultivation conditions. The protease with MCA was precipitated from the cultivated supernatant of wheat bran broth with ammonium sulfate and purified by anion-exchange chromatography. The molecular weight of the protease with MCA was determined as 35 kDa by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE and gelatin zymography. The cleavage site of the BD3526 protease with MCA in κ-casein was located at the Met106–Ala107 bond, as determined by mass spectrometry analysis.

  18. Dissecting the effect of genetic variation on the hepatic expression of drug disposition genes across the collaborative cross mouse strains

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    Aharon Nachshon

    2016-10-01

    Full Text Available A central challenge in pharmaceutical research is to investigate genetic variation in response to drugs. The Collaborative Cross (CC mouse reference population is a promising model for pharmacogenomic studies because of its large amount of genetic variation, genetic reproducibility, and dense recombination sites. While the CC lines are phenotypically diverse, their genetic diversity in drug disposition processes, such as detoxification reactions, is still largely uncharacterized. Here we systematically measured RNA-sequencing expression profiles from livers of 29 CC lines under baseline conditions. We then leveraged a reference collection of metabolic biotransformation pathways to map potential relations between drugs and their underlying expression quantitative trait loci (eQTLs. By applying this approach on proximal eQTLs, including eQTLs acting on the overall expression of genes and on the expression of particular transcript isoforms, we were able to construct the organization of hepatic eQTL-drug connectivity across the CC population. The analysis revealed a substantial impact of genetic variation acting on drug biotransformation, allowed mapping of potential joint genetic effects in the context of individual drugs, and demonstrated crosstalk between drug metabolism and lipid metabolism. Our findings provide a resource for investigating drug disposition in the CC strains, and offer a new paradigm for integrating biotransformation reactions to corresponding variations in DNA sequences.

  19. Salmonella Typhimurium induces SPI-1 and SPI-2 regulated and strain dependent downregulation of MHC II expression on porcine alveolar macrophages

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    Van Parys Alexander

    2012-06-01

    Full Text Available Abstract Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Circumvention of the host’s immune system by Salmonella might contribute to persistent infection of pigs. In the present study, we found that Salmonella Typhimurium strain 112910a specifically downregulated MHC II, but not MHC I, expression on porcine alveolar macrophages in a Salmonella pathogenicity island (SPI-1 and SPI-2 dependent way. Salmonella induced downregulation of MHC II expression and intracellular proliferation of Salmonella in macrophages were significantly impaired after opsonization with Salmonella specific antibodies prior to inoculation. Furthermore, the capacity to downregulate MHC II expression on macrophages differed significantly among Salmonella strains, independently of strain specific differences in invasion capacity, Salmonella induced cytotoxicity and altered macrophage activation status. The fact that strain specific differences in MHC II downregulation did not correlate with the extent of in vitro SPI-1 or SPI-2 gene expression indicates that other factors are involved in MHC II downregulation as well. Since Salmonella strain dependent interference with the pig’s immune response through downregulation of MHC II expression might indicate that certain Salmonella strains are more likely to escape serological detection, our findings are of major interest for Salmonella monitoring programs primarily based on serology.

  20. Differential mRNA expression of seven genes involved in cholesterol metabolism and transport in the liver of atherosclerosis-susceptible and -resistant Japanese quail strains

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    Li Xinrui

    2012-06-01

    Full Text Available Abstract Background Two atherosclerosis-susceptible and -resistant Japanese quail (Coturnix japonica strains obtained by divergent selection are commonly used as models to study atherosclerosis, but no genetic characterization of their phenotypic differences has been reported so far. Our objective was to examine possible differences in the expression of genes involved in cholesterol metabolism and transport in the liver between these two strains and to evaluate the value of this model to analyze the gene system affecting cholesterol metabolism and transport. Methods A factorial study with both strains (atherosclerosis-susceptible versus atherosclerosis-resistant and two diets (control versus cholesterol was carried out. The mRNA concentrations of four genes involved in cholesterol biosynthesis (HMGCR, FDFT1, SQLE and DHCR7 and three genes in cholesterol transport (ABCG5, ABCG8 and APOA1 were assayed using real-time quantitative PCR. Plasma lipids were also assayed. Results Expression of ABCG5 (control diet and ABCG8 (regardless of dietary treatment and expression of HMGCR, FDFT1 and SQLE (regardless of dietary treatment were significantly higher in the atherosclerosis-resistant than in the atherosclerosis-susceptible strain. Plasma triglyceride and LDL levels, and LDL/HDL ratio were significantly higher in the atherosclerosis-susceptible than in the atherosclerosis-resistant strain fed the cholesterol diet. In the atherosclerosis-susceptible strain, ABCG5 expression regressed significantly and positively on plasma LDL level, whereas DHCR7 and SQLE expression regressed significantly and negatively on plasma triglyceride level. Conclusions Our results provide support for the hypothesis that the atherosclerosis-resistant strain metabolizes and excretes cholesterol faster than the atherosclerosis-susceptible strain. We have also demonstrated that these quail strains are a useful model to study cholesterol metabolism and transport in relation with

  1. Expression of of VP60 gene from RHDV YL strain under control of ...

    African Journals Online (AJOL)

    zdx

    2012-06-19

    Jun 19, 2012 ... stability, bioactivity and favorable pharmacokinetics. (Gomord and Faye, 2004). So far, several plant species suitable for the expression of recombinant proteins with technical and pharmaceutical value have been described. Obvious advantages of plant-derived vaccines are the convenient storage of the.

  2. Gene expression profiling of a temperature-sensitive strain of Neospora caninum

    Science.gov (United States)

    To understand the genetic basis of virulence, gene expression profiles of a temperature-sensitive clone (NCts-8, relatively avirulent) and its wild type (NC-1) of Neospora caninum were characterized and compared using a high-density microarray with approximately 63,000 distinct oligonucleotides. Thi...

  3. Polygenic expression of teratozoospermia and normal fertility in B10.MOL-TEN1 mouse strain.

    Science.gov (United States)

    Hirawatari, Keitaro; Hanzawa, Naoto; Kuwahara, Maki; Aoyama, Hiroaki; Miura, Ikuo; Wakana, Shigeharu; Gotoh, Hideo

    2015-05-01

    Subfertility and infertility are two major reproductive health problems in human and domestic animals. The contribution of the genotype to these conditions is poorly understood. To examine the genetic basis of male subfertility, we analyzed its relationship to sperm morphology in B10.MOL-TEN1 mice, which shows high-frequencies (about 50%) of morphologically abnormal sperm. Drastic histological changes were also found in the testis of the B10.MOL-TEN1. Segregation analysis showed that the abnormal sperm phenotype in B10.MOL-TEN1 was inherited and was predictably controlled by at least three loci. We also found that male fertility of this strain was normal. These findings indicate a complicated relationship between sperm morphology and male subfertility. © 2015 The Authors. Congenital Anomalies published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Teratology Society.

  4. Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

    Science.gov (United States)

    Itoh, Takafumi; Hibi, Takao; Fujii, Yutaka; Sugimoto, Ikumi; Fujiwara, Akihiro; Suzuki, Fumiko; Iwasaki, Yukimoto; Kim, Jin-Kyung; Taketo, Akira; Kimoto, Hisashi

    2013-12-01

    Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

  5. Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost

    Directory of Open Access Journals (Sweden)

    Amore Antonella

    2012-12-01

    Full Text Available Abstract Background The use of lignocellulosic materials for second generation ethanol production would give several advantages such as minimizing the conflict between land use for food and fuel production, providing less expensive raw materials than conventional agricultural feedstock, allowing lower greenhouse gas emissions than those of first generation ethanol. However, cellulosic biofuels are not produced at a competitive level yet, mainly because of the high production costs of the cellulolytic enzymes. Therefore, this study was aimed at discovering new cellulolytic microorganisms and enzymes. Results Different bacteria isolated from raw composting materials obtained from vegetable processing industry wastes were screened for their cellulolytic activity on solid medium containing carboxymethylcellulose. Four strains belonging to the actinomycetes group were selected on the basis of their phenotypic traits and cellulolytic activity on solid medium containing carboxymethylcellulose. The strain showing the highest cellulolytic activity was identified by 16S rRNA sequencing as belonging to Streptomyces genus and it was designated as Streptomyces sp. strain G12. Investigating the enzymes responsible for cellulase activity produced by Streptomyces G12 by proteomic analyses, two endoglucanases were identified. Gene coding for one of these enzymes, named CelStrep, was cloned and sequenced. Molecular analysis showed that the celstrep gene has an open reading frame encoding a protein of 379 amino acid residues, including a signal peptide of 37 amino acid residues. Comparison of deduced aminoacidic sequence to the other cellulases indicated that the enzyme CelStrep can be classified as a family 12 glycoside hydrolase. Heterologous recombinant expression of CelStrep was carried out in Escherichia coli, and the active recombinant enzyme was purified from culture supernatant and characterized. It catalyzes the hydrolysis of carboxymethylcellulose

  6. Tetracycline alters gene expression in Salmonella strains that harbor the Tn10 transposon.

    Science.gov (United States)

    Hüttener, M; Prieto, A; Aznar, S; Dietrich, M; Paytubi, S; Juárez, A

    2018-04-01

    In this report, we show that bacterial plasmids that harbor the Tn10 transposon (i.e., the IncHI1 plasmid R27) modify expression of different Salmonella regulons responding to the presence of tetracycline (Tc) in the medium. By using as a model the Tc-dependent upregulation of the ibpAB operon (which belongs to the heat shock regulon), we have identified Tn10-tetA (coding for a Tc efflux pump) and adjacent tetC sequences as required for ibpAB upregulation. Characterization of transcripts in the tetAC region showed that tetA transcription can continue into tetC sequences, generating a long 3'UTR sequence, which can protect transcripts from RNA processing, thus increasing the expression of TetA protein. In the presence of Tc, the DnaK and IbpA chaperones are overexpressed and translocated to the periplasm and to the membrane fraction respectively. DnaK targeting unfolded proteins is known to induce heat shock by avoiding RpoH proteolysis. We correlate expression levels of Tn10-encoded TetA protein with heat shock induction in Salmonella, likely because TetA activity compromises protein secretion. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Differences in Shiga toxin and phage production among stx2g-positive STEC strains

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    Claudia Viviana Granobles Velandia

    2012-06-01

    Full Text Available Shigatoxigenic E. coli (STEC are characterized by the production of Shiga toxins (Stx encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness.The variant named stx2g was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx2g-positive strains isolated from humans, animals and environmental sources have shown that they have a close relationship. In this study, stx2g-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity.The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62 hybridized with a stx2-probe. Furthermore, the production of Stx was evaluated by EIA and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal with induced than in uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx2g-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected.Taking into account all the results, several differences could be found among stx2g-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx2-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx2g-positive strains Stx production is

  8. Expression of zinc transporter genes in rice as influenced by zinc-solubilizing Enterobacter cloacae strain ZSB14

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    Selvaraj eKrithika

    2016-04-01

    Full Text Available Zinc (Zn deficiency in major food crops has been considered as an important factor affecting the crop production and subsequently the human health. Rice (Oryza sativa is sensitive to Zn deficiency and thereby causes malnutrition to most of the rice-eating Asian populations. Application of zinc solubilizing bacteria (ZSB could be a sustainable agronomic approach to increase the soil available Zn which can mitigate the yield loss and consequently the nutritional quality of rice. Understanding the molecular interactions between rice and unexplored ZSB is useful for overcoming Zn deficiency problems. In the present study, the role of zinc solubilizing bacterial strain Enterobacter cloacae strain ZSB14 on regulation of Zn-regulated transporters and iron (Fe-regulated transporter-like protein (ZIP genes in rice under iron sufficient and deficient conditions was assessed by quantitative real-time reverse transcription PCR. The expression patterns of OsZIP1, OsZIP4 and OsZIP5 in root and shoot of rice were altered due to the Zn availability as dictated by Zn sources and ZSB inoculation. Fe sufficiency significantly reduced the root and shoot OsZIP1 expression, but not the OsZIP4 and OsZIP5 levels. Zinc oxide in the growth medium up-regulated all the assessed ZIP genes in root and shoot of rice seedlings. When ZSB was inoculated to rice seedlings grown with insoluble zinc oxide in the growth medium, the expression of root and shoot OsZIP1, OsZIP4 and OsZIP5 was reduced. In the absence of zinc oxide, ZSB inoculation up-regulated OsZIP1 and OsZIP5 expressions. Zinc nutrition provided to the rice seedling through ZSB-bound zinc oxide solubilization was comparable to the soluble zinc sulphate application which was evident through the ZIP genes’ expression and the Zn accumulation in root and shoot of rice seedlings. These results demonstrate that zinc solubilizing bacteria could play a crucial role in zinc fertilization and fortification of rice.

  9. A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

    Energy Technology Data Exchange (ETDEWEB)

    Thorgersen, Michael P. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Lancaster, W. Andrew [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Rajeev, Lara [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Ge, Xiaoxuan [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Vaccaro, Brian J. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Poole, Farris L. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Arkin, Adam P. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Mukhopadhyay, Aindrila [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Adams, Michael W. W. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology

    2016-12-02

    Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by

  10. Active Expression of Human Tissue Plasminogen Activator (t-PA) c-DNA from Pulmonary Metastases in the Methylotrophic Yeast Pichia Pastoris KM71H Strain

    Science.gov (United States)

    Mohseni, Amir Hossein; Soleimani, Mohammad; Majidzadeh-A, Keivan; Taghinezhad-S, Sedigheh; Keyvani, Hossein

    2017-08-27

    Background: Human tissue-type plasminogen activator (t-PA) is a key protease of the trypsin family. It catalyzes the activation of zymogen plasminogen to the fibrin-degrading proteinase, plasmin, leading to digestion of fibrin clots. The recombinant enzyme produced by recombinant technology issued to dissolve blood clots in treatment of various human diseases such as coronary artery thrombosis, pulmonary embolism, acute ischemic stroke (AIS). Pichia pastoris expression system is a unique system for the production of high level of recombinant proteins. GS115 and KM71H are two kinds of Pichia pastoris strains whilst production of recombinant proteins in these strains is not predictable. The aim of the study was evaluation of t-PA expression in KM71H strains. Methods: In this study, the cDNA of the t-PA gene was amplified by PCR, sequenced and cloned into Pichia pastoris KM71H host strain using pPICZalphaA expression vector that allows methanol-induced expression and secretion of the protein. Results: Dot blotting results confirmed the presence oft-PA in the cell supernatant. Western blotting test revealed the approximate size of 70 KDa for recombinant t-PA. Quantitative ELISA experiment showed 810 μg/L of t-PA in the supernatant samples. Zymography analysis confirmed the proteolytic activity and biological function of the expressed recombinant t-PA. Conclusions: Correspondingly, Pichia pastoris KM71H is an appropriate strain for production of active recombinant protein. Creative Commons Attribution License

  11. Oral immunization of mice with engineered Lactobacillus gasseri NM713 strain expressing Streptococcus pyogenes M6 antigen.

    Science.gov (United States)

    Mansour, Nahla M; Abdelaziz, Sahar A

    2016-08-01

    The aim of this in vivo study was to evaluate the effects of a recombinant probiotic strain, Lactobacillus gasseri NM713, which expresses the conserved region of streptococcal M6 protein (CRR6), as an oral vaccine against Streptococcus pyogenes. A dose of 10(9) cells of the recombinant strain in 150 μL PBS buffer was administered orally to a group of mice. One control group received an equivalent dose of Lb. gasseri NM613 (containing the empty plasmid without insert) or and another control group received PBS buffer. Each group contained 30 mice. The immunization protocol was followed on three consecutive days, after which two booster doses were administered at two week intervals. Fecal and serum samples were collected from the mice on Days 18, 32, 46, 58 after the first immunization and Day 0 prior to immunization. Anti-CRR6 IgA and IgG concentrations were measured by ELISA in fecal and sera samples, respectively, to assess immune responses. Vaccination with the recombinant Lb. gasseri NM713 strain induced significant protection after nasal challenge with S. pyogenes, only a small percentage of this group developing streptococcal infection (10%) or dying of it (3.3%) compared with the NM613 and PBS control groups, high percentages of which developed streptococcal infection (43.3% and 46.7%, respectively) and died of it (46.7% and 53%, respectively). These results indicate that recombinant Lb. gasseri NM713 has potential as an oral delivery vaccine against streptococcus group A. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  12. Marked inbred mouse strain difference in the expression of quinpirole induced compulsive like behavior based on behavioral pattern analysis.

    Science.gov (United States)

    de Haas, Ria; Seddik, Amir; Oppelaar, Hugo; Westenberg, Herman G M; Kas, Martien J H

    2012-09-01

    Obsessive-compulsive disorder (OCD) is a chronic and complex psychiatric disorder with a lifetime prevalence of 2-3%. Recent work has shown that OCD rituals were not only characterized by a high rate of repetition but also by an increased behavioral repertoire due to additional non-functional unique acts. These two behavioral characteristics may provide an ethological basis for studying compulsive behavior in an animal model of OCD. Here, quinpirole induced behavior (so far only investigated in rats) has been studied in A/J and C57BL/6J mice by using behavioral pattern analysis. The aim of this study is to investigate whether genetic background is mediating this behavior. Results showed that open field motor activity levels of saline treated C57BL/6J mice was significantly higher compared to A/J treated saline mice. Long-term quinpirole treatment increased open field motor activity levels in A/J, but not in C57BL/6J. Quinpirole treatment induced a strain dependent difference in behavioral repertoire. There was a dose dependent increase in the number of different behavioral patterns in A/J, whereas, in C57BL/6J there was a dose dependent decrease. This data suggest that genetic background is important in expressing quinpirole induced compulsive like behavior. Following quinpirole treatment, A/J mice express a greater behavioral repertoire with a high rate of repetition. This phenotype resembles that of OCD rituals in patients and indicates that this strain is very interesting to further validate for studying neurobiological mechanisms of compulsive behavior. Copyright © 2012. Published by Elsevier B.V.

  13. Molecular Cloning, Expression and Characterization of Plasmid Encoding Rhomboid 4 (ROM4 of Tachyzoite of Toxoplasma gondii RH Strain

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    Mohammad Taghi RAHIMI

    2017-12-01

    Full Text Available AbstractBackground: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 (ROM4 proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of T. gondii in an appropriate expression vector and eukaryotic cell for production of recombinant protein.Methods: Toxoplasma RNA was isolated from tachyzoites (RH strain and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on Toxoplasma ROM4 gene sequence with XhoI and EcoRI restriction sites at 5´ end of forward and reverse primers, respectively. ROM4 gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.Results: Cloning of ROM4 gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned ROM4 gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.Conclusion: This eukaryotic expression system is an appropriate system for high-level recombinant protein production of ROM4 gene from T. gondii tachyzoites used as antigenic component for serological assay and vaccine development.

  14. Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Egel-Mitani; Andersen; Diers; Hach; Thim; Hastrup; Vad

    2000-06-01

    Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.

  15. High density growth of T7 expression strains with auto-induction option

    Energy Technology Data Exchange (ETDEWEB)

    Studier, F. William (Stony Brook, NY)

    2010-07-20

    A bacterial growth medium for promoting auto-induction of transcription of cloned DNA in cultures of bacterial cells grown batchwise is disclosed. The transcription is under the control of a lac repressor. Also disclosed is a bacterial growth medium for improving the production of a selenomethionine-containing protein or polypeptide in a bacterial cell, the protein or polypeptide being produced by recombinant DNA techniques from a lac or T7lac promoter, the bacterial cell encoding a vitamin B12-dependent homocysteine methylase. Finally, disclosed is a bacterial growth medium for suppressing auto-induction of expression in cultures of bacterial cells grown batchwise, said transcription being under the control of lac repressor.

  16. GAL promoter-driven heterologous gene expression in Saccharomyces cerevisiae Δ strain at anaerobic alcoholic fermentation.

    Science.gov (United States)

    Ahn, Jungoh; Park, Kyung-Min; Lee, Hongweon; Son, Yeo-Jin; Choi, Eui-Sung

    2013-02-01

    The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P(GAL10) ) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. P(GAL10) in the Δgal80 mutant showed 0.8-fold (ADH1), fourfold (PDC1), and 50-fold (PGK) in promoter strength. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  17. Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins.

    Science.gov (United States)

    Vemulapalli, Ramesh; Sanakkayala, Neelima; Gulani, Jatinder; Schurig, Gerhardt G; Boyle, Stephen M; Lindsay, David S; Sriranganathan, Nammalwar

    2007-09-30

    Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.

  18. Asymptomatic bacteriuria Escherichia coli strain 83972 carries mutations in the foc locus and is unable to express F1C fimbriae

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Schembri, M.A.; Ulett, G.C.

    2006-01-01

    Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infection (UTI), very little is known about the mechanisms by which these strains colonize the urinary tract. Bacterial...... adhesion conferred by specific surface-associated adhesins is normally considered as a prerequisite for colonization of the urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. This study characterized the molecular...... this adhesin. The data imply that E. coli 83972 has lost its ability to express this important colonization factor as a result of host-driven evolution. The ancestor of the strain seems to have been a pyelonephritis strain of phylogenetic group B2. Strain 83972 therefore represents an example of bacterial...

  19. Expression of Cathepsin S in BCG converts it into a pro-apoptotic and highly immunogenic strain.

    Science.gov (United States)

    Lau, Alice; Singh, Vijender; Soualhine, Hafid; Hmama, Zakaria

    2017-04-11

    BCG vaccine, introduced almost 100years ago, is the only option to prevent TB disease. It effectively protects newborns from meningeal TB but fails to prevent adult pulmonary TB. TB kills 1.3million people annually in areas where BCG vaccination is widely practiced. Thus, more effective TB vaccines are urgently needed. Others and we have shown that BCG mimics features of virulent M. tuberculosis, in particular attenuation of essential macrophage functions such as phagosome maturation and antigen presentation. One of these studies revealed that defect in antigen presentation is largely due to down-regulation of the cysteine protease Cathepsin S (CatS), which prevents MHC II molecule maturation and proper antigen peptide loading. Recent studies also suggested a potential role for cysteine proteases in the regulation of apoptosis, a key cellular process used by the macrophage to (i) contain and process ingested bacteria and (ii) facilitate cross-talk antigen presentation between the macrophage and dendritic cells. To reverse the phenotype of vaccine-mediated macrophage attenuation, we engineered a novel BCG strain that expresses and secretes active CatS (rBCG-CatS) to examine its pro-apoptotic properties in vitro, and subsequently, immunogenicity in mice. Transcriptomic profiling of macrophages infected with rBCG-CatS, but not BCG, revealed upregulation of key pro-apoptotic genes and downregulation of anti-apoptotic genes, which were further confirmed by RT-qPCR analyses of expression of selected genes. Macrophages infected with rBCG-CatS undergo apoptosis as indicated by increased levels of annexin V staining and intracellular caspase-3 cleavage. Consistent with these findings, mice vaccinated with rBCG-CatS showed increased antigen-specific CD4 + T-cell responses, as well as enhanced cytokine production and proliferation in CD4 + upon ex vivo re-stimulation. Collectively, this study shows that a pro-apoptotic BCG strain alleviates adverse traits of the wild

  20. Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A-oryzae alpha-amylase

    DEFF Research Database (Denmark)

    Agger, Teit; Petersen, J.B.; O'Connor, S.M.

    2002-01-01

    The physiology of three strains of Aspergillus nidulans was examined-a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biom...

  1. Genomic expression catalogue of a global collection of BCG vaccine strains show evidence for highly diverged metabolic and cell-wall adaptations

    KAUST Repository

    Abdallah, Abdallah

    2015-10-21

    Although Bacillus Calmette-Guérin (BCG) vaccines against tuberculosis have been available for more than 90 years, their effectiveness has been hindered by variable protective efficacy and a lack of lasting memory responses. One factor contributing to this variability may be the diversity of the BCG strains that are used around the world, in part from genomic changes accumulated during vaccine production and their resulting differences in gene expression. We have compared the genomes and transcriptomes of a global collection of fourteen of the most widely used BCG strains at single base-pair resolution. We have also used quantitative proteomics to identify key differences in expression of proteins across five representative BCG strains of the four tandem duplication (DU) groups. We provide a comprehensive map of single nucleotide polymorphisms (SNPs), copy number variation and insertions and deletions (indels) across fourteen BCG strains. Genome-wide SNP characterization allowed the construction of a new and robust phylogenic genealogy of BCG strains. Transcriptional and proteomic profiling revealed a metabolic remodeling in BCG strains that may be reflected by altered immunogenicity and possibly vaccine efficacy. Together, these integrated-omic data represent the most comprehensive catalogue of genetic variation across a global collection of BCG strains.

  2. Effects of cell adhesion motif, fiber stiffness, and cyclic strain on tenocyte gene expression in a tendon mimetic fiber composite hydrogel.

    Science.gov (United States)

    Patel, Dharmesh; Sharma, Sadhana; Screen, Hazel R C; Bryant, Stephanie J

    2018-05-15

    We recently developed a fiber composite consisting of tenocytes seeded onto discontinuous fibers embedded within a hydrogel, designed to mimic physiological tendon micromechanics of tension and shear. This study examined if cell adhesion peptide (DGEA or YRGDS), fiber modulus (50 or 1300 kPa) and/or cyclic strain (5% strain, 1 Hz) influenced bovine tenocyte gene expression. Ten genes were analyzed and none were sensitive to peptide or fiber modulus in the absence of cyclic tensile strain. Genes associated with tendon (SCX and TNMD), collagens (COL1A1, COL3A1, COL11A1), and matrix remodelling (MMP1, MMP2, and TIMP3) were insensitive to cyclic strain. Contrarily, cyclic strain up-regulated IL6 by 30-fold and MMP3 by 10-fold in soft YRGDS fibers. IL6 expression in soft YRGDS fibers was 5.7 and 3.3-fold greater than in soft DGEA fibers and stiff RGD fibers, respectively, under cyclic strain. Our findings suggest that changes in the surrounding matrix can influence catabolic genes in tenocytes when cultured in a complex strain environment mimicking that of tendon, while having minimal effects on tendon and homeostatic genes. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Genomic expression catalogue of a global collection of BCG vaccine strains show evidence for highly diverged metabolic and cell-wall adaptations

    Science.gov (United States)

    Abdallah, Abdallah M.; Hill-Cawthorne, Grant A.; Otto, Thomas D.; Coll, Francesc; Guerra-Assunção, José Afonso; Gao, Ge; Naeem, Raeece; Ansari, Hifzur; Malas, Tareq B.; Adroub, Sabir A.; Verboom, Theo; Ummels, Roy; Zhang, Huoming; Panigrahi, Aswini Kumar; McNerney, Ruth; Brosch, Roland; Clark, Taane G.; Behr, Marcel A.; Bitter, Wilbert; Pain, Arnab

    2015-01-01

    Although Bacillus Calmette-Guérin (BCG) vaccines against tuberculosis have been available for more than 90 years, their effectiveness has been hindered by variable protective efficacy and a lack of lasting memory responses. One factor contributing to this variability may be the diversity of the BCG strains that are used around the world, in part from genomic changes accumulated during vaccine production and their resulting differences in gene expression. We have compared the genomes and transcriptomes of a global collection of fourteen of the most widely used BCG strains at single base-pair resolution. We have also used quantitative proteomics to identify key differences in expression of proteins across five representative BCG strains of the four tandem duplication (DU) groups. We provide a comprehensive map of single nucleotide polymorphisms (SNPs), copy number variation and insertions and deletions (indels) across fourteen BCG strains. Genome-wide SNP characterization allowed the construction of a new and robust phylogenic genealogy of BCG strains. Transcriptional and proteomic profiling revealed a metabolic remodeling in BCG strains that may be reflected by altered immunogenicity and possibly vaccine efficacy. Together, these integrated-omic data represent the most comprehensive catalogue of genetic variation across a global collection of BCG strains. PMID:26487098

  4. Two variants among Haemophilus influenzae serotype b strains with distinct bcs4, hcsA and hcsB genes display differences in expression of the polysaccharide capsule

    Directory of Open Access Journals (Sweden)

    Burger Marina

    2008-02-01

    Full Text Available Abstract Background Despite nearly complete vaccine coverage, a small number of fully vaccinated children in the Netherlands have experienced invasive disease caused by Haemophilus influenzae serotype b (Hib. This increase started in 2002, nine years after the introduction of nationwide vaccination in the Netherlands. The capsular polysaccharide of Hib is used as a conjugate vaccine to protect against Hib disease. To evaluate the possible rise of escape variants, explaining the increased number of vaccine failures we analyzed the composition of the capsular genes and the expressed polysaccharide of Dutch Hib strains collected before and after the introduction of Hib vaccination. Results The DNA sequences of the complete capsular gene clusters of 9 Dutch Hib strains were assessed and two variants, designated type I and type II were found. The two variants displayed considerable sequence divergence in the hcsA and hcsB genes, involved in transport of capsular polysaccharide to the cell surface. Application of hcsA type specific PCRs on 670 Hib strains collected from Dutch patients with invasive Hib disease showed that 5% of the strains collected before 1996 were type II. No endogenous type II Hib strains were isolated after 1995 and all type II strains were isolated from 0–4 year old, non-vaccinated children only. Analysis of a worldwide collection of Hib strains from the pre-vaccination era revealed considerable geographic differences in the distribution of the type I and type II strains with up to 73% of type II strains in the USA. NMR analysis of type I and type II capsule polysaccharides did not reveal structural differences. However, type I strains were shown to produce twice as much surface bound capsular polysaccharide. Conclusion Type II strains were only isolated during the pre-vaccination era from young, non-vaccinated individuals and displayed a lower expression of capsular polysaccharide than type I strains. The higher polysaccharide

  5. Oleuropein aglycone protects transgenic C. elegans strains expressing Aβ42 by reducing plaque load and motor deficit.

    Directory of Open Access Journals (Sweden)

    Luisa Diomede

    Full Text Available The presence of amyloid aggregates of the 42 amino acid peptide of amyloid beta (Aβ42 in the brain is the characteristic feature of Alzheimer's disease (AD. Amyloid beta (Aβ deposition is also found in muscle fibers of individuals affected by inclusion body myositis (sIBM, a rare muscular degenerative disease affecting people over 50. Both conditions are presently lacking an effective therapeutic treatment. There is increasing evidence to suggest that natural polyphenols may prevent the formation of toxic amyloid aggregates; this applies also to oleuropein aglycone (OLE, the most abundant polyphenol in extra virgin olive oil, previously shown to hinder amylin and Aβ aggregation. Here we evaluated the ability of OLE to interfere with Aβ proteotoxicity in vivo by using the transgenic CL2006 and CL4176 strains of Caenorhabditis elegans, simplified models of AD and of sIBM, which express human Aβ in the cytoplasm of body wall muscle cells. OLE-fed CL2006 worms displayed reduced Aβ plaque deposition, less abundant toxic Aβ oligomers, remarkably decreased paralysis and increased lifespan with respect to untreated animals. A protective effect was also observed in CL4176 worms but only when OLE was administered before the induction of the Aβ transgene expression. These effects were specific, dose-related, and not mediated by the known polyphenolic anti-oxidant activity, suggesting that, in this model organism, OLE interferes with the Aβ aggregation skipping the appearance of toxic species, as already shown in vitro for Aβ42.

  6. Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor.

    Science.gov (United States)

    Pérez, Adam A; Gajewski, John P; Ferlez, Bryan H; Ludwig, Marcus; Baker, Carol S; Golbeck, John H; Bryant, Donald A

    2017-02-01

    Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn 2+ -inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn 2+ uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA 7942 ) and its cognate SmtB 7942 repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA 6803 ) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of the Drosophila melanogaster β-carotene 15,15'-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology in Synechococcus sp. strain PCC 7002. This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn 2+ -inducible system, which is based on the

  7. An attenuated EIAV strain and its molecular clone strain differentially induce the expression of Toll-like receptors and type-I interferons in equine monocyte-derived macrophages.

    Science.gov (United States)

    Ma, Jian; Wang, Shan-Shan; Lin, Yue-Zhi; Liu, Hai-Fang; Wei, Hua-Mian; Du, Cheng; Wang, Xue-Feng; Zhou, Jian-Hua

    2013-09-27

    Activations of endosomal TLRs include TLR3, TLR7/8, and TLR9 stimulates the production of cytokines, such as type I interferons (IFNs), and therefore involves in virus-host interactions. In the present study, two equine anemia virus (EIAV) strains EIAVFDDV13 and EIAVFDDV3-8, which showed different induction on protective immunity, were compared regarding their ability to regulate the expression of endosomal TLRs, as well as type I IFNs, after infection of equine monocyte-derived macrophages (eMDMs). Our results showed that EIAVFDDV13 dramatically up-regulated the expression of TLR3 and IFNβ and less robustly up-regulated the expression of TRL9 and IFNα1, whereas EIAVFDDV3-8 induced significantly lower expression of type I IFN mRNA and protein and more strongly down-regulated the expression of TLR7 and TLR8. In addition, no significant differences in cell apoptosis were observed between these two strains. Given that the genomic variation of EIAVFDDV13 is considerably higher than that of molecular clone EIAVFDDV3-8, our results suggest that stronger TLR3 activation and increased INFβ production induced by the multi-species strain are associated with an effective vaccine-elicited protective immune response. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Heat shock and prolonged heat stress attenuate neurotoxin and sporulation gene expression in group I Clostridium botulinum strain ATCC 3502.

    Science.gov (United States)

    Selby, Katja; Mascher, Gerald; Somervuo, Panu; Lindström, Miia; Korkeala, Hannu

    2017-01-01

    Foodborne pathogenic bacteria are exposed to a number of environmental stresses during food processing, storage, and preparation, and in the human body. In order to improve the safety of food, the understanding of molecular stress response mechanisms foodborne pathogens employ is essential. Many response mechanisms that are activated during heat shock may cross-protect bacteria against other environmental stresses. To better understand the molecular mechanisms Clostridium botulinum, the causative agent of botulism, utilizes during acute heat stress and during adaptation to stressfully high temperature, the C. botulinum Group I strain ATCC 3502 was grown in continuous culture at 39°C and exposed to heat shock at 45°C, followed by prolonged heat stress at 45°C to allow adaptation of the culture to the high temperature. Growth in continuous culture was performed to exclude secondary growth phase effects or other environmental impacts on bacterial gene transcription. Changes in global gene expression profiles were studied using DNA microarray hybridization. During acute heat stress, Class I and III heat shock genes as well as members of the SOS regulon were activated. The neurotoxin gene botA and genes encoding the neurotoxin-associated proteins were suppressed throughout the study. Prolonged heat stress led to suppression of the sporulation machinery whereas genes related to chemotaxis and motility were activated. Induced expression of a large proportion of prophage genes was detected, suggesting an important role of acquired genes in the stress resistance of C. botulinum. Finally, changes in the expression of a large number of genes related to carbohydrate and amino acid metabolism indicated remodeling of the cellular metabolism.

  9. Gene sequencing, cloning, and expression of the recombinant L- Asparaginase of Pseudomonas aeruginosa SN4 strain in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Arastoo Badoei-dalfard

    2016-03-01

    Full Text Available Introduction: L- asparaginase is in an excessive demand in medical applications and in food treating industries, the request for this therapeutic enzyme is growing several folds every year. Materials and methods: In this study, a L- asparaginase gene from Pseudomonas aeruginosa strain SN4 was sequenced and cloned in E. coli. Primers were designed based on L- asparaginase from P. aeruginosa DSM 50071, which show high similarity to SN4 strain, according to 16S rRNA sequence. The L- asparaginase gene was exposed to restriction digestion with NdeI and XhoI enzymes and then ligated into pET21a plasmid. The ligated sample was transformed into competent E. coli (DE3 pLysS DH5a cells, according to CaCl2 method. The transformed E. coli cells were grown into LB agar plate containing 100 µg/ml ampicillin, IPTG (1 mM. Results: Recombinant L- asparaginase from E. coli BL21 induced after 9 h of incubation and showed high L- asparaginase activity about 93.4 IU/ml. Recombinant L- asparaginase sequencing and alignments showed that the presumed amino acid sequence composed of 350 amino acid residues showed high similarity with P. aeruginosa L- asparaginases about 99%. The results also indicated that SN4 L- asparaginase has the catalytic residues and conserve region similar to other L- asparaginases. Discussion and conclusion: This is the first report on cloning and expression of P. aeruginosa L- asparaginases in Escherichia coli. These results indicated a potent source of L- asparaginase for in vitro and in vivio anticancer consideration. 

  10. Structural defect linked to nonrandom mutations in the matrix gene of Biden strain subacute sclerosing panencephalitis virus defined by cDNA cloning and expression of chimeric genes

    International Nuclear Information System (INIS)

    Ayata, M.; Hirano, A.; Wong, T.C.

    1989-01-01

    Biken strain, a nonproductive measles viruslike agent isolated from a subacute sclerosing panencephalitis (SSPE) patient, contains a posttranscriptional defect affecting matrix (M) protein. A putative M protein was translated in vitro with RNA from Biken strain-infected cells. A similar protein was detected in vivo by an antiserum against a peptide synthesized from the cloned M gene of Edmonston strain measles virus. By using a novel method, full-length cDNAs of the Biken M gene were selectively cloned. The cloned Biken M gene contained an open reading frame which encoded 8 extra carboxy-terminal amino acid residues and 20 amino acid substitutions predicted to affect both the hydrophobicity and secondary structure of the gene product. The cloned gene was expressed in vitro and in vivo into a 37,500 M r protein electrophoretically and antigenically distinct from the M protein of Edmonston strain but identical to the M protein in Biken strain-infected cells. Chimeric M proteins synthesized in vitro and in vivo showed that the mutations in the carboxy-proximal region altered the local antigenicity and those in the amino region affected the overall protein conformation. The protein expressed from the Biken M gene was unstable in vivo. Instability was attributed to multiple mutations. These results offer insights into the basis of the defect in Biken strain and pose intriguing questions about the evolutionary origins of SSPE viruses in general

  11. Positional cloning reveals strain-dependent expression of Trim16 to alter susceptibility to bleomycin-induced pulmonary fibrosis in mice.

    Directory of Open Access Journals (Sweden)

    Anguel N Stefanov

    Full Text Available Pulmonary fibrosis is a disease of significant morbidity, with no effective therapeutics and an as yet incompletely defined genetic basis. The chemotherapeutic agent bleomycin induces pulmonary fibrosis in susceptible C57BL/6J mice but not in mice of the C3H/HeJ strain, and this differential strain response has been used in prior studies to map bleomycin-induced pulmonary fibrosis susceptibility loci named Blmpf1 and Blmpf2. In this study we isolated the quantitative trait gene underlying Blmpf2 initially by histologically phenotyping the bleomycin-induced lung disease of sublines of congenic mice to reduce the linkage region to 13 genes. Of these genes, Trim16 was identified to have strain-dependent expression in the lung, which we determined was due to sequence variation in the promoter. Over-expression of Trim16 by plasmid injection increased pulmonary fibrosis, and bronchoalveolar lavage levels of both interleukin 12/23-p40 and neutrophils, in bleomycin treated B6.C3H-Blmpf2 subcongenic mice compared to subcongenic mice treated with bleomycin only, which follows the C57BL/6J versus C3H/HeJ strain difference in these traits. In summary we demonstrate that genetic variation in Trim16 leads to its strain-dependent expression, which alters susceptibility to bleomycin-induced pulmonary fibrosis in mice.

  12. Global mRNA expression analysis in myosin II deficient strains of Saccharomyces cerevisiae reveals an impairment of cell integrity functions

    Directory of Open Access Journals (Sweden)

    Rivera-Molina Félix E

    2008-01-01

    Full Text Available Abstract Background The Saccharomyces cerevisiae MYO1 gene encodes the myosin II heavy chain (Myo1p, a protein required for normal cytokinesis in budding yeast. Myo1p deficiency in yeast (myo1Δ causes a cell separation defect characterized by the formation of attached cells, yet it also causes abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, increased chitin synthesis, and hypersensitivity to the chitin synthase III inhibitor Nikkomycin Z. To determine how differential expression of genes is related to these diverse cell wall phenotypes, we analyzed the global mRNA expression profile of myo1Δ strains. Results Global mRNA expression profiles of myo1Δ strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes (p ≤ 0.01 were identified with 263 up regulated and 284 down regulated genes in the myo1Δ strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. The SLT2/MPK1 gene was up regulated in the microarray, and a myo1Δslt2Δ double mutant was non-viable. Overexpression of ribosomal protein genes RPL30 and RPS31 suppressed the hypersensitivity to Nikkomycin Z and increased the levels of phosphorylated Slt2p in myo1Δ strains. Increased levels of phosphorylated Slt2p were also observed in wild type strains under these conditions. Conclusion Following this analysis of global mRNA expression in yeast myo1Δ strains, we conclude that 547 genes were differentially regulated in myo1Δ strains and that the stress response and protein biosynthesis gene categories were coordinately regulated in this mutant. The SLT2/MPK1 gene was confirmed to be essential for myo1Δ strain viability, supporting that the up

  13. Brucella abortus strain RB51 as a vector for heterologous protein expression and induction of specific Th1 type immune responses.

    Science.gov (United States)

    Vemulapalli, R; He, Y; Boyle, S M; Sriranganathan, N; Schurig, G G

    2000-06-01

    Brucella abortus strain RB51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Our ultimate goal is to develop strain RB51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong Th1 type of immune response is needed for effective protection. As a first step in that direction, we studied the expression of a foreign reporter protein, beta-galactosidase of Escherichia coli, and the 65-kDa heat shock protein (HSP65) of Mycobacterium bovis in strain RB51. We cloned the promoter sequences of Brucella sodC and groE genes in pBBR1MCS to generate plasmids pBBSODpro and pBBgroE, respectively. The genes for beta-galactosidase (lacZ) and HSP65 were cloned in these plasmids and used to transform strain RB51. An enzyme assay in the recombinant RB51 strains indicated that the level of beta-galactosidase expression is higher under the groE promoter than under the sodC promoter. In strain RB51 containing pBBgroE/lacZ, but not pBBSODpro/lacZ, increased levels of beta-galactosidase expression were observed after subjecting the bacteria to heat shock or following internalization into macrophage-like J774A.1 cells. Mice vaccinated with either of the beta-galactosidase-expressing recombinant RB51 strains developed specific antibodies of predominantly the immunoglobulin G2a (IgG2a) isotype, and in vitro stimulation of their splenocytes with beta-galactosidase induced the secretion of gamma interferon (IFN-gamma), but not interleukin-4 (IL-4). A Th1 type of immune response to HSP65, as indicated by the presence of specific serum IgG2a, but not IgG1, antibodies, and IFN-gamma, but not IL-4, secretion by the specific-antigen-stimulated splenocytes, was also detected in mice vaccinated with strain RB51 containing pBBgroE/hsp65. Studies with mice indicated that expression of beta-galactosidase or HSP65 did not alter either the attenuation characteristics of strain RB51 or

  14. Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate

    Science.gov (United States)

    Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

    2016-01-01

    Background and Objectives: Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Materials and Methods: Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. Results: The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. Conclusion: We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development. PMID:28210460

  15. Evaluation of Strains and Thicknesses of Pipe Elbows on the Basis of Expressions Resulting from the Eudirective for the Case of Large and Small Deformations

    Directory of Open Access Journals (Sweden)

    Śloderbach Z.

    2017-12-01

    Full Text Available The relations to calculate the maximum value of strains in processes of bending tubes on benders, in stretched layers of tubes, are presented in this work on the basis of the EU-Directive concerning production of pressure equipment. It has been shown that for large deformations that occur during bending of the pipes on knees, logarithmic strain measures (real and relative strain measures give different values of strain but equal wall thicknesses in the bending zone. Logarithmic measures are frequently used in engineering practice and are valid for large and small deformations. Reverse expressions were also derived to calculate the required initial wall thickness of the tube to be bent, in order to obtain the desired wall thickness of the knee after bending.

  16. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens

    2008-01-01

    . jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria...

  17. Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge.

    Science.gov (United States)

    Rajasekaran, Parthiban; Surendran, Naveen; Seleem, Mohamed N; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

    2011-04-12

    Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (PBrucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Genome sequences of lower Great Lakes Microcystis sp. reveal strain-specific genes that are present and expressed in western Lake Erie blooms.

    Directory of Open Access Journals (Sweden)

    Kevin Anthony Meyer

    Full Text Available Blooms of the potentially toxic cyanobacterium Microcystis are increasing worldwide. In the Laurentian Great Lakes they pose major socioeconomic, ecological, and human health threats, particularly in western Lake Erie. However, the interpretation of "omics" data is constrained by the highly variable genome of Microcystis and the small number of reference genome sequences from strains isolated from the Great Lakes. To address this, we sequenced two Microcystis isolates from Lake Erie (Microcystis aeruginosa LE3 and M. wesenbergii LE013-01 and one from upstream Lake St. Clair (M. cf aeruginosa LSC13-02, and compared these data to the genomes of seventeen Microcystis spp. from across the globe as well as one metagenome and seven metatranscriptomes from a 2014 Lake Erie Microcystis bloom. For the publically available strains analyzed, the core genome is ~1900 genes, representing ~11% of total genes in the pan-genome and ~45% of each strain's genome. The flexible genome content was related to Microcystis subclades defined by phylogenetic analysis of both housekeeping genes and total core genes. To our knowledge this is the first evidence that the flexible genome is linked to the core genome of the Microcystis species complex. The majority of strain-specific genes were present and expressed in bloom communities in Lake Erie. Roughly 8% of these genes from the lower Great Lakes are involved in genome plasticity (rapid gain, loss, or rearrangement of genes and resistance to foreign genetic elements (such as CRISPR-Cas systems. Intriguingly, strain-specific genes from Microcystis cultured from around the world were also present and expressed in the Lake Erie blooms, suggesting that the Microcystis pangenome is truly global. The presence and expression of flexible genes, including strain-specific genes, suggests that strain-level genomic diversity may be important in maintaining Microcystis abundance during bloom events.

  19. Down-regulatory effect of Thymus vulgaris L. on growth and Tri4 gene expression in Fusarium oxysporum strains.

    Science.gov (United States)

    Divband, Kolsum; Shokri, Hojjatollah; Khosravi, Ali Reza

    2017-03-01

    The aims of this study were to evaluate the efficacy of Thymus vulgaris (T. vulgaris) essential oil on the fungal growth and Tri4 gene expression in Fusarium oxysporum (F. oxysporum) strains. The oil was obtained by water-distillation using a Clevenger-type system. The chemical composition of the essential oil was obtained by gas chromatography- mass spectroscopy (GC-MS) and by retention indices. The antifungal activity was evaluated by broth microdilution assay. A quantitative real time RT-PCR (qRT-PCR) assay was also developed specific for F. oxysporum on the basis of trichothecene biosynthetic gene, Tri4, which allowed discrimination from F. oxysporum. Results showed thymol (32.67%) and p-cymene (16.68%) as the main components of T. vulgaris. Minimum inhibitory concentration (MIC) values varied from 5 to 20 μg/ml with T. vulgaris (mean: 10.50 μg/ml), while minimum fungicidal concentration (MFC) values ranged from 8 to 30 μg/ml with mean value of 16.20 μg/ml qRT-PCR results revealed a downregulation from 4.04 to 6.27 fold of Tri4 gene expression of the fungi exposed to T. vulgaris essential oil. The results suggest that T. vulgaris oil can be considered potential alternative natural fungicide to the synthetic chemicals that are currently used to prevent and control seed-borne diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Constitutively Opa-expressing and Opa-deficient neisseria gonorrhoeae strains differentially stimulate and survive exposure to human neutrophils.

    Science.gov (United States)

    Ball, Louise M; Criss, Alison K

    2013-07-01

    The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in frame, termed Opaless. Opaless Gc remained uniformly Opa negative (Opa(-)), whereas cultures of predominantly Opa(-) parental Gc and an intermediate lacking the "translucent" subset of opa genes (ΔopaBEGK) stochastically gave rise to Opa-positive (Opa(+)) bacterial colonies. Loss of Opa expression did not affect Gc growth. Opaless Gc survived exposure to primary human PMNs and suppressed the PMN oxidative burst akin to parental, Opa(-) bacteria. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. When a non-phase-variable, in-frame allele of FA1090 opaD was reintroduced into Opaless Gc, the bacteria induced the PMN oxidative burst, and OpaD(+) Gc survived less well after exposure to PMNs compared to Opa(-) bacteria. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology.

  1. Quantitative metabolomics of a xylose-utilizing Saccharomyces cerevisiae strain expressing the Bacteroides thetaiotaomicron xylose isomerase on glucose and xylose.

    Science.gov (United States)

    Mert, M J; Rose, S H; la Grange, D C; Bamba, T; Hasunuma, T; Kondo, A; van Zyl, W H

    2017-10-01

    The yeast Saccharomyces cerevisiae cannot utilize xylose, but the introduction of a xylose isomerase that functions well in yeast will help overcome the limitations of the fungal oxido-reductive pathway. In this study, a diploid S. cerevisiae S288c[2n YMX12] strain was constructed expressing the Bacteroides thetaiotaomicron xylA (XI) and the Scheffersomyces stipitis xyl3 (XK) and the changes in the metabolite pools monitored over time. Cultivation on xylose generally resulted in gradual changes in metabolite pool size over time, whereas more dramatic fluctuations were observed with cultivation on glucose due to the diauxic growth pattern. The low G6P and F1,6P levels observed with cultivation on xylose resulted in the incomplete activation of the Crabtree effect, whereas the high PEP levels is indicative of carbon starvation. The high UDP-D-glucose levels with cultivation on xylose indicated that the carbon was channeled toward biomass production. The adenylate and guanylate energy charges were tightly regulated by the cultures, while the catabolic and anabolic reduction charges fluctuated between metabolic states. This study helped elucidate the metabolite distribution that takes place under Crabtree-positive and Crabtree-negative conditions when cultivating S. cerevisiae on glucose and xylose, respectively.

  2. Genomic analysis of the biocontrol strain Pseudomonas fluorescens Pf29Arp with evidence of T3SS and T6SS gene expression on plant roots.

    Science.gov (United States)

    Marchi, Muriel; Boutin, Morgane; Gazengel, Kévin; Rispe, Claude; Gauthier, Jean-Pierre; Guillerm-Erckelboudt, Anne-Yvonne; Lebreton, Lionel; Barret, Matthieu; Daval, Stéphanie; Sarniguet, Alain

    2013-06-01

    Several bacterial strains of the Pseudomonas genus provide plant growth stimulation, plant protection against pests or bioremediation. Among these bacteria, P. fluorescens Pf29Arp reduces the severity of take-all, a disease caused by the pathogenic fungus Gaeumannomyces graminis var. tritici (Ggt) on wheat roots. In this study, we obtained a draft genome of Pf29Arp and subsequent comparative genomic analyses have revealed that this bacterial strain is closely related to strains of the 'P. brassicacearum-like' subgroup including P. brassicacearum ssp. brassicacearum NFM421 and P. fluorescens F113. Despite an overall chromosomal organization similar to these strains, a number of features including antibiotic synthesis gene clusters from secondary metabolism are not found in the Pf29Arp genome. But Pf29Arp possesses different protein secretion systems including type III (T3SS) and type VI (T6SS) secretion systems. Pf29Arp is the first Pseudomonas sp. strain described with four T6SS clusters (cluster I, II, III and IV). In addition, some protein-coding genes involved in the assembly of these secretion systems are basally expressed during Pf29Arp colonization of healthy wheat roots and display different expression patterns on necrotized roots caused by Ggt. These data suggest a role of T3SS and T6SS in the Pf29Arp adaptation to different root environments. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

  3. Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene rosa in virulent and attenuated strains of Renibacterium salmoninarum

    Science.gov (United States)

    O'Farrell, C. L.; Strom, M.S.

    1999-01-01

    Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5, to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of rosa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization, and total p57 expression between 33209 anti MT 239 are not due to differences in rosa sequence or differences in steady state transcript levels.

  4. Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia.

    Science.gov (United States)

    Rodas, Claudia; Klena, John D; Nicklasson, Matilda; Iniguez, Volga; Sjöling, Asa

    2011-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP(bol) in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP(bol)) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.

  5. Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51 expressing N. caninum protective antigens.

    Science.gov (United States)

    Ramamoorthy, Sheela; Sanakkayala, Neelima; Vemulapalli, Ramesh; Jain, Neeta; Lindsay, David S; Schurig, Gerhardt S; Boyle, Stephen M; Sriranganathan, Nammalwar

    2007-11-01

    Bovine abortions caused by the apicomplexan parasite Neospora caninum have been responsible for severe economic losses to the cattle industry. Infected cows either experience abortion or transmit the parasite transplacentally at a rate of up to 95%. Neospora caninum vaccines that can prevent vertical transmission and ensure disruption in the life cycle of the parasite greatly aid in the management of neosporosis in the cattle industry. Brucella abortus strain RB51, a commercially available vaccine for bovine brucellosis, can also be used as a vector to express plasmid-encoded proteins from other pathogens. Neospora caninum protective antigens MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in strain RB51. Female C57BL/6 mice were vaccinated with a recombinant strain RB51 expressing N. caninum antigen or irradiated tachyzoites, boosted 4 weeks later and then bred. Antigen-specific IgG, IFN-gamma and IL-10 were detected in vaccinated pregnant mice. Vaccinated mice were challenged with 5 x 10(6)N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups 3 weeks after birth and examined for the presence of N. caninum by real-time PCR. The RB51-MIC3, RB51-GRA6, irradiated tachyzoite vaccine, pooled strain RB51-Neospora vaccine, RB51-MIC1 and RB51-SRS2 vaccines elicited approximately 6-38% protection against vertical transmission. However, the differences in parasite burden in brain tissue of pups from the control and vaccinated groups were highly significant for all groups. Thus, B. abortus strain RB51 expressing the specific N. caninum antigens induced substantial protection against vertical transmission of N. caninum in mice.

  6. Prevention of lethal experimental infection of C57BL/6 mice by vaccination with Brucella abortus strain RB51 expressing Neospora caninum antigens.

    Science.gov (United States)

    Ramamoorthy, Sheela; Sanakkayala, Neelima; Vemulapalli, Ramesh; Duncan, Robert B; Lindsay, David S; Schurig, Gerhart S; Boyle, Stephen M; Kasimanickam, Ramanathan; Sriranganathan, Nammalwar

    2007-11-01

    Bovine abortions caused by the intracellular protozoal parasite Neospora caninum are a major concern to cattle industries worldwide. A strong Th1 immune response is required for protection against N. caninum. Brucella abortus strain RB51 is currently used as a live, attenuated vaccine against bovine brucellosis. Strain RB51 can also be used as an expression vector for heterologous protein expression. In this study, putative protective antigens of N. caninum MIC1, MIC3, GRA2, GRA6 and SRS2, were expressed individually in B. abortus strain RB51. The ability of each of the recombinant RB51 strains to induce N. caninum-specific immunity was assessed in C57BL/6 mice. Mice were immunised by two i.p. inoculations, 4 weeks apart. Five weeks after the second immunisation, spleen cells from the vaccinated mice secreted high levels of IFN-gamma and IL-10 upon in vitro stimulation with N. caninum whole cell lysate antigens. N. caninum-specific antibodies of both IgG1 and IgG2a subtypes were detected in the serum of the vaccinated mice. Mice in the vaccinated and control groups were challenged with 2 x 10(7)N. caninum tachyzoites i.p. and observed for 28 days after vaccination. All unvaccinated control mice died within 7 days. Mice in the MIC1 and GRA6 vaccine groups were completely protected while the mice in the SRS2, GRA2 and MIC3 vaccinated groups were partially protected and experienced 10-50% mortality. The non-recombinant RB51 vector control group experienced an average protection of 69%. These results suggest that expression of protective antigens of N. caninum in B. abortus strain RB51 is a novel approach towards the development of a multivalent vaccine against brucellosis and neosporosis.

  7. The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium

    Directory of Open Access Journals (Sweden)

    Hannah M. Read

    2016-06-01

    Full Text Available Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC and enterohaemorrhagic E. coli (EHEC infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169 in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments.

  8. Daily expression pattern of protein-encoding genes and small noncoding RNAs in synechocystis sp. strain PCC 6803.

    Science.gov (United States)

    Beck, Christian; Hertel, Stefanie; Rediger, Anne; Lehmann, Robert; Wiegard, Anika; Kölsch, Adrian; Heilmann, Beate; Georg, Jens; Hess, Wolfgang R; Axmann, Ilka M

    2014-09-01

    Many organisms harbor circadian clocks with periods close to 24 h. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. These rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes. The oscillator of photosynthetic cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, linked to a complex regulatory network. Synechocystis sp. strain PCC 6803 possesses the standard cyanobacterial kaiABC gene cluster plus multiple kaiB and kaiC gene copies and antisense RNAs for almost every kai transcript. However, there is no clear evidence of circadian rhythms in Synechocystis sp. PCC 6803 under various experimental conditions. It is also still unknown if and to what extent the multiple kai gene copies and kai antisense RNAs affect circadian timing. Moreover, a large number of small noncoding RNAs whose accumulation dynamics over time have not yet been monitored are known for Synechocystis sp. PCC 6803. Here we performed a 48-h time series transcriptome analysis of Synechocystis sp. PCC 6803, taking into account periodic light-dark phases, continuous light, and continuous darkness. We found that expression of functionally related genes occurred in different phases of day and night. Moreover, we found day-peaking and night-peaking transcripts among the small RNAs; in particular, the amounts of kai antisense RNAs correlated or anticorrelated with those of their respective kai target mRNAs, pointing toward the regulatory relevance of these antisense RNAs. Surprisingly, we observed that the amounts of 16S and 23S rRNAs in this cyanobacterium fluctuated in light-dark periods, showing maximum accumulation in the dark phase. Importantly, the amounts of all transcripts, including small noncoding RNAs, did not show any rhythm under continuous light or darkness, indicating the absence

  9. A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase α3β1 isoform.

    Science.gov (United States)

    Hirz, Melanie; Richter, Gerald; Leitner, Erich; Wriessnegger, Tamara; Pichler, Harald

    2013-11-01

    The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure-function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [(3)H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.

  10. Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Growth yields and morphological characterization

    DEFF Research Database (Denmark)

    Robin, Jarno Jacky Christian; Lettier, G.; Mcintyre, Mhairi

    2003-01-01

    it is fed with adipic acid. The biomass yield and maintenance coefficients for the strain were similar to those found for penicillin-producing strains of Penicillium chrysogenum. The maximum specific growth rate in the chemostat was found to be 0.11 h(-1). Metabolic degradation of adipate was found to take...

  11. Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice

    International Nuclear Information System (INIS)

    Gentschev, Ivaylo; Fensterle, Joachim; Schmidt, Andreas; Potapenko, Tamara; Troppmair, Jakob; Goebel, Werner; Rapp, Ulf R

    2005-01-01

    Serine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models. The antigen C-Raf has been fused to the C-terminal secretion signal of Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays. C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas. The combination of the C-Raf antigen, hemolysin secretion system and Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies

  12. 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.

  13. Expression of efflux pump MexAB-OprM and OprD of Pseudomonas aeruginosa strains isolated from clinical samples using qRT-PCR.

    Science.gov (United States)

    Arabestani, Mohammad Reza; Rajabpour, Mojtaba; Yousefi Mashouf, Rasoul; Alikhani, Mohammad Yousef; Mousavi, Seyed Masoud

    2015-02-01

    Pseudomonas aeruginosa is one of the most common nosocomial pathogens with high mortality rates. Organisms such as Pseudomonas aeruginosa have the ability to develop high level MDR (Multi drug resistance). The MexAB-OprM system is one of the largest multi-drug resistant efflux pumps with high levels of expression and the first finding of the RND (Resistance-nodulation-division) family in P. aeruginosa. For better understanding of the antibiotic resistance mechanism in P. aeruginosa, this study was conducted to determine the expression of the genes encoding these efflux pumps in 100 strains of P. aeruginosa isolated from patients admitted to various hospitals in Hamadan using quantitative Real-Time PCR (qRT-PCR). This study examined 100 strains of P. aeruginosa isolated from patients admitted to various hospitals in Hamadan. Then, 31 samples were selected based on collected specimen type and their antibiotic susceptibility pattern; i.e., the samples with reduced susceptibility to antibiotics, particularly carbapenems. Antibiotic disk diffusion method was performed for aminoglycoside, quinolone and carbapenem. Furthermore, MIC method was performed for ciprofloxacin, gentamicin and imipenem. Finally, qRT-PCR was used for determining the efflux pump genes expression.  Among eight selected antibiotics, the greatest resistance was to levofloxacin (61.2%, n = 19) and the lowest one to imipenem (9.6%, n = 3). All isolates (100%, n = 31) exhibited efflux pump MexAB-OprM genes but different expression was observed in different strains. The result of gene expression indicated that significant differences in expression of MexR (P value = 0.003), OprD (P value resistance mechanisms is very complicated. Although efflux pump MexAB-OprM plays an important role in antibiotic resistance in P. aeruginosa, because of acting the efflux pumps on antibiotics in a non-specific manner, it is elusive to consider or describe an antibiotic resistance based on the presence or absence of an

  14. Cloning of alpha- and beta-galactosidase genes from an extreme thermophile, Thermus strain T2, and their expression in Thermus thermophilus HB27.

    Science.gov (United States)

    Koyama, Y; Okamoto, S; Furukawa, K

    1990-01-01

    The genes encoding thermostable alpha- and beta-galactosidases from an extremely thermophilic bacterium, Thermus strain T2, were cloned in Escherichia coli. The alpha-galactosidase gene was located just downstream from the beta-galactosidase gene. The genes were introduced into Thermus thermophilus HB27 with the aid of Thermus cryptic plasmid pTT8, and beta-galactosidases were expressed constitutively. PMID:2167630

  15. Yoghurt fermentation at elevated temperatures by strains of Streptococcus thermophilus expressing a small heat-shock protein: application of a two-plasmid system for constructing food-grade strains of Streptococcus thermophilus.

    Science.gov (United States)

    El Demerdash, Hassan A; Oxmann, Julian; Heller, Knut J; Geis, Arnold

    2006-04-01

    Streptococcus thermophilus S4 expressing a small heat-shock protein from the plasmid pSt04-encoded copy of shsp, is able to carry out fermentation at elevated temperature, i.e., at 50 degrees C. In yoghurt culture together with Lactobacillus delbrueckii subsp. bulgaricus, fermentation at elevated temperature results in a mild yoghurt with low post-acidification and improved stability of the starter bacteria during storage at 4 degrees C. To transfer pSt04 into commercial S. thermophilus yoghurt starter strains, a two-plasmid system was constructed. A helper plasmid providing a selectable antibiotic marker, but relying on the repA gene of pSt04, was transformed together with pSt04. After isolation of transformants, the helper plasmid was readily lost upon incubation of transformants in antibiotic-free medium, thus yielding food-grade strains carrying pSt04 only. Successful application of the system was demonstrated.

  16. Genetics of the α 1,6-dextran response: expression of the QUPC52 idiotype in different inbred and congenic strains of mice

    International Nuclear Information System (INIS)

    D'Hoostelaere, L.; Potter, M.

    1982-01-01

    Antibodies to dextran B512 were raised in various strains of mice and were assayed by a radioimunoassay procedure. Idiotypic antibodies to the IgA(k) dextran B512 binding myeloma proteins QUOC52 and W3129 of BALB/c origin were prepred in rabbits. After adsorption, each antiserum was specific for the immunizing myeloma protein and did not react with hundreds of other myeloma proteins; nonetheless, antibodies to dextran B512 from various strains of mice cross-reacted in these test systems. Of the 2 idiotypes tested, the W3129 idiotype was more universally expressed in different strains of mice. The QUPC52 idiotype was the predominant idiotype in BALB/c anti-dextran B512 antibodies and was found in only a few other inbred strains. Using a battery of congenic and inbred strains, it was shown that the QUPC52 idiotype was controlled by genes linked to the Igh complex locus (chromosome 12) and to the Ig k complex locus (chromosome 6). The W3129 idiotype was found in a number of stocks of mice in the genus Mus recently isolated from the wild. The QUPC52 idiotype thus far was found only in inbred mice

  17. Constitutive expression of the DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation.

    Science.gov (United States)

    Wu, Dianhui; Li, Xiaomin; Lu, Jian; Chen, Jian; Zhang, Liang; Xie, Guangfa

    2016-01-01

    Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Allele frequency and gene expression of a putative carboxylesterase-encoding gene in a pyrethroid resistant strain of the tick Boophilus microplus.

    Science.gov (United States)

    Hernandez, R; Guerrero, F D; George, J E; Wagner, G G

    2002-09-01

    We utilized RNA Northern blot analysis and ribonuclease protection assays (RPA) to study the mRNA expression level of a putative carboxylesterase-encoding gene from several strains of Boophilus microplus (Canestrini). Both the Northern analysis and RPAs indicated that an esterase transcript was more abundant in the pyrethroid resistant strain, Coatzacoalcos (Cz), compared to a susceptible control strain and a resistant strain whose pyrethroid resistance is mediated through a target site insensitivity mechanism. A PCR-based assay was designed to identify the presence of a previously reported point mutation in this B. microplus esterase gene. The reported G-->A substitution at nucleotide 1120 creates an EcoR I site in the mutant allele which can be detected by EcoR I digestion of the amplification products. The PCR assays showed that the frequency of the mutant allele was highest in the Cz-resistant strain, which has been shown to have an esterase-mediated resistance mechanism. The PCR assay can be performed either on individual tick larvae or hemolymph from adults.

  19. Differential MicroRNA Expression in Human Macrophages with Mycobacterium tuberculosis Infection of Beijing/W and Non-Beijing/W Strain Types.

    Directory of Open Access Journals (Sweden)

    Lin Zheng

    Full Text Available The role of microRNAs in association with Mycobacterium tuberculosis (MTB infection and the immunology regulated by microRNAs upon MTB infection have not been fully unravelled. We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of Beijing/W and non-Beijing/W clinical strains. We also studied the microRNA profiles of the host macrophages by microarray in a small cohort with active MTB disease, latent infection (LTBI, and from healthy controls.The results revealed that 14 microRNAs differentiated infections of Beijing/W from non-Beijing/W strains (P<0.05. A unique signature of 11 microRNAs in human macrophages was identified to differentiate active MTB disease from LTBI and healthy controls. Pathway analyses of these differentially expressed miRNAs suggest that the immune-regulatory interactions involving TGF-β signalling pathway take part in the dysregulation of critical TB processes in the macrophages, resulting in active expression of both cell communication and signalling transduction systems.We showed for the first time that the Beijing/W TB strains repressed a number of miRNAs expressions which may reflect their virulence characteristics in altering the host response. The unique signatures of 11 microRNAs may deserve further evaluation as candidates for biomarkers in the diagnosis of MTB and Beijing/W infections.

  20. Differential MicroRNA Expression in Human Macrophages with Mycobacterium tuberculosis Infection of Beijing/W and Non-Beijing/W Strain Types.

    Science.gov (United States)

    Zheng, Lin; Leung, Eric; Lee, Nelson; Lui, Grace; To, Ka-Fai; Chan, Raphael C Y; Ip, Margaret

    2015-01-01

    The role of microRNAs in association with Mycobacterium tuberculosis (MTB) infection and the immunology regulated by microRNAs upon MTB infection have not been fully unravelled. We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of Beijing/W and non-Beijing/W clinical strains. We also studied the microRNA profiles of the host macrophages by microarray in a small cohort with active MTB disease, latent infection (LTBI), and from healthy controls. The results revealed that 14 microRNAs differentiated infections of Beijing/W from non-Beijing/W strains (PmicroRNAs in human macrophages was identified to differentiate active MTB disease from LTBI and healthy controls. Pathway analyses of these differentially expressed miRNAs suggest that the immune-regulatory interactions involving TGF-β signalling pathway take part in the dysregulation of critical TB processes in the macrophages, resulting in active expression of both cell communication and signalling transduction systems. We showed for the first time that the Beijing/W TB strains repressed a number of miRNAs expressions which may reflect their virulence characteristics in altering the host response. The unique signatures of 11 microRNAs may deserve further evaluation as candidates for biomarkers in the diagnosis of MTB and Beijing/W infections.

  1. Expression and Immunological Analysis of the Plasmid-Borne mlp Genes of Borrelia burgdorferi Strain B31

    OpenAIRE

    Porcella, Stephen F.; Fitzpatrick, Cecily A.; Bono, James L.

    2000-01-01

    A lipoprotein gene family first identified in Borrelia burgdorferi strain 297, designated 2.9 LP and recently renamed mlp, was found on circular and linear plasmids in the genome sequence of B. burgdorferi strain B31-M1. Sequence analyses of the B31 mlp genes and physically linked variant gene families indicated that mlp gene heterogeneity is unique and unrelated to location or linkage to divergent sequences. Evidence of recombination between B31 mlp alleles was also detected. Northern blot a...

  2. Strain and cocaine-induced differential opioid gene expression may predispose Lewis but not Fischer rats to escalate cocaine self-administration.

    Science.gov (United States)

    Valenza, Marta; Picetti, Roberto; Yuferov, Vadim; Butelman, Eduardo R; Kreek, Mary Jeanne

    2016-06-01

    The aim of the present study was to investigate alterations in gene expression of opioid system components induced by extended access (18 h) cocaine self-administration and to determine the impact of genetic background in the vulnerability to escalate cocaine intake. Comparing two inbred rat strains, we previously reported that Lewis rats progressively escalated cocaine consumption compared to Fischer rats, in a new translational model of intravenous cocaine self-administration, which included 14 sessions of 18-h operant sessions in which rats were allowed to select the cocaine unit dose to self-administer. We compare here Fischer and Lewis rats in the gene expression of endogenous opioid peptides (Pomc, Penk, Pdyn) and cognate receptors (Oprm, Oprk and Oprd) in reward-related brain regions, after exposure to either cocaine self-administration or yoked-saline, in the aforementioned translational paradigm. We performed a correlation analysis between the mRNA level, found in the Dorsal Striatum (DS), Nucleus accumbens (NAcc) shell and core respectively, and individual cocaine intake. Our findings show that the gene expression of all the aforementioned opioid genes exhibit strain-dependent differences in the DS, in absence of cocaine exposure. Also, different strain-specific cocaine-induced mRNA expression of Oprm and Oprk was found in DS. Only few differences were found in the ventral parts of the striatum. Moreover, gene expression level of Pdyn, Penk, Oprk, and Oprm in the DS was significantly correlated with cocaine intake only in Fischer rats. Overall, these data shed light on potential genetic differences which may predispose of subjects to initiate and escalate cocaine consumption. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Expression levels and subcellular localization of Bcy1p in Candida albicans mutant strains devoid of one BCY1 allele results in a defective morphogenetic behavior.

    Science.gov (United States)

    Giacometti, Romina; Souto, Guadalupe; Silberstein, Susana; Giasson, Luc; Cantore, María Leonor; Passeron, Susana

    2006-01-01

    We investigated expression, functionality and subcellular localization of C. albicans Bcy1p, the PKA regulatory subunit, in mutant strains having one BCY1 allele fused to a green fluorescent protein (GFP). DE-52 column chromatography of soluble extracts of yeast cells from strains bearing one BCY1 allele (fused or not to GFP) showed co-elution of Bcy1p and Bcy1p-GFP with phosphotransferase activity, suggesting that interaction between regulatory and catalytic subunits was not impaired by the GFP tag. Subcellular localization of Bcy1p-GFP supports our previous hypothesis on the nuclear localization of the regulatory subunit, which can thus tether the PKA catalytic subunit to the nucleus. Protein modeling of CaBcy1p, showed that the fusion of the GFP tag to Bcy1p C-terminus did not significantly disturb its proper folding. Bcy1p levels in mutant strains having one or both BCY1 alleles, led us to establish a direct correlation between the amount of protein and the number of alleles, indicating that deletion of one BCY1 allele is not fully compensated by overexpression of the other. The morphogenetic behavior of several C. albicans mutant strains bearing one or both BCY1 alleles, in a wild-type and in a TPK2 null genetic background, was assessed in N-acetylglucosamine (GlcNAc) liquid medium at 37 degrees C. Strains with one BCY1 allele tagged or not, behaved similarly, displaying pseudohyphae and true hyphae. In contrast, hyphal morphology was almost exclusive in strains having both BCY1 alleles, irrespective of the GFP insertion. It can be inferred that a tight regulation of PKA activity is needed for hyphal growth.

  4. Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Rollinson David

    2008-12-01

    Full Text Available Abstract Background Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. Results We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs libraries, suppression subtractive hybridization (SSH libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7

  5. Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

    Science.gov (United States)

    Lockyer, Anne E; Spinks, Jenny; Kane, Richard A; Hoffmann, Karl F; Fitzpatrick, Jennifer M; Rollinson, David; Noble, Leslie R; Jones, Catherine S

    2008-01-01

    Background Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. Results We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1

  6. Cell fusion assay by expression of respiratory syncytial virus (RSV) fusion protein to analyze the mutation of palivizumab-resistant strains.

    Science.gov (United States)

    Yasui, Yosuke; Yamaji, Yoshiaki; Sawada, Akihito; Ito, Takashi; Nakayama, Tetsuo

    2016-05-01

    Respiratory syncytial virus (RSV) consists of fusion (F), glyco (G), and small hydrophobic (SH) proteins as envelope proteins, and infects through cell fusion. F protein is expressed on the surface of infected cells, and induces cell fusion. In the present report, expression plasmids of the F, G and SH proteins were constructed and cell fusion activity was investigated under T7 RNA polymerase. F protein alone induced cell fusion at a lower concentration than previously reported, and co-expression of F and SH proteins induced cell fusion more efficiently than F protein alone. Palivizumab is the only prophylactic agent against RSV infection. Palivizumab-resistant strains having mutations of the F protein of K272E and S275F were reported. These mutations were introduced into an F-expression plasmid, and exhibited no inhibition of cell fusion with palivizumab. Among the RSV F protein mutants, N276S has been reported to have partial resistance against palivizumab, but the F expression plasmid with the N276S mutation showed a reduction in cell fusion in the presence of palivizumab, showing no resistance to palivizumab. The present expression system was useful for investigating the mechanisms of RSV cell fusion. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Recombinant canine distemper virus strain snyder hill expressing green or red fluorescent proteins causes meningoencephalitis in the ferret

    NARCIS (Netherlands)

    M. Ludlow (Martin); D.T. Nguyen (Tien); D. Silin; O. Lyubomska; R.D. de Vries (Rory); V. von Messling; S. McQuaid (Stephen); R.L. de Swart (Rik); W.P. Duprex (Paul)

    2012-01-01

    textabstractThe propensity of canine distemper virus (CDV) to spread to the central nervous system is one of the primary features of distemper. Therefore, we developed a reverse genetics system based on the neurovirulent Snyder Hill (SH) strain of CDV (CDVSH) and show that this virus rapidly

  8. Differential expression of hepatic genes involved in cholesterol homeostasis in high- and low-responding strains of laboratory opossums

    NARCIS (Netherlands)

    Chan, Jeannie; Donalson, Lisa M.; Kushwaha, Rampratap S.; Ferdinandusse, Sacha; Vandeberg, Jane F.; Vandeberg, John L.

    2008-01-01

    Plasma very low-density lipoprotein and low-density lipoprotein (VLDL+LDL) cholesterol levels of 2 partially inbred strains of opossums (Monodelphis domestica) differ markedly when they are fed a high-cholesterol and low-fat (HCLF) diet. High-responding opossums exhibit a dramatic increase

  9. Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV).

    Science.gov (United States)

    Dulwich, Katherine L; Giotis, Efstathios S; Gray, Alice; Nair, Venugopal; Skinner, Michael A; Broadbent, Andrew J

    2017-12-01

    Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called 'very virulent' (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B-cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B-cell activation and signalling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.

  10. Effects of dietary supplementation of four strains of lactic acid bacteria on growth, immune-related response and genes expression of the juvenile sea cucumber Apostichopus japonicus Selenka.

    Science.gov (United States)

    Li, Chao; Ren, Yichao; Jiang, Senhao; Zhou, Shun; Zhao, Jinshan; Wang, Renjie; Li, Yongmei

    2018-03-01

    A feeding experiment was conducted to evaluate the effects of four strains of lactic acid bacteria (LAB) [i.e. Lactobacillus plantarum LL11 (LP), Weissella confuse LS13 (WC), Lactococcus lactis LH8 (LL) and Enterococcus faecalis LC3 (ES)] isolated from marine fish on growth, immune response and expression levels of immune-related gens in body wall of juvenile sea cucumber Apostichopus japonicus. As a result, sea cucumber had better growth performance fed supplementation of LP and ES than the control group (P  .05). For the gene expression levels, different expression patterns were observed among four groups, heat shock proteins (HSP60, HSP70 and HSP90) and caspase-2 showed dramatic up-regulation at 30 d while NF-kappa-B transcription factor p65 was down-regulated at 15 d and up-regulated at 30 d, and nitric oxide synthase was down-regulated at both timepoints in almost all the four groups. In conclusion, the four LAB strains screened from marine fish supplemented in diets indicated positive effects on immune response for A. japonicus, especially, the L. plantarum LL11 and E. faecalis LC3 indicated better growth performance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Protection against California 2002 NDV strain afforded by adenovirus vectored vaccine expressing Fusion or Hemagglutination-neuraminidase genes

    Science.gov (United States)

    Vectored vaccines expressing the combination of the hemagglutinin-neuraminidase (HN) and fusion (F) genes generally have better clinical protection against Newcastle disease virus (NDV) than when either the F and HN genes are expressed alone. Interestingly, the protection induced by F is usually bet...

  12. Cloning and expression of the superoxide dismutase gene of the feline strain of Porphyromonas gingivalis: immunological recognition of the protein by cats with periodontal disease.

    Science.gov (United States)

    Love, Daria N; Redwin, Judith; Norris, Jacqueline M

    2002-05-01

    Recent evidence suggests that feline members of the genus Porphyromonas are of consequence in periodontal disease in cats. Several possible virulence factors from feline strains of Porphyromonas gingivalis have been described that have similarities to those of human P. gingivalis. Both human and feline strains of P. gingivalis produce superoxide dismutase (SOD) which has been proposed as modulator of the inflammatory response during infection. The objective of this study was to clone the superoxide dismutase gene of feline P. gingivalis, to compare the characteristics of its product with that of the native enzyme and to determine its immunoreactivity in cats with periodontal disease. The sod gene of the feline strain Veterinary Pathology and Bacteriology (VPB) 3457 of P. gingivalis was amplified by PCR and cloned in frame with the alpha-peptide of the LacZ gene of E. coli in plasmid pUC19. This construct expressed SOD activity in E. coli with characteristics similar to those of the native SOD enzyme of P. gingivalis human strain 381 and the parent feline strain VPB 3457. The recombinant SOD had an apparent molecular weight of 54,700+/-1300 (S.E.M.) and was inactivated by 5mM hydrogen peroxide but not by 2mM KCN. There was a significant association (P=0.005) between the immunoreactivity of cats to P. gingivalis VPB 3457 soluble whole cell proteins on immunoblots and their responsiveness to the SOD protein. This suggests that cats showing a marked serum responsiveness to P. gingivalis itself, react to the SOD enzyme and further supports the role of feline P. gingivalis in periodontal disease.

  13. Central nervous system Toll-like receptor expression in response to Theiler's murine encephalomyelitis virus-induced demyelination disease in resistant and susceptible mouse strains

    Directory of Open Access Journals (Sweden)

    Turrin Nicolas P

    2008-12-01

    Full Text Available Abstract Background In immunopathological diseases, such as multiple sclerosis (MS, genetic and environmental factors that contribute to the initiation and progression of the disease are often discussed. The Theiler murine encephalomyelitis virus-induced demyelination disease (TMEV-IDD model used to study MS reflects this: genetically susceptible mice infected intra-cerebrally with TMEV develop a chronic demyelination disease. TMEV-IDD can be induced in resistant mouse strains by inducing innate immunity with lipopolysaccharide (LPS. Interestingly, Toll-like receptor 4 (TLR4 is the cognate receptor for LPS and its activation can induces up-regulation of other TLRs, such as TLR7 (the receptor for TMEV and 9, known to be involved in autoimmunity. Up-regulation of TLRs could be involved in precipitating an autoimmune susceptible state. Consequently, we looked at TLR expression in the susceptible (SJL/J and resistant (C57BL/6 strains of mice infected with TMEV. The resistant mice were induced to develop TMEV-IDD by two LPS injections following TMEV infection. Results Both strains were found to up-regulate multiple TLRs (TLR2, 7 and 9 following the TMEV infection. Expression of these TLRs and of viral mRNA was significantly greater in infected SJL/J mice. The susceptible SJL/J mice showed up-regulation of TLR3, 6 and 8, which was not seen in C57BL/6 mice. Conclusion Expression of TLRs by susceptible mice and the up-regulation of the TLRs in resistant mice could participate in priming the mice toward an autoimmune state and develop TMEV-IDD. This could have implications on therapies that target TLRs to prevent the emergence of conditions such as MS in patients at risk for the disease.

  14. Expression of chicken interleukin-2 by a highly virulent strain of Newcastle disease virus leads to decreased systemic viral load but does not significantly affect mortality in chickens.

    Science.gov (United States)

    Susta, Leonardo; Diel, Diego G; Courtney, Sean; Cardenas-Garcia, Stivalis; Sundick, Roy S; Miller, Patti J; Brown, Corrie C; Afonso, Claudio L

    2015-08-08

    In mammals, interleukin 2 (IL-2) has been shown to decrease replication or attenuate pathogenicity of numerous viral pathogens (herpes simplex virus, vaccinia virus, human respiratory syncytial virus, human immunodeficiency virus) by activating natural killer cells (NK), cytotoxic T lymphocytes and expanding subsets of memory cells. In chickens, IL-2 has been shown to activate T cells, and as such it might have the potential to affect replication and pathogenesis of Newcastle disease virus (NDV). To assess the effect of IL-2 during NDV infection in chickens, we produced a recombinant virulent NDV strain expressing chicken IL-2 (rZJ1-IL2). The effects of IL-2 expression were investigated in vivo using the intracerebral pathogenicity index (ICPI) in day-old chicks and pathogenesis experiments in 4-week-old chickens. In these studies, rZJ1-IL2 was compared to a control virus expressing the green fluorescent protein (rZJ1-GFP). Assessed parameters included survival curves, detailed histological and immunohistochemical grading of lesions in multiple organs, and virus isolation in blood, spleen and mucosal secretions of infected birds. At the site of infection (eyelid), expression of IL-2 was demonstrated in areas of rZJ-IL2 replication, confirming IL-2 production in vivo. Compared to rZJ1-GFP strain, rZJ1-IL2 caused milder lesions and displayed decreased viral load in blood, spleen and mucosal secretions of infected birds. In the rZJ1-IL2-infected group, virus level in the blood peaked at day 4 post-infection (pi) (10(3.46) EID50 /0.1 ml) and drastically decreased at day 5 pi (10(0.9) EID50/0.1 ml), while in the rZJ1-GFP-infected group virus levels in the blood reached 10(5.35) EID50/0.1 ml at day 5. However, rZJ1-IL2-infected groups presented survival curves similar to control birds infected with rZJ1-GFP, with comparable clinical signs and 100 % mortality. Further, expression of IL-2 did not significantly affect the ICPI scores, compared to rZJ1-GFP strain. Increased

  15. Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

    OpenAIRE

    Itoh, Takafumi; Hibi, Takao; Fujii, Yutaka; Sugimoto, Ikumi; Fujiwara, Akihiro; Suzuki, Fumiko; Iwasaki, Yukimoto; Kim, Jin-Kyung; Taketo, Akira; Kimoto, Hisashi

    2013-01-01

    Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly di...

  16. Constitutively Opa-Expressing and Opa-Deficient Neisseria gonorrhoeae Strains Differentially Stimulate and Survive Exposure to Human Neutrophils

    OpenAIRE

    Ball, Louise M.; Criss, Alison K.

    2013-01-01

    The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in frame, termed Opaless. Opaless Gc remained uniforml...

  17. Characterization of the expression of the thcB gene, coding for a pesticide-degrading cytochrome P-450 in Rhodococcus strains.

    OpenAIRE

    Shao, Z Q; Behki, R

    1996-01-01

    A cytochrome P-450 system in Rhodococcus strains, encoded by thcB, thcC, and thcD, participates in the degradation of thiocarbamates and several other pesticides. The regulation of the system was investigated by fusing a truncated lacZ in frame to thcB, the structural gene for the cytochrome P-450 monooxygenase. Analysis of the thcB-lacZ fusion showed that the expression of thcB was 10-fold higher in the presence of the herbicide EPTC (s-ethyl dipropylthiocarbamate). Similar enhancement of th...

  18. Metabolomic and 13C-Metabolic Flux Analysis of a Xylose-Consuming Saccharomyces cerevisiae Strain Expressing Xylose Isomerase

    Science.gov (United States)

    Wasylenko, Thomas M.; Stephanopoulos, Gregory

    2016-01-01

    Over the past two decades significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative PPP is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. PMID:25311863

  19. Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

    Science.gov (United States)

    Wasylenko, Thomas M; Stephanopoulos, Gregory

    2015-03-01

    Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. © 2014 Wiley Periodicals, Inc.

  20. Expression of endogenous ALV antigens and susceptibility to subgroup E ALV in three strains of chickens (Endogenous avian C. type virus)

    International Nuclear Information System (INIS)

    Robinson, H.L.; Lamoreux, W.F.

    1976-01-01

    Cells from three strains of Kimber Farms chickens, K16, K18, and K28, have been characterized for the expression of endogenous avian C-type virus (ALV) antigens and for susceptibility to subgroup E ALV. In K16 the coordinate dominant expression of chick helper factor (chf ), the type specific antigen of endogenous subgroup E ALV, and the group specific (gs) antigen of ALV was observed. The expression of chf and gs antigen in K16 chf + gs + cells was similar to that observed in SPAFAS and H and N gs + cells. In K18 chf but not gs antigen was expressed. The expression of chf in K18 chf + gs + cells was distinct from that observed for the chf + gs + pedigrees but similar to that found in SPAFAS chf + gs - helper extremely high (h/sub E/) cells. Cells from K16 and K18 chickens were uniformly resistant to subgroup E ALV. In cells from K28 chickens, susceptibility to subgroup B virus correlated with susceptibility to subgroup E virus. The efficiency of plating of subgroup E virus on susceptible K28 cells with 10 3 --10 4 -fold lower on chf + cells than on chf - cells

  1. Cloning of gag gene of HIV-1 subtype c (Indian strain into a mammalian expression vector and in vitro expression studies

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    Chugh P

    2003-01-01

    Full Text Available PURPOSE: Acquired immunodeficiency syndrome caused by HIV-1 is one of the biggest health problems we are facing today. It is required to concentrate efforts towards designing a safe, effective and affordable vaccine candidate in the context of the growing epidemic worldwide. Recently the approach of DNA based immunogen has evoked a lot of enthusiasm in the preclinical models. METHODS: This study was designed towards a subtype C based gag DNA construct in the expression vector pJW4304. The gag and protease genes of HIV-1 subtype C were cloned into a mammalian expression vector pJW4304. The cloning strategy was designed so as to express a naturally processed form of the protein. Expression of gag protein by the construct pJWgagprotease49587 was evaluated by western blotting, p24 antigen capture ELISA and electron microscopy. RESULTS: Gag p24 was detected both in the supernatant and in the transfected cells. Extra cellular p24 protein was estimated by p24 antigen capture ELISA. Immunoblotting using HIV positive polyclonal sera further confirmed the expression and processing of gag gene. The 24kDa band has been observed in cell lysates, which indicates that the proper processing is taking place in the presence of protease. Virus like particles were seen budding from the cell membrane 24 and 48 hours post transfection by transmission electron microscopy. CONCLUSIONS: The recombinant construct pJWgagprotease49587 has shown good expression in vitro and therefore is a good candidate to study immunogenicity of the construct. Immunogenicity testing in mice is being carried out currently with this construct.

  2. Recombinant infectious bronchitis virus (IBV) H120 vaccine strain expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) protects chickens against IBV and NDV challenge.

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    Yang, Xin; Zhou, Yingshun; Li, Jianan; Fu, Li; Ji, Gaosheng; Zeng, Fanya; Zhou, Long; Gao, Wenqian; Wang, Hongning

    2016-05-01

    Infectious bronchitis (IB) and Newcastle disease (ND) are common viral diseases of chickens, which are caused by infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), respectively. Vaccination with live attenuated strains of IBV-H120 and NDV-LaSota are important for the control of IB and ND. However, conventional live attenuated vaccines are expensive and result in the inability to differentiate between infected and vaccinated chickens. Therefore, there is an urgent need to develop new efficacious vaccines. In this study, using a previously established reverse genetics system, we generated a recombinant IBV virus based on the IBV H120 vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of NDV. The recombinant virus, R-H120-HN/5a, exhibited growth dynamics, pathogenicity and viral titers that were similar to those of the parental IBV H120, but it had acquired hemagglutination activity from NDV. Vaccination of SPF chickens with the R-H120-HN/5a virus induced a humoral response at a level comparable to that of the LaSota/H120 commercial bivalent vaccine and provided significant protection against challenge with virulent IBV and NDV. In summary, the results of this study indicate that the IBV H120 strain could serve as an effective tool for designing vaccines against IB and other infectious diseases, and the generation of IBV R-H120-HN/5a provides a solid foundation for the development of an effective bivalent vaccine against IBV and NDV.

  3. Effect of phenolic compounds and osmotic stress on the expression of penicillin biosynthetic genes from Penicillium chrysogenum var. halophenolicum strain

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    Sumaya Ferreira Guedes

    2012-01-01

    Full Text Available Phenol and phenolic compounds are aromatic pollutants that inhibit biological treatment of wastewaters. Penicillium chrysogenum var. halophenolicum is a halotolerant fungus that previously showed the ability to degrade phenol and resorcinol in high salinity conditions. The presence of the penicillin biosynthetic cluster in P. chrysogenum var. halophenolicum was recently described. In this article, we examined the expression of pcbAB, pcbC and penDE, genes responsible for δ-(L-α-aminoadipyl-L-cysteinyl-D-valine synthetase, isopenicillin N synthase and isopenicillin N acyltransferase activities, respectively, in P. chrysogenum var. halophenolicum. A quantitative PCR (qPCR approach was used to determine how these genes were expressed in media with 2% and 5.9% NaCl supplemented with phenol, catechol, hydroquinone and resorcinol as the sole carbon source. The effect of salt on the capability of P. chrysogenum var. halophenolicum to degrade aromatic compounds was measured using HPLC. qPCR analysis of RNA extracted from P. chrysogenum var. halophenolicum indicated that the expression levels of pcbAB, pcbC and penDE decreased in high saline concentrations compared to the levels expressed in media with glucose. High concentrations of salt significantly repress the expression of pcbAB and penDE. The pcbC gene was expressed differentially in catechol containing medium. There was no evident relationship between the expression levels of penicillin biosynthetic genes and yields of penicillin. Meanwhile, the presence of phenol and phenolic compounds seems to positively influence the antibiotic production; high concentrations of salt stimulated penicillin production. These results support the hypothesis that phenol, phenolic compounds and high concentrations of salt could act like a stress factor for P. chrysogenum var. halophenolicum resulting in higher yields of β-lactam antibiotic production.

  4. Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

    Science.gov (United States)

    Heinemann, Ute; Engels, Dirk; Bürger, Sibylle; Kiziak, Christoph; Mattes, Ralf; Stolz, Andreas

    2003-01-01

    The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents. PMID:12902216

  5. Construction of a recombinant Lactococcus lactis strain expressing a fusion protein of Omp22 and HpaA from Helicobacter pylori for oral vaccine development.

    Science.gov (United States)

    Zhang, Rongguang; Duan, Guangcai; Shi, Qingfeng; Chen, Shuaiyin; Fan, Qingtang; Sun, Nan; Xi, Yuanlin

    2016-11-01

    To develop orally administrated anti-Helicobacter pylori vaccination, a Lactococcus lactis strain was genetically constructed for fusion expression of H. pylori protective antigens HpaA and Omp22. The fusion gene of omp22 and hpaA with an adapter encoding three glycines was cloned from a plasmid pMAL-c2x-omp22-hpaA into Escherichia coli MC1061 and L. lactis NZ3900 successively using a shutter vector pNZ8110. Expression of the fusion gene in L. lactis was induced with nisin resulting in production of proteins with molecular weights of 50 and 28 kDa. Both of them were immunoreactive with mouse anti-H. pylori sera as determined via western blotting. Oral vaccination of BALB/c mice using the L. lactis strain carrying pNZ8110-omp22-hpaA elicited significant systematic humoral immune response (P lactis with immunogenicity. This is a considerable step towards H. pylori vaccines.

  6. Hypothalamic expression of Peg3 gene is associated with maternal care differences between SM/J and LG/J mouse strains.

    Science.gov (United States)

    Chiavegatto, Silvana; Sauce, Bruno; Ambar, Guilherme; Cheverud, James M; Peripato, Andrea C

    2012-07-01

    Maternal care is essential in mammals, and variations in the environment provided by mothers may directly influence the viability of newborns and emotional behavior later in life. A previous study investigated genetic variations associated with maternal care in an intercross of LG/J and SM/J inbred mouse strains and identified two single-locus QTLs (quantitative trait loci). Here, we selected three candidate genes located within these QTLs intervals; Oxt on chromosome 2, and FosB and Peg3 on chromosome 7 and tested their association with maternal care. LG/J females showed impaired postpartum nest building and pup retrieval, a one-day delay in milk ejection, reduced exploratory activity, and higher anxiety-like behavior when compared to SM/J females. The nucleotide sequences of Oxt and FosB were similar between strains, as were their hypothalamic expression levels. Conversely, Peg3 nucleotide sequences showed four nonsynonymous replacement substitutions on LG/J dams, T11062G, G13744A, A13808G, and G13813A, and a 30 base pair (10 aa) in tandem repeat in the coding region with three copies in SM/J and five copies in LG/J. Maternal care impaired LG/J mothers express 37% lower Peg3 mRNA levels in the hypothalamus on the second postpartum day. We also found an association of the Peg3 repeat-variant and poor maternal care in F(2) heterozygote females derived from a LG/J × SM/J intercross. These results may suggest that the maternally imprinted Peg3 gene is responsible for the single-locus QTL on chromosome 7 that has been shown to influence maternal care in these strains. Furthermore, these data provide additional support for an epigenetic regulation of maternal behavior.

  7. Strain-dependent effects of sub-chronically infused losartan against kainic acid-induced seizures, oxidative stress, and heat shock protein 72 expression.

    Science.gov (United States)

    Tchekalarova, Jane; Ivanova, Natasha; Pechlivanova, Daniela; Ilieva, Kalina; Atanasova, Milena

    2014-01-01

    We studied the involvement of angiotensin (Ang) II AT1 receptors in the pathophysiology of kainate (KA)-induced neurotoxicity, focusing on the regulation of the oxidative stress state and expression of HSP 72 in the frontal cortex and hippocampus in two strains, spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. The KA injection was executed after the rats were infused subcutaneously via osmotic mini-pumps with losartan (10 mg/kg day) for 14 days. Losartan delayed the onset of KA-induced seizures in SHRs but not in Wistar rats without affecting the seizure intensity score. This selective AT1 receptor antagonist decreased the lipid peroxidation only in naive SHRs. However, it attenuated the KA-induced increase in lipid peroxidation in both SHRs and Wistar rats. The adaptive enhancement of cytosolic superoxide dismutase (SOD) activity in KA-treated SHRs was recovered to control level after sub-chronic losartan infusion while no change in mitochondrial SOD activity was detected in the two strains. Both losartan and KA produced a higher expression of HSP 72 in the hippocampus of the two strains compared to naive rats infused with vehicle. Taken together, our findings demonstrate that the efficacy of a sub-chronic systemic losartan infusion in preventing the KA-induced seizure activity and neurotoxicity is more pronounced in SHRs, considered as a model of essential hypertension, than in normotenisve Wistar rats. The results suggest that the blockade of AT1 receptors, commonly used as a strategy for prevention of high blood pressure, may be useful as an adjunctive treatment in status epilepticus to reduce oxidative stress and neurotoxicity.

  8. New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells

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    Davide Campoccia

    2018-04-01

    Full Text Available Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI, and internalised bacteria. The investigation included nine Staphylococcus aureus, seven Staphylococcus epidermidis, five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

  9. New Parameters to Quantitatively Express the Invasiveness of Bacterial Strains from Implant-Related Orthopaedic Infections into Osteoblast Cells.

    Science.gov (United States)

    Campoccia, Davide; Montanaro, Lucio; Ravaioli, Stefano; Cangini, Ilaria; Testoni, Francesca; Visai, Livia; Arciola, Carla Renata

    2018-04-03

    Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI), and internalised bacteria. The investigation included nine Staphylococcus aureus , seven Staphylococcus epidermidis , five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

  10. Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

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    Joan Joseph

    2010-01-01

    Full Text Available Mycobacterium bovis Bacillus Calmette-Guérin (BCG as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261 and Mycobacteria spp. α-antigen promoter (in plasmid pJH222. Among 14 rBCG:HIV-1gp120 (pMV261 colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222 colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors.

  11. Gene expression profiles of the thermotolerant yeast Saccharomyces cerevisiae strain KKU-VN8 during high-temperature ethanol fermentation using sweet sorghum juice.

    Science.gov (United States)

    Techaparin, Atiya; Thanonkeo, Pornthap; Klanrit, Preekamol

    2017-10-01

    To investigate gene expression profiles of the thermotolerant yeast Saccharomyces cerevisiae strain KKU-VN8, a potential high-ethanol producer, in response to various stresses during high-temperature ethanol fermentation using sweet sorghum juice (SSJ) under optimal conditions. The maximal ethanol concentration obtained by S. cerevisiae KKU-VN8 using SSJ at 40 °C was 66.6 g/l, with a productivity of 1.39 g/l/h and a theoretical ethanol yield of 81%. Quantitative RT-PCR assays were performed to investigate the gene expression profiles of S. cerevisiae KKU-VN8. Differential expression of genes encoding heat-shock proteins (HSP82, HSP104, SSA4), genes involved in trehalose metabolism (TPS1, TPS2, NTH1) and genes involved the glycolytic pathway (ADH1, ADH2, CDC19) at various time points during fermentation was observed. The expression levels of HSP82, HSP104, SSA4, ADH1 and CDC19 were significantly higher than those of the controls (10.2-, 4-, 8-, 8.9- and 5.9-fold higher, respectively). In contrast, the expression levels of TPS1, TPS2, NTH1 and ADH2 were approx. 2-fold less than those of the controls. The highly expressed genes encoding heat-shock proteins, HSP82 and SSA4, potentially play an important role in helping S. cerevisiae KKU-VN8 cope with various stresses that occur during high-temperature fermentation, leading to higher ethanol production efficiency.

  12. Immunobiotic Lactobacillus strains augment NLRP3 expression in newborn and adult porcine gut-associated lymphoid tissues.

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    Tohno, Masanori; Shimosato, Takeshi; Aso, Hisashi; Kitazawa, Haruki

    2011-12-15

    We isolated cDNA encoding porcine nucleotide-binding domain-like receptor family, pryin domain containing 3 (NLRP3) from Peyer's patches. The complete nucleotide open reading frame of porcine NLRP3 contains 3108-bp encoding a deduced polypeptide of 1036-amino acid residues. The porcine NLRP3 amino acid sequence is more similar to the longest isoform of human than the mouse counterpart. The predicted amino acid sequence of porcine NLRP3 presented nine C-terminal leucine-rich repeat domains. In newborn swine, the expression of NLRP3 was detected at higher levels in spleen and mesenteric lymph nodes, while lower levels were observed in intestinal tissues. In adult swine, NLRP3 was strongly expressed in Peyer's patches and the mesenteric lymph nodes, and the expression level in the lower intestinal tissues was comparable to that in spleen. Toll-like receptor and nucleotide-binding domain ligands, as well as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus gasseri, enhanced NLRP3 expression in gut-associated lymphoid tissues (GALT) of newborn and adult swine. Our results should aid in understanding the intestinal immunoregulatory mechanisms underlying NLRP3 activation and the priming ability of immunobiotic lactic acid bacteria in porcine GALT. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Human rotavirus strain Wa downregulates NHE1 and NHE6 expressions in rotavirus-infected Caco-2 cells.

    Science.gov (United States)

    Chen, Honglang; Song, Lijun; Li, Guixian; Chen, Wenfeng; Zhao, Shumin; Zhou, Ruoxia; Shi, Xiaoying; Peng, Zhenying; Zhao, Wenchang

    2017-06-01

    Rotavirus (RV) is the most common cause of severe gastroenteritis and fatal dehydration in human infants and neonates of different species. However, the pathogenesis of rotavirus-induced diarrhea is poorly understood. Secretory diarrhea caused by rotavirus may lead to a combination of excessive secretion of fluid and electrolytes into the intestinal lumen. Fluid absorption in the small intestine is driven by Na + -coupled transport mechanisms at the luminal membrane, including Na + /H + exchanger (NHE). Here, we performed qRT-PCR to detect the transcription of NHEs. Western blotting was employed for protein detection. Furthermore, immunocytochemistry was used to validate the NHE's protein expression. Finally, intracellular Ca 2+ concentration was detected by confocal laser scanning microscopy. The results demonstrated that the NHE6 mRNA and protein expressed in the human colon adenocarcinoma cell line (Caco-2). Furthermore, RV-Wa induced decreased expression of the NHE1 and NHE6 in Caco-2 cell in a time-dependent manner. In addition, intracellular Ca 2+ concentration in RV-Wa-infected Caco-2 cells was higher than that in the mock-infected cells. Furthermore, RV-Wa also can downregulate the expression of calmodulin (CaM) and calmodulin kinase II (CaMKII) in Caco-2 cells. These findings provides important insights into the mechanisms of rotavirus-induced diarrhea. Further studies on the underlying pathophysiological mechanisms that downregulate NHEs in RV-induced diarrhea are required.

  14. FLO11 expression and lipid biosynthesis are required for air-liquid biofilm formation in a Saccharomyces cerevisiae flor strain.

    Science.gov (United States)

    Zara, Giacomo; Goffrini, Paola; Lodi, Tiziana; Zara, Severino; Mannazzu, Ilaria; Budroni, Marilena

    2012-11-01

    Air-liquid biofilm formation is largely dependent on Flo11p and seems related to cell lipid content and composition. Here, it is shown that in the presence of cerulenin, a known inhibitor of the fatty acid synthase complex, biofilm formation is inhibited together with FLO11 transcription in a flor strain of Saccharomyces cerevisiae, while the administration of saturated fatty acids to cerulenin-containing medium restores biofilm formation and FLO11 transcription. It is also shown that, in biofilm cells, the FLO11 transcription is accompanied by the transcription of ACC1, ACS1 and INO1 key genes in lipid biosynthesis and that biofilm formation is affected by the lack of inositol in flor medium. These results are compatible with the hypothesis that the air-liquid biofilm formation depends on FLO11 transcription levels as well as on fatty acids biosynthesis. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  15. Biomimetic fetal rotation bioreactor for engineering bone tissues-Effect of cyclic strains on upregulation of osteogenic gene expression.

    Science.gov (United States)

    Ravichandran, Akhilandeshwari; Wen, Feng; Lim, Jing; Chong, Mark Seow Khoon; Chan, Jerry K Y; Teoh, Swee-Hin

    2018-01-03

    Cells respond to physiological mechanical stresses especially during early fetal development. Adopting a biomimetic approach, it is necessary to develop bioreactor systems to explore the effects of physiologically relevant mechanical strains and shear stresses for functional tissue growth and development. This study introduces a multimodal bioreactor system that allows application of cyclic compressive strains on premature bone grafts that are cultured under biaxial rotation (chamber rotation about 2 axes) conditions for bone tissue engineering. The bioreactor is integrated with sensors for dissolved oxygen levels and pH that allow real-time, non-invasive monitoring of the culture parameters. Mesenchymal stem cells-seeded polycaprolactone-β-tricalcium phosphate scaffolds were cultured in this bioreactor over 2 weeks in 4 different modes-static, cyclic compression, biaxial rotation, and multimodal (combination of cyclic compression and biaxial rotation). The multimodal culture resulted in 1.8-fold higher cellular proliferation in comparison with the static controls within the first week. Two weeks of culture in the multimodal bioreactor utilizing the combined effects of optimal fluid flow conditions and cyclic compression led to the upregulation of osteogenic genes alkaline phosphatase (3.2-fold), osteonectin (2.4-fold), osteocalcin (10-fold), and collagen type 1 α1 (2-fold) in comparison with static cultures. We report for the first time, the independent and combined effects of mechanical stimulation and biaxial rotation for bone tissue engineering using a bioreactor platform with non-invasive sensing modalities. The demonstrated results show leaning towards the futuristic vision of using a physiologically relevant bioreactor system for generation of autologous bone grafts for clinical implantation. Copyright © 2018 John Wiley & Sons, Ltd.

  16. Strain-specific Plasmodium falciparum multifunctional CD4(+) T cell cytokine expression in Malian children immunized with the FMP2.1/AS02A vaccine candidate.

    Science.gov (United States)

    Graves, Shawna F; Kouriba, Bourema; Diarra, Issa; Daou, Modibo; Niangaly, Amadou; Coulibaly, Drissa; Keita, Yamoussa; Laurens, Matthew B; Berry, Andrea A; Vekemans, Johan; Ripley Ballou, W; Lanar, David E; Dutta, Sheetij; Gray Heppner, D; Soisson, Lorraine; Diggs, Carter L; Thera, Mahamadou A; Doumbo, Ogobara K; Plowe, Christopher V; Sztein, Marcelo B; Lyke, Kirsten E

    2016-05-17

    Based on Plasmodium falciparum (Pf) apical membrane antigen 1 (AMA1) from strain 3D7, the malaria vaccine candidate FMP2.1/AS02A showed strain-specific efficacy in a Phase 2 clinical trial in 400 Malian children randomized to 3 doses of the AMA1 vaccine candidate or control rabies vaccine on days 0, 30 and 60. A subset of 10 Pf(-) (i.e., no clinical malaria episodes) AMA1 recipients, 11 Pf(+) (clinical malaria episodes with parasites with 3D7 or Fab9-type AMA1 cluster 1 loop [c1L]) AMA1 recipients, and 10 controls were randomly chosen for analysis. Peripheral blood mononuclear cells (PBMCs) isolated on days 0, 90 and 150 were stimulated with full-length 3D7 AMA1 and c1L from strains 3D7 (c3D7) and Fab9 (cFab9). Production of IFN-γ, TNF-α, IL-2, and/or IL-17A was analyzed by flow cytometry. Among AMA1 recipients, 18/21 evaluable samples stimulated with AMA1 demonstrated increased IFN-γ, TNF-α, and IL-2 derived from CD4(+) T cells by day 150 compared to 0/10 in the control group (pvaccines, CD4(+) cells expressing both TNF-α and IL-2 were increased in Pf(-) children compared to Pf(+) children. When PBMCs were stimulated with c3D7 and cFab9 separately, 4/18 AMA1 recipients with an AMA1-specific CD4(+) response had a significant response to one or both c1L. This suggests that recognition of the AMA1 antigen is not dependent upon c1L alone. In summary, AMA1-specific T cell responses were notably increased in children immunized with an AMA1-based vaccine candidate. The role of CD4(+)TNF-α(+)IL-2(+)-expressing T cells in vaccine-induced strain-specific protection against clinical malaria requires further exploration. Clinicaltrials.gov Identifier: NCT00460525. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Construction of strain engineered for expression of porcine β-defensin-2/cecropin P1 fusion antimicrobial peptides and its growth-promoting effect and antimicrobial activity

    Directory of Open Access Journals (Sweden)

    Jian Xu

    2017-04-01

    Full Text Available Objective To generate recombinant Bacillus subtilis (B. subtilis engineered for expression of porcine β-defensin-2 (pBD-2 and cecropin P1 (CP1 fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli. The weight gain and diarrhea incidence of piglets were measured after challenge. Results The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis and gram-positive bacteria (Staphylococcus aureus. Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial

  18. [Expression, structure and antigenicity analysis of N51 derived from the N-terminal heptad repeat domain in gp41 of HIV-1 CRF07_BC strain].

    Science.gov (United States)

    Shao, Jiping; Jiang, Shibo; Liu, Shuwen

    2012-12-01

    To express N51 derived from the N-terminal heptad repeat (NHR) domain in gp41 of the HIV-1 CRF07_BC strain and analyze its molecular structure and antigenicity. Overlapping PCR was used to amplify the DNA fragment encoding N51Fd gene, which was then subcloned into the vector pFUSE-hIgG1-Fc2. The construct was confirmed by DNA sequencing. The structure and antigenicity of the recombinant protein N51FdFc-BC were analyzed using bioinformatic software, circular dichroism, and Western blotting. A recombinant expression vector pFUSE/N51Fd-BC was successfully constructed. N51FdFc-BC recombinant protein with a relative molecular mass of about 35 000 was effectively expressed in mammalian 293T cells and could be recognized by rabbit antibodies against HIV-1 gp41 N/C peptides as shown by Western blotting. Bioinformatic analysis showed that the recombinant protein N51FdFc-BC, with a relative molecular mass of 34 315.1 and a PI of 7.59, formed a secondary structure of random coil to allow its interactions as an antigen with antibodies. Circular dichroism measurement confirmed the random coil structure of N51FdFc-BC protein. The recombinant protein N51FdFc-BC has a random coil structure and can be used as an immunogen for development of HIV-1 subunit vaccine.

  19. The consistent differential expression of genetic pathways following exposure of an industrial Pseudomonas aeruginosa strain to preservatives and a laundry detergent formulation.

    Science.gov (United States)

    Green, Angharad E; Amézquita, Alejandro; Le-Marc, Yvan; Bull, Matthew J; Connor, Thomas R; Mahenthiralingam, Eshwar

    2018-03-14

    Pseudomonas aeruginosa (P. aeruginosa) is a common contaminant associated with product recalls in the home and personal care (HPC) industry. Preservation systems are used to prevent spoilage and protect consumers, but greater knowledge is needed of preservative resistance mechanisms used by P. aeruginosa contaminants. We aimed to identify genetic pathways associated with preservative exposure by using an industrial P. aeruginosa strain and implementing RNA-Seq to understand gene expression changes in response to industry relevant conditions. The consistent differential expression of five genetic pathways during exposure to multiple industrial growth conditions associated with benzisothiazolone (BIT) and phenoxyethanol (POE) preservatives, and a laundry detergent (LD) formulation, was observed. A MexPQ-OpmE Resistance Nodulation Division (RND) efflux pump system was commonly up-regulated in response to POE, a combination of BIT and POE, and LD together with BIT. In response to all industry conditions, a putative sialic acid transporter and isoprenoid biosynthesis gnyRDBHAL operon demonstrated consistent up-regulation. Two operons phnBA and pqsEDCBA involved in PQS production and quorum-sensing, were also consistently down-regulated during exposure to all the industry conditions. The ability to identify consistently differentially expressed genetic pathways in P. aeruginosa can inform the development of future targeted preservation systems that maintain product safety and minimise resistance development.

  20. Molecular cloning, sequence analysis, prokaryotic expression, and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain.

    Science.gov (United States)

    Pan, H C; Yang, H Q; Zhao, F X; Qian, X C

    2014-08-28

    The cDNA sequence of foot-specific peroxidase PPOD1 from the Chinese strain of Hydra magnipapillata was cloned by reverse transcription-polymerase chain reaction. The cDNA sequence contained a coding region with an 873-bp open reading frame, a 31-bp 5'-untranslated region, and a 36-bp 3'-untranslated region. The structure prediction results showed that PPOD1 contains 10.34% of α-helix, 38.62% of extended strand, 12.41% of β-turn, and 38.62% of random coil. The structural core was α-helix at the N terminus. The GenBank protein blast server showed that PPOD1 contains 2 fascin-like domains. In addition, high-level PPOD1 activity was only present in the ectodermal epithelial cells located on the edge of the adhesive face of the basal disc, and that these cells extended lamellipodia and filopodia when the basal disc was tightly attached to a glass slide. The fascin-like domains of Hydra PPOD1 might contribute to the bundling of the actin filament of these cells, and hence, the formation of filopodia. In conclusion, these cells might play an important role in strengthening the adsorbability of the basal disc to substrates.

  1. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  2. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    Energy Technology Data Exchange (ETDEWEB)

    Kraschnefski, Mark J.; Scott, Stacy A. [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia); Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von [Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010 (Australia); Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia)

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.

  3. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    International Nuclear Information System (INIS)

    Kraschnefski, Mark J.; Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von; Blanchard, Helen

    2005-01-01

    The carbohydrate-binding component (VP8* 64–223 ) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8* 64–223 structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3 2 21 and monoclinic P2 1 ) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8* 64–223 structure by molecular replacement

  4. Genetic variation in hippocampal microRNA expression differences in C57BL/6 J X DBA/2 J (BXD recombinant inbred mouse strains

    Directory of Open Access Journals (Sweden)

    Parsons Michael J

    2012-09-01

    Full Text Available Abstract Background miRNAs are short single-stranded non-coding RNAs involved in post-transcriptional gene regulation that play a major role in normal biological functions and diseases. Little is currently known about how expression of miRNAs is regulated. We surveyed variation in miRNA abundance in the hippocampus of mouse inbred strains, allowing us to take a genetic approach to the study of miRNA regulation, which is novel for miRNAs. The BXD recombinant inbred panel is a very well characterized genetic reference panel which allows quantitative trait locus (QTL analysis of miRNA abundance and detection of correlates in a large store of brain and behavioural phenotypes. Results We found five suggestive trans QTLs for the regulation of miRNAs investigated. Further analysis of these QTLs revealed two genes, Tnik and Phf17, under the miR-212 regulatory QTLs, whose expression levels were significantly correlated with miR-212 expression. We found that miR-212 expression is correlated with cocaine-related behaviour, consistent with a reported role for this miRNA in the control of cocaine consumption. miR-31 is correlated with anxiety and alcohol related behaviours. KEGG pathway analysis of each miRNA’s expression correlates revealed enrichment of pathways including MAP kinase, cancer, long-term potentiation, axonal guidance and WNT signalling. Conclusions The BXD reference panel allowed us to establish genetic regulation and characterize biological function of specific miRNAs. QTL analysis enabled detection of genetic loci that regulate the expression of these miRNAs. eQTLs that regulate miRNA abundance are a new mechanism by which genetic variation influences brain and behaviour. Analysis of one of these QTLs revealed a gene, Tnik, which may regulate the expression of a miRNA, a molecular pathway and a behavioural phenotype. Evidence of genetic covariation of miR-212 abundance and cocaine related behaviours is strongly supported by previous

  5. Systemically administered DNA and fowlpox recombinants expressing four vaccinia virus genes although immunogenic do not protect mice against the highly pathogenic IHD-J vaccinia strain.

    Science.gov (United States)

    Bissa, Massimiliano; Pacchioni, Sole Maria; Zanotto, Carlo; De Giuli Morghen, Carlo; Illiano, Elena; Granucci, Francesca; Zanoni, Ivan; Broggi, Achille; Radaelli, Antonia

    2013-12-26

    The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully eradicated the disease worldwide. Therefore, it was not administered any more after 1980, as smallpox no longer existed as a natural infection. However, emerging threats by terrorist organisations has prompted new programmes for second-generation vaccine development based on attenuated VV strains, which have been shown to cause rare but serious adverse events in immunocompromised patients. Considering the closely related animal poxviruses that might also be used as bioweapons, and the increasing number of unvaccinated young people and AIDS-affected immunocompromised subjects, a safer and more effective smallpox vaccine is still required. New avipoxvirus-based vectors should improve the safety of conventional vaccines, and protect from newly emerging zoonotic orthopoxvirus diseases and from the threat of deliberate release of variola or monkeypox virus in a bioterrorist attack. In this study, DNA and fowlpox recombinants expressing the L1R, A27L, A33R and B5R genes were constructed and evaluated in a pre-clinical trial in mouse, following six prime/boost immunisation regimens, to compare their immunogenicity and protective efficacy against a challenge with the lethal VV IHD-J strain. Although higher numbers of VV-specific IFNγ-producing T lymphocytes were observed in the protected mice, the cytotoxic T-lymphocyte response and the presence of neutralising antibodies did not always correlate with protection. In spite of previous successful results in mice, rabbits and monkeys, where SIV/HIV transgenes were expressed by the fowlpox vector, the immune response elicited by these recombinants was low, and most of the mice were not protected. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Occurrence and characterization of stx and/or eae-positive Escherichia coli isolated from wildlife, including a typical EPEC strain from a wild boar.

    Science.gov (United States)

    Alonso, Carla Andrea; Mora, Azucena; Díaz, Dafne; Blanco, Miguel; González-Barrio, David; Ruiz-Fons, Francisco; Simón, Carmen; Blanco, Jorge; Torres, Carmen

    2017-08-01

    Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains are food-borne pathogens associated with acute diarrhea. Haemolytic-uremic syndrome (HUS) is often a complication of STEC infection. In order to examine the occurrence, serotypes, virulence and antimicrobial-resistance profiles of STEC and EPEC in wildlife, 326 faecal E. coli strains from 304 clinically healthy animals were analyzed. For this approach stx 1 , stx 2 and eae genes, as well as accessory virulence determinants (ehx, hlyA, saa, tia, bfp, subAB) were PCR-screened and sequenced. Serotyping was performed employing all available O (O1-O185) and H (H1-H56) antisera. Genetic diversity was analyzed by XbaI-PFGE and phylotyping. Thirteen STEC (4.3%) and 10 EPEC (3.3%) were identified among 12 deer, 3 mouflon, 6 wild boars and 2 birds. Nine STEC showed seropathotypes B (O145:[H28]) and C (O22:H8, O128:[H2]) associated with HUS, and D (O110:H28, O146:H21, O146:[H28], ONT:H8) associated with human diarrhea. Although most isolates harbored stx 2b and stx 1c variants, stx 2a and stx 1a (related with severe disease) were also detected. Additionally, the eae gene was present in one stx 2a -positive O145:[H28] STEC from a deer and 11 STEC harbored subAB genes (mainly the subAB 2 variant). EPEC isolates showed 7 different intimin variants (β1, β2, γ1, ε1, ζ1, ι1-A, κ). Interestingly, the O49:[H10] eae-κ EPEC isolated from a wild boar was bfpA-positive showing a combination of serotype/virulence profile previously detected among human clinical tEPEC. Based on present results, wild ruminants, wild boars and to a lesser extent birds would be carriers of potentially pathogenic STEC and EPEC strains. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Annotation of Differential Gene Expression in Small Yellow Follicles of a Broiler-Type Strain of Taiwan Country Chickens in Response to Acute Heat Stress.

    Science.gov (United States)

    Cheng, Chuen-Yu; Tu, Wei-Lin; Wang, Shih-Han; Tang, Pin-Chi; Chen, Chih-Feng; Chen, Hsin-Hsin; Lee, Yen-Pai; Chen, Shuen-Ei; Huang, San-Yuan

    2015-01-01

    This study investigated global gene expression in the small yellow follicles (6-8 mm diameter) of broiler-type B strain Taiwan country chickens (TCCs) in response to acute heat stress. Twelve 30-wk-old TCC hens were divided into four groups: control hens maintained at 25°C and hens subjected to 38°C acute heat stress for 2 h without recovery (H2R0), with 2-h recovery (H2R2), and with 6-h recovery (H2R6). Small yellow follicles were collected for RNA isolation and microarray analysis at the end of each time point. Results showed that 69, 51, and 76 genes were upregulated and 58, 15, 56 genes were downregulated after heat treatment of H2R0, H2R2, and H2R6, respectively, using a cutoff value of two-fold or higher. Gene ontology analysis revealed that these differentially expressed genes are associated with the biological processes of cell communication, developmental process, protein metabolic process, immune system process, and response to stimuli. Upregulation of heat shock protein 25, interleukin 6, metallopeptidase 1, and metalloproteinase 13, and downregulation of type II alpha 1 collagen, discoidin domain receptor tyrosine kinase 2, and Kruppel-like factor 2 suggested that acute heat stress induces proteolytic disintegration of the structural matrix and inflamed damage and adaptive responses of gene expression in the follicle cells. These suggestions were validated through gene expression, using quantitative real-time polymerase chain reaction. Functional annotation clarified that interleukin 6-related pathways play a critical role in regulating acute heat stress responses in the small yellow follicles of TCC hens.

  8. Diversities of interaction of murine macrophages with three strains of Candida albicans represented by MyD88, CARD9 gene expressions and ROS, IL-10 and TNF-α secretion

    OpenAIRE

    Zhang, Xiaohuan; Ge, Yanping; Li, Wenqing; Hu, Yan

    2014-01-01

    Aim: To explore the mechanisms underlying the different responses of macrophages to distinct Candida albicans strains. Methods: Bone marrow was collected from mice. Macrophages were independently incubated with 3 Candida albicans strains. Results: MyD88 expression in Candida albicans 3683 group was significantly higher than that in Candida albicans 3630 group and Candida albicans SC5314 group, and marked difference was also observed between later two groups (P

  9. Control of Virulence Gene Expression by the Master Regulator, CfaD, in the Prototypical Enterotoxigenic Escherichia coli Strain, H10407

    Directory of Open Access Journals (Sweden)

    Carla Hodson

    2017-08-01

    Full Text Available Enterotoxigenic Escherichia coli (ETEC is the most common bacterial cause of diarrhea in children in developing countries, as well as in travelers to these countries. To cause disease, ETEC needs to produce a series of virulence proteins including enterotoxins, colonization factors and secretion pathways, which enable this pathogen to colonize the human small intestine and deliver enterotoxins to epithelial cells. Previously, a number of studies have demonstrated that CfaD, an AraC-like transcriptional regulator, plays a key role in virulence gene expression by ETEC. In this study, we carried out a transcriptomic analysis of ETEC strain, H10407, grown under different conditions, and determined the complete set of genes that are regulated by CfaD. In this way, we identified a number of new target genes, including rnr-1, rnr-2, etpBAC, agn43, flu, traM and ETEC_3214, whose expression is strongly activated by CfaD. Using promoter-lacZ reporters, primer extension and electrophoretic mobility shift assays, we characterized the CfaD-mediated activation of several selected target promoters. We also showed that the gut-associated environmental signal, sodium bicarbonate, stimulates CfaD-mediated upregulation of its virulence target operons. Finally, we screened a commercial small molecule library and identified a compound (CH-1 that specifically inhibited the regulatory function of CfaD, and by 2-D analoging, we identified a second inhibitor (CH-2 with greater potency.

  10. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yang-De, E-mail: zhangyd1960@yahoo.com.cn; Li, Hao [National Hepatobiliary and Enteric Surgery Research Center of The Ministry of Health, Xiangya Hospital, Central South University, Hunan Province (China); Liu, Hui; Pan, Yi-Feng [Biochemistry Laboratory, Institution of Biomedical Engineering, Central South University, Hunan Province (China); National Hepatobiliary and Enteric Surgery Research Center of The Ministry of Health, Xiangya Hospital, Central South University, Hunan Province (China)

    2007-02-01

    Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2{sub 1}2{sub 1}2{sub 1} and tetragonal P4{sub 1}2{sub 1}2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*{sub 65–224} structure was determined by molecular replacement.

  11. A hemolytic-uremic syndrome-associated strain O113:H21 Shiga toxin-producing Escherichia coli specifically expresses a transcriptional module containing dicA and is related to gene network dysregulation in Caco-2 cells

    Science.gov (United States)

    Bando, Silvia Yumi; Iamashita, Priscila; Guth, Beatriz E.; dos Santos, Luis F.; Fujita, André; Abe, Cecilia M.; Ferreira, Leandro R.

    2017-01-01

    Shiga toxin-producing (Stx) Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some of these strains may cause hemolytic uremic syndrome (HUS). The molecular mechanism underlying this capacity and the differential host cell response to HUS-causing strains are not yet completely understood. In Brazil O113:H21 strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here we conducted comparative gene co-expression network (GCN) analyses of two O113:H21 STEC strains: EH41, reference strain, isolated from HUS patient in Australia, and Ec472/01, isolated from cattle feces in Brazil. These strains were cultured in fresh or in Caco-2 cell conditioned media. GCN analyses were also accomplished for cultured Caco-2 cells exposed to EH41 or Ec472/01. Differential transcriptome profiles for EH41 and Ec472/01 were not significantly changed by exposure to fresh or Caco-2 conditioned media. Conversely, global gene expression comparison of both strains cultured in conditioned medium revealed a gene set exclusively expressed in EH41, which includes the dicA putative virulence factor regulator. Network analysis showed that this set of genes constitutes an EH41 specific transcriptional module. PCR analysis in Ec472/01 and in other 10 Brazilian cattle-isolated STEC strains revealed absence of dicA in all these strains. The GCNs of Caco-2 cells exposed to EH41 or to Ec472/01 presented a major transcriptional module containing many hubs related to inflammatory response that was not found in the GCN of control cells. Moreover, EH41 seems to cause gene network dysregulation in Caco-2 as evidenced by the large number of genes with high positive and negative covariance interactions. EH41 grows slowly than Ec472/01 when cultured in Caco-2 conditioned medium and fitness-related genes are hypoexpressed in that strain. Therefore, EH41 virulence may be derived from its capacity for dysregulating enterocyte genome functioning and its

  12. Evaluation of a LaSota strain-based recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of avian metapneumovirus (aMPV) subgroup A or B as a bivalent vaccine in turkeys

    Science.gov (United States)

    To develop a bivalent vaccine candidate, a LaSota strain-based recombinant Newcastle disease virus (NDV) clone expressing the glycoprotein (G) of avian metapneumovirus (aMPV) subgroup A or B was generated using reverse genetics. Vaccination of turkeys with the NDV/aMPV-A G or NDV/aMPV-B G recombinan...

  13. Cloning, characterization and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain SWK1, native to Himalayan valley Kashmir.

    Science.gov (United States)

    Reyaz, A L; Arulselvi, P Indra

    2016-05-01

    Bacillus thuringiensis (Bt) is a gram positive bacterium which is effectively being used in pest management strategies as an eco-friendly bioinsecticide. In the present study a new cry2A gene was cloned from a promising indigenous B. thuringiensis SWK1 strain previously characterized for its toxicity against Spodoptera litura and Helicoverpa armigera larvae. The nucleotide sequence of the cloned cry2A gene pointed out that the open reading frame has 1902 bases encoding a polypeptide of 634 amino acid residues with a probable molecular weight of 70kDa. Homology comparisons showed that the deduced amino acid sequence of Cry2A had a similarity of 94% compared to that of the known Cry2Aa protein in the NCBI database and this gene has been named as cry2Al1 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry2Al1 was ligated into pET 22b vector and expressed in Escherichia coli BL21 (DE3) pLysS under the control of T7 promoter induced by isopropyl-beta-d-thiogalactopyranoside (IPTG). SDS-PAGE analysis confirmed the expression of cry2Al1 as ∼65kDa protein. Insect pest bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 are toxic to S. litura and H. armigera with LC50 2.448μg/ml and H. armigera with 3.374μg/ml respectively. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Cloning and expression of cry2Aa from native Bacillus thuringiensis strain SY49-1 and its insecticidal activity against Culex pipiens (Diptera: Culicidae).

    Science.gov (United States)

    Yilmaz, Semih; Azizoglu, Ugur; Ayvaz, Abdurrahman; Temizgul, Ridvan; Atciyurt, Zehra Büşra; Karabörklü, Salih

    2017-04-01

    Bacillus thuringiensis (Berliner) (Bt) is well known for having toxicity against pest insects because of their ability to form endospores and broad-range activity of their parasporal inclusions. In this study, a new member of cry2A gene from previously characterized native B. thuringiensis SY49-1 strain was cloned, expressed and used for its activity against Culex pipiens (Diptera: Culicidae) larvae. The sequence analysis of the cloned cry2A gene revealed that it encodes a polypeptide of 633 aa residues with 99% identity to Cry2Aa protein with expected molecular weight of 70.7 kDa. Bacillus thuringiensis delta-endotoxin nomenclature committee designed our sequence as Cry2Aa18 being a new member of Bt toxins. Bioassays against last instar larvae of C. pipiens indicated that Cry2Aa18 has considerable toxicity with LC 50 of 630 μg ml -1 . In order to prevent the spread of infectious diseases mediated by C. pipiens, this newly characterized cry2Aa18 gene could constitute as an important biological control tool for controlling mosquito larvae living in freshwater systems and can be used as a good alternative for minimizing the use of chemicals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Infection of the upper respiratory tract of hamsters by the bovine parainfluenza virus type 3 BN-1 strain expressing enhanced green fluorescent protein.

    Science.gov (United States)

    Ohkura, Takashi; Minakuchi, Moeko; Sagai, Mami; Kokuho, Takehiro; Konishi, Misako; Kameyama, Ken-Ichiro; Takeuchi, Kaoru

    2015-02-01

    Bovine parainfluenza virus type 3 (BPIV3) is an important pathogen associated with bovine respiratory disease complex (BRDC). We have generated a recombinant BPIV3 expressing enhanced green fluorescent protein (rBPIV3-EGFP) based on the BN-1 strain isolated in Japan. After intranasal infection of hamsters with rBPIV3-EGFP, EGFP fluorescence was detected in the upper respiratory tract including the nasal turbinates, pharynx, larynx, and trachea. In the nasal turbinates, rBPIV3-EGFP attained high titers (>10(6) TCID50/g of tissue) 2-4 days after infection. Ciliated epithelial cells in the nasal turbinates and trachea were infected with rBPIV3-EGFP. Histopathological analysis indicated that mucosal epithelial cells in bronchi were shed by 6 days after infection, leaving non-ciliated cells, which may have increased susceptibility to bacterial infection leading to the development of BRDC. These data indicate that rBPIV3-EGFP infection of hamsters is a useful small animal model for studying the development of BPIV3-associated BRDC. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Effect of ethanol and low pH on citrulline and ornithine excretion and arc gene expression by strains of Lactobacillus brevis and Pediococcus pentosaceus.

    Science.gov (United States)

    Araque, Isabel; Bordons, Albert; Reguant, Cristina

    2013-02-01

    The accumulation of citrulline and ornithine in wine or beer as a result of the arginine catabolism of some lactic acid bacteria (LAB) species increases the risk of ethyl carbamate and putrescine formation, respectively. Several LAB species, which are found as spoilage bacteria in alcoholic beverages, have been reported to be arginine degrading. This study evaluates the effect of ethanol content and low pH on the excretion of citrulline and ornithine by two strains belonging to the potential contaminant species Lactobacillus brevis and Pediococcus pentosaceus. In the conditions that most affected cell viability, arginine consumption per cell increased noticeably, indicating that arginine utilization may be a stress responsive mechanism. L. brevis showed a higher accumulation of ornithine in the media than P. pentosaceus. In the presence of ethanol, a higher expression of the arcC gene was found in P. pentosaceus, which resulted in a lower excretion of citrulline and ornithine than in L. brevis. This suggests that L. brevis is more likely to produce these amino acids, which are precursors of ethyl carbamate and putrescine. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. RcsB determines the locus of enterocyte effacement (LEE) expression and adherence phenotype of Escherichia coli O157 : H7 spinach outbreak strain TW14359 and coordinates bicarbonate-dependent LEE activation with repression of motility.

    Science.gov (United States)

    Morgan, Jason K; Vendura, Khoury W; Stevens, Stanley M; Riordan, James T

    2013-11-01

    The 2006 US spinach outbreak of Escherichia coli O157 : H7, characterized by unusually severe disease, has been attributed to a strain (TW14359) with enhanced pathogenic potential, including elevated virulence gene expression, robust adherence and the presence of novel virulence factors. This study proposes a mechanism for the unique virulence expression and adherence phenotype of this strain, and further expands the role for regulator RcsB in control of the E. coli locus of enterocyte effacement (LEE) pathogenicity island. Proteomic analysis of TW14359 revealed a virulence proteome consistent with previous transcriptome studies that included elevated levels of the LEE regulatory protein Ler and type III secretion system (T3SS) proteins, secreted T3SS effectors and Shiga toxin 2. Basal levels of the LEE activator and Rcs phosphorelay response regulator, RcsB, were increased in strain TW14359 relative to O157 : H7 strain Sakai. Deletion of rcsB eliminated inherent differences between these strains in ler expression, and in T3SS-dependent adherence. A reciprocating regulatory pathway involving RcsB and LEE-encoded activator GrlA was identified and predicted to co-ordinate LEE activation with repression of the flhDC flagellar regulator and motility. Overexpression of grlA was shown to increase RcsB levels, but did not alter expression from promoters driving rcsB transcription. Expression of rcsDB and RcsB was determined to increase in response to physiological levels of bicarbonate, and bicarbonate-dependent stimulation of the LEE was shown to be dependent on an intact Rcs system and ler activator grvA. The results of this study significantly broaden the role for RcsB in enterohaemorrhagic E. coli virulence regulation.

  18. Optimising expression of the recombinant fusion protein biopesticide ω-hexatoxin-Hv1a/GNA in Pichia pastoris: sequence modifications and a simple method for the generation of multi-copy strains.

    Science.gov (United States)

    Pyati, Prashant; Fitches, Elaine; Gatehouse, John A

    2014-08-01

    Production of recombinant protein bio-insecticides on a commercial scale can only be cost effective if host strains with very high expression levels are available. A recombinant fusion protein containing an arthropod toxin, ω-hexatoxin-Hv1a, (from funnel web spider Hadronyche versuta) linked to snowdrop lectin (Galanthus nivalis agglutinin; GNA) is an effective oral insecticide and candidate biopesticide. However, the fusion protein was vulnerable to proteolysis during production in the yeast Pichia pastoris. To prevent proteolysis, the Hv1a/GNA fusion expression construct was modified by site-directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. To obtain a high expressing clone of P. pastoris to produce recombinant Hv1a/GNA, a straightforward method was used to produce multi-copy expression plasmids, which does not require multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. Removal of the Kex2 site resulted in increased levels of intact fusion protein expressed in wild-type P. pastoris strains, improving levels of intact recombinant protein recoverable. Incorporation of a C-terminal (His)6 tag enabled single step purification of the fusion protein. These modifications did not affect the insecticidal activity of the recombinant toxin towards lepidopteran larvae. Introduction of multiple expression cassettes increased the amount of secreted recombinant fusion protein in a laboratory scale fermentation by almost tenfold on a per litre of culture basis. Simple modifications in the expression construct can be advantageous for the generation of high expressing P. pastoris strains for production of a recombinant protein, without altering its functional properties.

  19. The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of Bacillus cereus strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNAAsn

    LENUS (Irish Health Repository)

    Foy, Niall

    2010-07-22

    Abstract Background Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression. Results A global study of 891 completely sequenced bacterial genomes identified T box elements associated with control of LysRS expression in only four bacterial species: B. cereus, B. thuringiensis, Symbiobacterium thermophilum and Clostridium beijerinckii. Here we investigate the T box element found in the regulatory region of the lysK gene in B. cereus strain 14579. We show that this T box element is functional, responding in a canonical manner to an increased level of uncharged tRNALys but, unusually, also responding to an increased level of uncharged tRNAAsn. We also show that B. subtilis strains with T box regulated expression of the endogenous lysS or the heterologous lysK genes are viable. Conclusions The T box element controlling lysK (encoding LysRS1) expression in B. cereus strain 14579 is functional, but unusually responds to depletion of charged tRNALys and tRNAAsn. This may have the advantage of making LysRS1 expression responsive to a wider range of nutritional stresses. The viability of B. subtilis strains with a single LysRS1 or LysRS2, whose expression is controlled by this T box element, makes the rarity of the occurrence of such control of LysRS expression puzzling.

  20. Human intestinal mucosa-associated Lactobacillus and Bifidobacterium strains with probiotic properties modulate IL-10, IL-6 and IL-12 gene expression in THP-1 cells.

    Science.gov (United States)

    Čitar, M; Hacin, B; Tompa, G; Štempelj, M; Rogelj, I; Dolinšek, J; Narat, M; Matijašić, B Bogovič

    2015-01-01

    Lactobacilli and bifidobacteria are considered one of the permanent genera of the physiological human intestinal microbiota and represent an enormous pool of potential probiotic candidates. Approximately 450 isolates of presumptive Lactobacillus or Bifidobacterium strains were obtained from bioptic samples of colonic and ileal mucosa from 15 adolescents aged 12 to 18 years. On the basis of randomly amplified polymorphic DNA (RAPD)-PCR analysis, 20 strains were selected for further taxonomic classification and characterisation, as well as assessment of probiotic properties and safety. Importantly, selected strains showed the capability of colonising different parts of the intestine. The most frequently isolated species was Lactobacillus paracasei followed by Lactobacillus fermentum. The majority of isolates were susceptible to antimicrobials of human and veterinary importance, however, tetracycline and/or erythromycin resistance was observed in Lactobacillus plantarum and L. fermentum strains. Thirteen strains were able to ferment more than 19 different carbon sources and three out of five tested strains exerted antagonistic activity against several different indicator strains. Two Lactobacillus isolates (L. paracasei L350 and L. fermentum L930 bb) and one Bifidobacterium isolate (Bifidobacterium animalis subsp. animalis IM386) fulfilled in vitro selection criteria for probiotic strains and exhibited strong downregulation of pro-inflammatory cytokines IL-6 and IL-12 and upregulation of anti-inflammatory IL-10. The selected strains represent suitable candidates for further studies regarding their positive influence on host health and could play an important role in ameliorating the symptoms of inflammatory bowel diseases.

  1. Genome sequence of the deltaproteobacterial strain NaphS2 and analysis of differential gene expression during anaerobic growth on naphthalene.

    Directory of Open Access Journals (Sweden)

    Raymond J DiDonato

    2010-11-01

    Full Text Available Anaerobic polycyclic hydrocarbon (PAH degradation coupled to sulfate reduction may be an important mechanism for in situ remediation of contaminated sediments. Steps involved in the anaerobic degradation of 2-methylnaphthalene have been described in the sulfate reducing strains NaphS3, NaphS6 and N47. Evidence from N47 suggests that naphthalene degradation involves 2-methylnaphthalene as an intermediate, whereas evidence in NaphS2, NaphS3 and NaphS6 suggests a mechanism for naphthalene degradation that does not involve 2-methylnaphthalene. To further characterize pathways involved in naphthalene degradation in NaphS2, the draft genome was sequenced, and gene and protein expression examined.Draft genome sequencing, gene expression analysis, and proteomic analysis revealed that NaphS2 degrades naphthoyl-CoA in a manner analogous to benzoyl-CoA degradation. Genes including the previously characterized NmsA, thought to encode an enzyme necessary for 2-methylnaphthalene metabolism, were not upregulated during growth of NaphS2 on naphthalene, nor were the corresponding protein products. NaphS2 may possess a non-classical dearomatizing enzyme for benzoate degradation, similar to one previously characterized in Geobacter metallireducens. Identification of genes involved in toluene degradation in NaphS2 led us to determine that NaphS2 degrades toluene, a previously unreported capacity. The genome sequence also suggests that NaphS2 may degrade other monoaromatic compounds.This study demonstrates that steps leading to the degradation of 2-naphthoyl-CoA are conserved between NaphS2 and N47, however while NaphS2 possesses the capacity to degrade 2-methylnaphthalene, naphthalene degradation likely does not proceed via 2-methylnaphthalene. Instead, carboxylation or another form of activation may serve as the first step in naphthalene degradation. Degradation of toluene and 2-methylnaphthalene, and the presence of at least one bss-like and bbs-like gene cluster

  2. Dioxin-induced retardation of development through a reduction in the expression of pituitary hormones and possible involvement of an aryl hydrocarbon receptor in this defect: A comparative study using two strains of mice with different sensitivities to dioxin

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Tomoki; Taura, Junki; Hattori, Yukiko; Ishii, Yuji; Yamada, Hideyuki, E-mail: hyamada@phar.kyushu-u.ac.jp

    2014-08-01

    We have previously revealed that treating pregnant rats with 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) reduces the expression of gonadotropins and growth hormone (GH) in the fetal and neonatal pituitary. A change in gonadotropin expression impairs the testicular expression of steroidogenic proteins in perinatal pups, and imprint defects in sexual behavior after reaching maturity. In this study, we examined whether TCDD also affects the expression of gonadotropin and GH in mice using C57BL/6J and DBA/2J strains which express the aryl hydrocarbon receptor (Ahr) exhibiting a different affinity for TCDD. When pregnant C57BL/6J mice at gestational day (GD) 12 were given oral TCDD (0.2–20 μg/kg), all doses significantly attenuated the pituitary expression of gonadotropin mRNAs in fetuses at GD18. On the other hand, in DBA/2J mice, a much higher dose of TCDD (20 μg/kg) was needed to produce a significant attenuation. Such reduction in the C57BL/6J strain continued until at least postnatal day (PND) 4. In agreement with this, TCDD reduced the testicular expression of steroidogenic proteins in C57BL/6J neonates at PND2 and 4, although the same did not occur in the fetal testis and ovary. Furthermore, TCDD reduced the perinatal expression of GH, litter size and the body weight of newborn pups only in the C57BL/6J strain. These results suggest that 1) also in mice, maternal exposure to TCDD attenuates gonadotropin-regulated steroidogenesis and GH expression leading to the impairment of pup development and sexual immaturity; and 2) Ahr activation during the late fetal and early postnatal stages is required for these defects. - Highlights: • The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on mouse growth was studied. • TCDD reduced the levels of luteinizing hormone and growth hormone in perinatal pups. • Maternal exposure to TCDD also attenuated testicular steroidogenesis in pups. • The above effects of TCDD were more pronounced in C57BL/6J than in DBA/2J

  3. Dioxin-induced retardation of development through a reduction in the expression of pituitary hormones and possible involvement of an aryl hydrocarbon receptor in this defect: A comparative study using two strains of mice with different sensitivities to dioxin

    International Nuclear Information System (INIS)

    Takeda, Tomoki; Taura, Junki; Hattori, Yukiko; Ishii, Yuji; Yamada, Hideyuki

    2014-01-01

    We have previously revealed that treating pregnant rats with 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) reduces the expression of gonadotropins and growth hormone (GH) in the fetal and neonatal pituitary. A change in gonadotropin expression impairs the testicular expression of steroidogenic proteins in perinatal pups, and imprint defects in sexual behavior after reaching maturity. In this study, we examined whether TCDD also affects the expression of gonadotropin and GH in mice using C57BL/6J and DBA/2J strains which express the aryl hydrocarbon receptor (Ahr) exhibiting a different affinity for TCDD. When pregnant C57BL/6J mice at gestational day (GD) 12 were given oral TCDD (0.2–20 μg/kg), all doses significantly attenuated the pituitary expression of gonadotropin mRNAs in fetuses at GD18. On the other hand, in DBA/2J mice, a much higher dose of TCDD (20 μg/kg) was needed to produce a significant attenuation. Such reduction in the C57BL/6J strain continued until at least postnatal day (PND) 4. In agreement with this, TCDD reduced the testicular expression of steroidogenic proteins in C57BL/6J neonates at PND2 and 4, although the same did not occur in the fetal testis and ovary. Furthermore, TCDD reduced the perinatal expression of GH, litter size and the body weight of newborn pups only in the C57BL/6J strain. These results suggest that 1) also in mice, maternal exposure to TCDD attenuates gonadotropin-regulated steroidogenesis and GH expression leading to the impairment of pup development and sexual immaturity; and 2) Ahr activation during the late fetal and early postnatal stages is required for these defects. - Highlights: • The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on mouse growth was studied. • TCDD reduced the levels of luteinizing hormone and growth hormone in perinatal pups. • Maternal exposure to TCDD also attenuated testicular steroidogenesis in pups. • The above effects of TCDD were more pronounced in C57BL/6J than in DBA/2J

  4. Multiepitope fusion antigen induces broadly protective antibodies that prevent adherence of Escherichia coli strains expressing colonization factor antigen I (CFA/I), CFA/II, and CFA/IV.

    Science.gov (United States)

    Ruan, Xiaosai; Knudsen, David E; Wollenberg, Katie M; Sack, David A; Zhang, Weiping

    2014-02-01

    Diarrhea is the second leading cause of death in children younger than 5 years and continues to be a major threat to global health. Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacteria causing diarrhea in developing countries. ETEC strains are able to attach to host small intestinal epithelial cells by using bacterial colonization factor antigen (CFA) adhesins. This attachment helps to initiate the diarrheal disease. Vaccines that induce antiadhesin immunity to block adherence of ETEC strains that express immunologically heterogeneous CFA adhesins are expected to protect against ETEC diarrhea. In this study, we created a CFA multiepitope fusion antigen (MEFA) carrying representative epitopes of CFA/I, CFA/II (CS1, CS2, and CS3), and CFA/IV (CS4, CS5, and CS6), examined its immunogenicity in mice, and assessed the potential of this MEFA as an antiadhesin vaccine against ETEC. Mice intraperitoneally immunized with this CFA MEFA exhibited no adverse effects and developed immune responses to CFA/I, CFA/II, and CFA/IV adhesins. Moreover, after incubation with serum of the immunized mice, ETEC or E. coli strains expressing CFA/I, CFA/II, or CFA/IV adhesins were significantly inhibited in adherence to Caco-2 cells. Our results indicated this CFA MEFA elicited antibodies that not only cross-reacted to CFA/I, CFA/II and CFA/IV adhesins but also broadly inhibited adherence of E. coli strains expressing these seven adhesins and suggested that this CFA MEFA could be a candidate to induce broad-spectrum antiadhesin protection against ETEC diarrhea. Additionally, this antigen construction approach (creating an MEFA) may be generally used in vaccine development against heterogenic pathogens.

  5. Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo

    Directory of Open Access Journals (Sweden)

    Littman Dan R

    2009-01-01

    Full Text Available Abstract Background Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1. Results Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. Conclusion Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.

  6. Influence of the adipate and dissolved oxygen concentrations on the beta-lactam production during continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus

    DEFF Research Database (Denmark)

    Robin, Jarno Jacky Christian; Bonneau, S.; Schipper, D.

    2003-01-01

    The influence of adipate concentration and dissolved oxygen on production of adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) by a recombinant strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was studied in glucose-limited continuous cultures....... from 15 to 7%AS, r(p) (total) increased to 25 mumol g DW-1 h(-1), mainly due to a two-fold increase in the adipoyl-6-aminopenicillanic acid (ad-6-APA) specific productivity....

  7. Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains.

    Science.gov (United States)

    Khalil, Rowaida K S; Skinner, Craig; Patfield, Stephanie; He, Xiaohua

    2016-07-01

    Enterobacter cloacae M12X01451 strain recently identified from a clinical specimen produces a new Stx1 subtype (Stx1e) that was not neutralized by existing anti-Stx1 monoclonal antibodies. Acquisition of stx by Ent. cloacae is rare and origin/stability of stx1e in M12X01451 is not known. In this study, we confirmed the ability of Stx1a- and Stx1e-converting phages from an Escherichia coli O157:H7 strain RM8530 and M12X01451 respectively to infect several E. coli and Ent. cloacae strains. stx1e was detected in 97.5% and 72.5% of progenies of strains lysogenized by stx1e phage after 10 (T10) and 20 (T20) subcultures, versus 65% and 17.5% for stx1a gene. Infection of M12X01451 and RM8530 with each other's phages generated double lysogens containing both phages. stx1a was lost after T10, whereas the stx1e was maintained even after T20 in M12X01451 lysogens. In RM8530 lysogens, the acquired stx1e was retained with no mutations, but 20% of stx1a was lost after T20 ELISA and western blot analyses demonstrated that Stx1e was produced in all strains lysogenized by stx1e phage; however, Stx1a was not detected in any lysogenized strain. The study results highlight the potential risks of emerging Stx-producing strains via bacteriophages either in the human gastrointestinal tract or in food production environments, which are matters of great concern and may have serious impacts on human health. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Complete Draft Genome Sequence of Escherichia coli KRX, a Strain for Efficient Cloning and High-Yield Expression of Proteins under Control of the T7 RNA Polymerase

    OpenAIRE

    Schwarzhans, Jan-Philipp; Wibberg, Daniel; Winkler, Anika; Kalinowski, Jörn; Friehs, Karl

    2017-01-01

    ABSTRACT Escherichia coli KRX is a strain offering both a high transformation efficiency and the possibility to produce the target protein to high yields in one host, avoiding additional cloning steps. Here, the draft genome sequence of E. coli KRX is presented and provides the genetic basis for additional biotechnological applications.

  9. Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7- aminodeacetoxycephalosporanic acid production

    DEFF Research Database (Denmark)

    Robin, Jarno Jacky Christian; Jakobsen, M.; Beyer, M.

    2001-01-01

    The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this pr...

  10. Comparative analysis of the virulence characteristics of epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from Chinese children: ST59 MRSA highly expresses core gene-encoded toxin.

    Science.gov (United States)

    Li, Shipeng; Sun, Jing; Zhang, Jianzhong; Li, Xiangmei; Tao, Xiaoxia; Wang, Lijuan; Sun, Mingjiao; Liu, Yingchao; Li, Juan; Qiao, Yanhong; Yu, Sangjie; Yao, Kaihu; Yang, Yonghong; Shen, Xuzhuang

    2014-02-01

    This study aims to investigate the prevalence of a novel cell wall-anchored protein gene, sasX, and to obtain information on the genetic basis for the pathogenic potential of the MRSA strains isolated from Chinese children. The molecular and virulence characteristics of the clinical strains were analyzed. Twenty-two sequence types (STs) were obtained, with six epidemic clones ST59, ST239, ST1, ST910, ST88, and ST338 accounting for 35.8, 22, 6.6, 6.6, 5.3, and 4.1% respectively. The expression levels of hla, psmα, and RNAIII were higher in ST59 than in other STs (p MRSA isolates. ST239-MRSA-SCCmecIII-t037 (61.5%) was the predominant sasX-positive MRSA clone. The expressions of PSMα and RNAIII were higher in sasX-positive ST239 isolates than in sasX-negative ST239 ones (p MRSA was higher than that by sasX-negative ST239 MRSA (p = 0.008). This study indicated that ST59 was the predominant clone in the MRSA isolates obtained from Chinese children and might have stronger pathogenic potential. The prevalence of the sasX gene in the MRSA isolates from children was relatively low. Furthermore, the sasX gene might be related to the expressions of PSMα and RNAIII and infection invasiveness. © 2013 APMIS Published by John Wiley & Sons Ltd.

  11. A replicating modified vaccinia tiantan strain expressing an avian-derived influenza H5N1 hemagglutinin induce broadly neutralizing antibodies and cross-clade protective immunity in mice.

    Directory of Open Access Journals (Sweden)

    Haixia Xiao

    Full Text Available To combat the possibility of a zoonotic H5N1 pandemic in a timely fashion, it is necessary to develop a vaccine that would confer protection against homologous and heterologous human H5N1 influenza viruses. Using a replicating modified vaccinia virus Tian Tan strain (MVTT as a vaccine vector, we constructed MVTTHA-QH and MVTTHA-AH, which expresses the H5 gene of a goose-derived Qinghai strain A/Bar-headed Goose/Qinghai/1/2005 or human-derived Anhui Strain A/Anhui/1/2005. The immunogenicity profiles of both vaccine candidates were evaluated. Vaccination with MVTTHA-QH induced a significant level of neutralizing antibodies (Nabs against a homologous strain and a wide range of H5N1 pseudoviruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4. Neutralization tests (NT and Haemagglutination inhibition (HI antibodies inhibit the live autologous virus as well as a homologous A/Xingjiang/1/2006 and a heterologous A/Vietnam/1194/2004, representing two human isolates from clade 2.2 and clade 1, respectively. Importantly, mice vaccinated with intranasal MVTTHA-QH were completely protected from challenge with lethal dosages of A/Bar-headed Goose/Qinghai/1/2005 and the A/Viet Nam/1194/2004, respectively, but not control mice that received a mock MVTTS vaccine. However, MVTTHA-AH induced much lower levels of NT against its autologous strain. Our results suggest that it is feasible to use the H5 gene from A/Bar-headed Goose/Qinghai/1/2005 to construct an effective vaccine, when using MVTT as a vector, to prevent infections against homologous and genetically divergent human H5N1 influenza viruses.

  12. Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens

    OpenAIRE

    Wang, Jichun; Ge, Aimin; Xu, Mengwei; Wang, Zhisheng; Qiao, Yongfeng; Gu, Yiqi; Liu, Chang; Liu, Yamei; Hou, Jibo

    2015-01-01

    Background Highly pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry. Duck enteritis virus (DEV)-vectored vaccines expressing AIV H5N1 hemagglutinin (HA) may be viable AIV and DEV vaccine candidates. Methods To facilitate the generation and further improvement of DEV-vectored HA(H5) vaccines, we first constructed an infectious clone of DEV Chinese vaccine strain C-KCE (DEVC-KCE). Then, we generated a DEV-vectored HA(H5) vaccine (DEV-H5(UL55)) based on the bacteria...

  13. Gene Cloning, Expression and Activity Analysis of Manganese Superoxide Dismutase from Two Strains of Gracilaria lemaneiformis (Gracilariaceae, Rhodophyta under Heat Stress

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    2012-04-01

    Full Text Available Manganese superoxide dismutase (Mn-SOD plays a crucial role in antioxidant responses to environmental stress. To determine whether Mn-SOD affects heat resistance of Gracilaria lemaneiformis, we cloned Mn-SOD cDNA sequences of two strains of this red alga, wild type and cultivar 981. Both cDNA sequences contained an ORF of 675 bp encoding 224 amino acid residues. The cDNA sequences and the deduced amino acid sequences of the two strains shared relatively high identity (more than 99%. No intron existed in genomic DNA of Mn-SOD in G. lemaneiformis. Southern blotting indicated that there were multiple copies, possibly four, of Mn-SOD in both strains. Both in the wild type and cultivar 981, SOD mRNA transcription and SOD activity increased under high temperature stress, while cultivar 981 was more heat resistant based on its SOD activity. This research suggests that there may be a direct relationship between SOD activity and the heat resistance of G. lemaneiformis.

  14. Comparative proteomic analysis of extracellular secreted proteins expressed by two pathogenic Acanthamoeba castellanii clinical isolates and a non-pathogenic ATCC strain.

    Science.gov (United States)

    Huang, Jian-Ming; Lin, Wei-Chen; Li, Sung-Chou; Shih, Min-Hsiu; Chan, Wen-Ching; Shin, Jyh-Wei; Huang, Fu-Chin

    2016-07-01

    Acanthamoeba keratitis (AK) is a serious ocular disease caused by pathogenic Acanthamoeba gaining entry through wounds in the corneal injury; generally, patients at risk for contracting AK wear contact lenses, usually over a long period of time. Moreover, pathogenic Acanthamoeba causes serious consequences: it makes the cornea turbid and difficult to operate on, including procedures such as enucleation of the eyeball. At present, diagnosis of this disease is not straightforward, and treatment is very demanding. We have established the comparative transcriptome and extracellular secreted proteomic database according to the non-pathogenic strain ATCC 30010 and the pathogenic strains NCKU_B and NCKU_D. We identified 44 secreted proteins successfully, 10 consensus secreted proteins and 34 strain-specific secreted proteins. These proteins may provide targets for therapy and immuno-diagnosis of Acanthamoeba infections. This study shows a suitable approach to identify secreted proteins in Acanthamoeba and provides new perspectives for the study of molecules potentially involved in the AK. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Expression of the marA, soxS, acrB and ramA genes related to the AcrAB/TolC efflux pump in Salmonella entérica strains with and without quinolone resistance-determining regions gyrA gene mutations

    Directory of Open Access Journals (Sweden)

    Rafaela Gomes Ferrari

    Full Text Available Several studies have been conducted in recent years to elucidate the structure, function and significance of AcrB, MarA, SoxS and RamA in Salmonella enterica. In this study, the relative quantification of acrB, soxS, marA and ramA genes expression was evaluated in 14 strains of S. enterica, with or without accompanying mutations in the quinolone resistance-determining regions of the gyrA gene, that were exposed to ciprofloxacin during the exponential growth phase. The presence of ciprofloxacin during the log phase of bacterial growth activated the genes marA, soxS, ramA and acrB in all S. enterica strains analyzed in this study. The highest expression levels for acrB were observed in strains with gyrA mutation, and marA showed the highest expression in the strains without mutation. Considering only the strains with ciprofloxacin minimum inhibitory concentration values 0.125 [1]g/mL (low susceptibility, with and without mutations in gyrA, the most expressed gene was marA. In this study, we observed that strains resistant to nalidixic acid may express genes associated with the efflux pump and the expression of the AcrAB-TolC pump genes seems to occur independently of mutations in gyrA.

  16. Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, A. Patricia [University of Washington, Department of Medicinal Chemistry, School of Pharmacy (United States); Spyracopoulos, Leo [Department of Biochemistry (Canada); Irvin, Randall T. [University of Alberta, Department of Medical Microbiology and Immunology (Canada); Sykes, Brian D. [Department of Biochemistry (Canada)

    2000-07-15

    The backbone dynamics of a {sup 15}N-labeled recombinant PAK pilin peptide spanning residues 128-144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys{sup 128}-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys{sup 144}) were probed by measurements of {sup 15}N NMR relaxation. This PAK(128-144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The {sup 15}N longitudinal (T{sub 1}) and transverse (T{sub 2}) relaxation rates and the steady-state heteronuclear {l_brace}{sup 1}H{r_brace}-{sup 15}N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 deg. C ) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the 'model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153-162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II {beta}-turns spanning residues Asp{sup 134}-Glu-Gln-Phe{sup 137} and Pro{sup 139}-Lys-Gly-Cys{sup 142}, respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.

  17. Development of an anhydrotetracycline-inducible gene expression system for solvent-producing Clostridium acetobutylicum: A useful tool for strain engineering.

    Science.gov (United States)

    Dong, Hongjun; Tao, Wenwen; Zhang, Yanping; Li, Yin

    2012-01-01

    Clostridium acetobutylicum is an important solvent (acetone-butanol-ethanol) producing bacterium. However, a stringent, effective, and convenient-to-use inducible gene expression system that can be used for regulating the gene expression strength in C. acetobutylicum is currently not available. Here, we report an anhydrotetracycline-inducible gene expression system for solvent-producing bacterium C. acetobutylicum. This system consists of a functional chloramphenicol acetyltransferase gene promoter containing tet operators (tetO), Pthl promoter (thiolase gene promoter from C. acetobutylicum) controlling TetR repressor expression cassette, and the chemical inducer anhydrotetracycline (aTc). The optimized system, designated as pGusA2-2tetO1, allows gene regulation in an inducer aTc concentration-dependent way, with an inducibility of over two orders of magnitude. The stringency of TetR repression supports the introduction of the genes encoding counterselective marker into C. acetobutylicum, which can be used to increase the mutant screening efficiency. This aTc-inducible gene expression system will thus increase the genetic manipulation capability for engineering C. acetobutylicum. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Characterization of Bromadiolone Resistance in a Danish Strain of Norway Rats, Rattus norvegicus, by Hepatic Gene Expression Profiling of VKORC1 and Calumenin

    DEFF Research Database (Denmark)

    Markussen, Mette Drude; Heiberg, Ann-Charlotte; Fredholm, Merete

    2007-01-01

    indicate that bromadiolone resistance does not involve an over-expression of calumenin. We observed a low VKORC1 mRNA expression in resistant rats compared to susceptible rats, which may explain pleiotropic effects of resistance, such as a low VKOR activity and an enhanced need for vitamin K, observed......Anticoagulant agents, such as warfarin and bromadiolone, are used to control populations of Norway rats (Rattus norvegicus). The anticoagulants compromise the blood-coagulation process by inhibiting the vitamin K2,3 epoxide reductase enzyme complex (VKOR). Mutations in the VKORC1 gene, encoding...

  19. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens

    2008-01-01

    Background Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression...

  20. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

    Science.gov (United States)

    Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O,A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutral...

  1. Antimicrobial peptides effectively kill a broad spectrum of Listeria monocytogenes and Staphylococcus aureus strains independently of origin, sub-type, or virulence factor expression

    DEFF Research Database (Denmark)

    Gottlieb, Caroline Trebbien; Thomsen, L.E.; Ingmer, H.

    2008-01-01

    Background Host defense peptides (HDPs), or antimicrobial peptides (AMPs), are important components of the innate immune system that bacterial pathogens must overcome to establish an infection and HDPs have been suggested as novel antimicrobial therapeutics in treatment of infectious diseases......-type, and phenotypic behavior. Strains within each species were equally sensitive to HDPs and oxidative stress representing important components of the innate immune defense system. Four non-human peptides (protamine, plectasin, novicidin, and novispirin G10) were similar in activity profile (MIC value spectrum...... Caenorhabditis elegans. For L. monocytogenes, proliferation in whole blood was paralleled by high invasion in Caco-2 cells and fast killing of C. elegans, however, no such pattern in phenotypic behavior was observed for S. aureus and none of the phenotypic differences were correlated to sensitivity to HDPs...

  2. Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

    Science.gov (United States)

    Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

    2016-04-01

    Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Expressão dos genes nodC, nodW e nopP em Bradyrhizobium japonicum estirpe CPAC 15 avaliada por RT-qPCR Expression of nodC, nodW and nopP genes in Bradyrhizobium japonicum CPAC 15 strain evaluated by RT-qPCR

    Directory of Open Access Journals (Sweden)

    Simone Bortolan

    2009-11-01

    Full Text Available O objetivo deste trabalho foi avaliar a expressão, por RT-qPCR, dos genes de nodulação nodC e nodW e do gene nopP da estirpe CPAC 15, que provavelmente atuam na infecção das raízes da soja. Foram realizados dois experimentos. No primeiro, a expressão dos genes foi avaliada nas células após a incubação com genisteína por 15 min, 1, 4 e 8 horas. Os resultados revelaram que os três genes apresentaram maior expressão imediatamente após o contato com o indutor (15 min. No segundo experimento, a bactéria foi cultivada na presença de indutores (genisteína ou exsudatos de sementes de soja por 48 horas. A expressão dos três genes foi maior na presença de genisteína, com valores de expressão para nodC, nodW e nopP superiores ao controle. Os resultados obtidos confirmam a funcionalidade dos três genes na estirpe CPAC 15, com ênfase para o nopP, cuja funcionalidade em Bradyrhizobium japonicum foi descrita pela primeira vez.The objective of this work was to evaluate, by RT-qPCR, the expression of the nodC and nodW nodulation genes and of the nopP gene of the CPAC 15 strain, which probably play a role in the infection of soybean roots. Two experiments were done. In the first, the gene expression was evaluated in cells after incubation with genistein for 15 min, 1, 4 and 8 hours. Results showed that the three genes showed higher expression immediately after contact with the inducer (15 min. In the second experiment, the bacterium was grown in the presence of inducers (genistein or soybean seed exudates for 48 hours. The expression of the three genes was greater when induced by genistein, and the expression of nodC, nodW and nopP had higher values than the control. The results confirm the functionality of the three genes in the CPAC 15 strain, with an emphasis on the nopP, whose functionality in Bradyrhizobium japonicum was described for the first time.

  4. Heat-killed whole-cell products of the probiotic Pseudomonas aeruginosa VSG2 strain affect in vitro cytokine expression in head kidney macrophages of Labeo rohita.

    Science.gov (United States)

    Giri, Sib Sankar; Sen, Shib Sankar; Jun, Jin Woo; Park, Se Chang; Sukumaran, V

    2016-03-01

    Present study was undertaken to investigate the efficiency of heat-killed whole-cell products (HKWCPs) of probiotic Pseudomonas aeruginosa VSG2 strain in stimulating the cytokine responses in the head kidney (HK) macrophages of Labeo rohita. The HK macrophages were incubated with HKWCPs or lipopolysaccharide (LPS), and the responses of cytokine genes, namely interleukin-10 (IL-10), IL-1β, IL-p35, IL-12p40, tumour necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), interferon-alpha (IFN-α), and interferon-gamma (IFN-γ) were assessed by quantitative real-time PCR (qRT-PCR) at 2, 8, 16, 24, 48, 72 h post-stimulation (hps). Among proinflammatory cytokines, significantly higher expression of IL-1β and TNF-α was observed at 8-24 hps, and 2-16 hps with HKWCPs, respectively, as compared to controls. However, COX-2 and NF-κB displayed strong expression (P production) and humoral (lysozyme) immune parameters of treated HK macrophages confirmed the induction of inflammatory response. Thus, our results indicated that HKWCPs of probiotic P. aeruginosa VSG2 had greater potential for stimulating the in vitro expression of cytokines in fish and that these HKWCPs may be used as vaccine adjuvants in aquaculture. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 β-glucosidase by a diploid strain of Saccharomyces cerevisiae.

    Science.gov (United States)

    Mendoza-Aguayo, David J; Poggi-Varaldo, Héctor M; García-Mena, Jaime; Ramos-Valdivia, Ana C; Salgado, Luis M; de la Torre-Martínez, Mayra; Ponce-Noyola, Teresa

    2014-01-01

    The catalytic fraction of the Cellulomonas flavigena PN-120 oligomeric β-glucosidase (BGLA) was expressed both intra- and extracellularly in a recombinant diploid of Saccharomyces cerevisiae, under limited nutrient conditions. The recombinant enzyme (BGLA¹⁵) expressed in the supernatant of a rich medium showed 582 IU/L and 99.4 IU/g dry cell, with p-nitrophenyl-β-D-glucopyranoside as substrate. BGLA¹⁵ displayed activity against cello-oligosaccharides with 2-5 glucose monomers, demonstrating that the protein is not specific for cellobiose and that the oligomeric structure is not essential for β-D-1,4-bond hydrolysis. Native β-glucosidase is inhibited almost completely at 160 mM glucose, thus limiting cellobiose hydrolysis. At 200 mM glucose concentration, BGLA¹⁵ retained more than 50 % of its maximal activity, and even at 500 mM glucose concentration, more than 30 % of its activity was preserved. Due to these characteristics of BGLA¹⁵ activity, recombinant S. cerevisiae is able to utilize cellulosic materials (cello-oligosaccharides) to produce bioethanol.

  6. Differential expression of maize chitinases in the presence or absence of Trichoderma harzianum strain T22 and indications of a novel exo- endo-heterodimeric chitinase activity

    Directory of Open Access Journals (Sweden)

    Harman Gary E

    2010-07-01

    Full Text Available Abstract Background The interaction of plants with endophytic symbiotic fungi in the genus Trichoderma alters the plant proteome and transcriptome and results in enhanced plant growth and resistance to diseases. In a previous study, we identified the numerous chitinolytic enzyme families and individual enzymes in maize which are implicated in plant disease resistance and other plant responses. Results We examined the differential expression of the entire suite of chitinolytic enzymes in maize plants in the presence and absence of T. harzianum. Expression of these enzymes revealed a band of chitinolytic enzyme activity that had greater mass than any known chitinase. This study reports the characterization of this large protein. It was found to be a heretofore undiscovered heterodimer between an exo- and an endo-enzyme, and the endo portion differed between plants colonized with T. harzianum and those grown in its absence and between shoots and roots. The heterodimeric enzymes from shoots in the presence and absence of T. harzianum were purified and characterized. The dimeric enzyme from Trichoderma-inoculated plants had higher specific activity and greater ability to inhibit fungal growth than those from control plants. The activity of specific chitinolytic enzymes was higher in plants grown from Trichoderma treated seeds than in control plants. Conclusions This is the first report of a dimer between endo- and exochitinase. The endochitinase component of the dimer changed post Trichoderma inoculation. The dimer originating from Trichoderma inoculated plants had a higher antifungal activity than the comparable enzyme from control plants.

  7. Differential expression of maize chitinases in the presence or absence of Trichoderma harzianum strain T22 and indications of a novel exo- endo-heterodimeric chitinase activity

    Science.gov (United States)

    2010-01-01

    Background The interaction of plants with endophytic symbiotic fungi in the genus Trichoderma alters the plant proteome and transcriptome and results in enhanced plant growth and resistance to diseases. In a previous study, we identified the numerous chitinolytic enzyme families and individual enzymes in maize which are implicated in plant disease resistance and other plant responses. Results We examined the differential expression of the entire suite of chitinolytic enzymes in maize plants in the presence and absence of T. harzianum. Expression of these enzymes revealed a band of chitinolytic enzyme activity that had greater mass than any known chitinase. This study reports the characterization of this large protein. It was found to be a heretofore undiscovered heterodimer between an exo- and an endo-enzyme, and the endo portion differed between plants colonized with T. harzianum and those grown in its absence and between shoots and roots. The heterodimeric enzymes from shoots in the presence and absence of T. harzianum were purified and characterized. The dimeric enzyme from Trichoderma-inoculated plants had higher specific activity and greater ability to inhibit fungal growth than those from control plants. The activity of specific chitinolytic enzymes was higher in plants grown from Trichoderma treated seeds than in control plants. Conclusions This is the first report of a dimer between endo- and exochitinase. The endochitinase component of the dimer changed post Trichoderma inoculation. The dimer originating from Trichoderma inoculated plants had a higher antifungal activity than the comparable enzyme from control plants. PMID:20594307

  8. Sequence analysis of the large (L) polymerase gene and trailer of the peste des petits ruminants virus vaccine strain Nigeria 75/1: expression and use of the L protein in reverse genetics.

    Science.gov (United States)

    Minet, C; Yami, M; Egzabhier, B; Gil, P; Tangy, F; Brémont, M; Libeau, G; Diallo, A; Albina, E

    2009-10-01

    The large (L) polymerase gene and the 5'-terminal UTR of the genome of peste des petits ruminants virus (PPRV), vaccine strain Nigeria 75/1, were cloned and sequenced. The L protein was also expressed in eukaryotic cells and its polymerase activity was quantitatively measured in a PPR reverse genetics assay using a reporter minigenome. Comparative sequence analysis of this functional L gene with corresponding genes of other morbilliviruses showed a degree of conservation exceeding 70%. The multiple sequence alignment and the phylogenetic study of L gene discriminated the morbilliviruses in 6 clusters, which are more closely related to Tupaia and Henipaviruses than to other paramyxoviruses. Important protein domains and functional motifs of the L polymerase of the PPRV Nigeria 75/1 vaccine were also identified by using different bioinformatics tools.

  9. Real-time RT-PCR expression analysis of chitinase and endoglucanase genes in the three-way interaction between the biocontrol strain Clonostachys rosea IK726, Botrytis cinera and strawberry

    DEFF Research Database (Denmark)

    Mamarabadi, Mojtaba; Jensen, Birgit; Jensen, Søren Dan Funck

    2008-01-01

    Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two...... endoglucanase genes from C. rosea strain IK726 was analyzed using real-time RT-PCR in vitro and in strawberry leaves during interaction with B. cinerea. Specific primers were designed for ß-tubulin genes from C. rosea and B. cinerea, respectively, and a gene encoding a DNA-binding protein (DBP) from strawberry......, allowing in situ activity assessment of each fungus in vitro and during their interaction on strawberry leaves. Growth of B. cinerea was inhibited in all pathogen-antagonist interactions while the activity of IK726 was slightly increased. In all in vitro interactions, four of the six genes were upregulated...

  10. Clinical expression profiles of complex regional pain syndrome, fibromyalgia and a-specific repetitive strain injury: more common denominators than pain?

    Science.gov (United States)

    Marinus, Johan; Van Hilten, Jacobus J

    2006-03-30

    To systematically evaluate and compare the clinical manifestations, disease course, risk factors and demographic characteristics of Complex Regional Pain Syndrome type 1 (CRPS), fibromyalgia (FM) and a-specific Repetitive Strain Injury (RSI). A literature search was performed using terms related to the aforementioned topics and diseases. Only original clinical studies that included at least 20 subjects were eligible. Fifty-nine studies on CRPS, 73 on FM and 7 on a-specific RSI were identified. The diseases show similarities in age distribution, male-female ratio, pain characteristics and sensory signs and symptoms. Motor, autonomic and trophic changes are frequently reported in CRPS, but only occasionally in FM and RSI. Systemic symptoms are found in patients with CRPS and FM, and in a subgroup of patients with RSI. In all three disorders, symptoms usually start locally, but may spread to other body regions later, which, in the case of FM, is a prerequisite for diagnosis. Disease onset is always, usually, or occasionally of traumatic origin in RSI, CRPS and FM, respectively. Anxiety and depression are more frequent in patients compared to controls, but probably not very different from patients with other pain conditions or chronic diseases. Apart from some obvious differences between CRPS, FM and RSI, the similarities are conspicuous. The common features of CRPS, FM and a-specific RSI may suggest that a common pathway is involved, but until patients with these type of symptoms are assessed with a uniform assessment procedure, a thorough comparison cannot be made. A systematic evaluation of patients with a suspected diagnosis of CRPS, FM or RSI, may lead to a better appreciation of the differences and similarities in these diseases and help to unravel the underlying mechanisms.

  11. A phase 1 study of a group B meningococcal native outer membrane vesicle vaccine made from a strain with deleted lpxL2 and synX and stable expression of opcA.

    Science.gov (United States)

    Keiser, Paul B; Gibbs, Barnett T; Coster, Trinka S; Moran, E Ellen; Stoddard, Mark B; Labrie, Joseph E; Schmiel, Deborah H; Pinto, Valerian; Chen, Ping; Zollinger, Wendell D

    2010-10-08

    This phase 1 clinical trial assessed the safety and immunogenicity of a native outer membrane vesicle (NOMV) vaccine prepared from a lpxL2(-) synX(-) mutant of strain 44/76 with opcA expression stabilized. Thirty-four volunteers were assigned to one of the three dose groups (25 mcg, 25 mcg with aluminum hydroxide adjuvant, and 50 mcg) to receive three intramuscular injections at 0, 6 and 24 weeks. Specific local and systemic adverse events (AEs) were solicited by diary and at visits on days 1, 2, 7 and 14 after each vaccination and at the end of the study at 30 weeks. Blood chemistries, complete blood count, and coagulation studies were measured on each vaccination day and again two days later. Blood for antibody measurements and bactericidal assays were drawn 0, 14, and 42 days after each vaccination. The proportion of volunteers who developed a fourfold or greater increase in serum bactericidal activity (SBA) to the wild-type parent of the vaccine strain with high opcA expression at 6 weeks after the third dose was 12/26 (0.46, 95% confidence interval 0.27-0.65). Antibody levels to OpcA were significantly higher in vaccine responders than in non-responders (p=0.008), and there was a trend for higher antibody levels to the lipooligosaccharide (LOS) (p=0.059). Bactericidal depletion assays on sera from volunteers with high-titer responses also indicate a major contribution of anti-OpcA and anti-LOS antibodies to the bactericidal response.These results suggest that genetically modified NOMV vaccines can induce protection against group B meningococcus. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Modulating activity of vancomycin and daptomycin on the expression of autolysis cell-wall turnover and membrane charge genes in hVISA and VISA strains.

    Directory of Open Access Journals (Sweden)

    Viviana Cafiso

    Full Text Available Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA and Vancomycin-Intermediate-Staphylococcus aureus (VISA], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM, cell-wall turnover (sceD, membrane charges (mprF-dltA and regulatory mechanisms (agr-locus-graRS-walKR, in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA, responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar

  13. Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.

    Science.gov (United States)

    Pichery, Mélanie; Mirey, Emilie; Mercier, Pascale; Lefrancais, Emma; Dujardin, Arnaud; Ortega, Nathalie; Girard, Jean-Philippe

    2012-04-01

    IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.

  14. The Genome of the Toluene-Degrading Pseudomonas veronii Strain 1YdBTEX2 and Its Differential Gene Expression in Contaminated Sand.

    Directory of Open Access Journals (Sweden)

    Marian Morales

    Full Text Available The natural restoration of soils polluted by aromatic hydrocarbons such as benzene, toluene, ethylbenzene and m- and p-xylene (BTEX may be accelerated by inoculation of specific biodegraders (bioaugmentation. Bioaugmentation mainly involves introducing bacteria that deploy their metabolic properties and adaptation potential to survive and propagate in the contaminated environment by degrading the pollutant. In order to better understand the adaptive response of cells during a transition to contaminated material, we analyzed here the genome and short-term (1 h changes in genome-wide gene expression of the BTEX-degrading bacterium Pseudomonas veronii 1YdBTEX2 in non-sterile soil and liquid medium, both in presence or absence of toluene. We obtained a gapless genome sequence of P. veronii 1YdBTEX2 covering three individual replicons with a total size of 8 Mb, two of which are largely unrelated to current known bacterial replicons. One-hour exposure to toluene, both in soil and liquid, triggered massive transcription (up to 208-fold induction of multiple gene clusters, such as toluene degradation pathway(s, chemotaxis and toluene efflux pumps. This clearly underlines their key role in the adaptive response to toluene. In comparison to liquid medium, cells in soil drastically changed expression of genes involved in membrane functioning (e.g., lipid composition, lipid metabolism, cell fatty acid synthesis, osmotic stress response (e.g., polyamine or trehalose synthesis, uptake of potassium and putrescine metabolism, highlighting the immediate response mechanisms of P. veronii 1YdBTEX2 for successful establishment in polluted soil.

  15. Individual variation in p53 and Cip1 expression profiles in normal human fibroblast strains following exposure to high-let radiation

    International Nuclear Information System (INIS)

    Carpenter, T.R.; Johnson, N.F.; Gilliland, F.D.

    1995-01-01

    Exposure to α-particles emitted by radon progeny appears to be the second-leading cause of lung cancer mortality. However, individual susceptibility to the carcinogenic effects of α-particles remains poorly characterized. Variation in susceptibility to cancer produced by certian classes of DNA-damaging chemicals is suspected to involve differences in metabolic activation and detoxication. Susceptibility to α-particle-induced cancer may involve variations in capacity or opportunity to repair DNA damage. Subtle variations in DNA repair capacity would more likely explain radon-related lung cancer susceptibility. The p53 tumor suppressor protein accumulates as a cellular response to DNA damage from ionizing radiation and regulates arrest in the G 1 portion of the cell cycle. Arrest in G 1 portion of the cell cycle. While upstream regulation of p53 protein stability is poorly understood, variations in the ability to accumulate p53 following DNA damage represent potential variations in lung cancer susceptibility related to radon progeny. Further, transcription of the cell-cycle regulatory gene Cip1 is regulated by p53 and increases following ionizing radiation. Therefore, variations in the expression of Cip1 following α-particle exposure may also be a susceptibility factor in radon-related lung cancers. The purpose of the present investigation was to measure p53 and Cip1 protein induction following α-particle exposure of fibroblast lines from nine individuals to determine if there were significant variations. The expression of Cip1 protein indicates the differences in response are biologically relevant

  16. Individual variation in p53 and Cip1 expression profiles in normal human fibroblast strains following exposure to high-let radiation

    Energy Technology Data Exchange (ETDEWEB)

    Carpenter, T.R.; Johnson, N.F.; Gilliland, F.D. [Univ. of New Mexico, Albuquerque, NM (United States)] [and others

    1995-12-01

    Exposure to {alpha}-particles emitted by radon progeny appears to be the second-leading cause of lung cancer mortality. However, individual susceptibility to the carcinogenic effects of {alpha}-particles remains poorly characterized. Variation in susceptibility to cancer produced by certian classes of DNA-damaging chemicals is suspected to involve differences in metabolic activation and detoxication. Susceptibility to {alpha}-particle-induced cancer may involve variations in capacity or opportunity to repair DNA damage. Subtle variations in DNA repair capacity would more likely explain radon-related lung cancer susceptibility. The p53 tumor suppressor protein accumulates as a cellular response to DNA damage from ionizing radiation and regulates arrest in the G{sub 1} portion of the cell cycle. Arrest in G{sub 1} portion of the cell cycle. While upstream regulation of p53 protein stability is poorly understood, variations in the ability to accumulate p53 following DNA damage represent potential variations in lung cancer susceptibility related to radon progeny. Further, transcription of the cell-cycle regulatory gene Cip1 is regulated by p53 and increases following ionizing radiation. Therefore, variations in the expression of Cip1 following {alpha}-particle exposure may also be a susceptibility factor in radon-related lung cancers. The purpose of the present investigation was to measure p53 and Cip1 protein induction following {alpha}-particle exposure of fibroblast lines from nine individuals to determine if there were significant variations. The expression of Cip1 protein indicates the differences in response are biologically relevant.

  17. Intramuscular Immunization of Mice with the Live-Attenuated Herpes Simplex Virus 1 Vaccine Strain VC2 Expressing Equine Herpesvirus 1 (EHV-1) Glycoprotein D Generates Anti-EHV-1 Immune Responses in Mice.

    Science.gov (United States)

    Liu, Shiliang A; Stanfield, Brent A; Chouljenko, Vladimir N; Naidu, Shan; Langohr, Ingeborg; Del Piero, Fabio; Ferracone, Jacqueline; Roy, Alma A; Kousoulas, Konstantin G

    2017-06-15

    Vaccination remains the best option to combat equine herpesvirus 1 (EHV-1) infection, and several different strategies of vaccination have been investigated and developed over the past few decades. Herein, we report that the live-attenuated herpes simplex virus 1 (HSV-1) VC2 vaccine strain, which has been shown to be unable to enter into neurons and establish latency in mice, can be utilized as a vector for the heterologous expression of EHV-1 glycoprotein D (gD) and that the intramuscular immunization of mice results in strong antiviral humoral and cellular immune responses. The VC2-EHV-1-gD recombinant virus was constructed by inserting an EHV-1 gD expression cassette under the control of the cytomegalovirus immediate early promoter into the VC2 vector in place of the HSV-1 thymidine kinase (UL23) gene. The vaccines were introduced into mice through intramuscular injection. Vaccination with both the VC2-EHV-1-gD vaccine and the commercially available vaccine Vetera EHV XP 1/4 (Vetera; Boehringer Ingelheim Vetmedica) resulted in the production of neutralizing antibodies, the levels of which were significantly higher in comparison to those in VC2- and mock-vaccinated animals ( P vaccination with the VC2-EHV-1-gD vaccine stimulated robust IgG1 and IgG2a antibodies after three vaccinations ( P vaccinated mice produced significantly higher levels of IgM than mice in the other groups before and after challenge ( P Vaccination with VC2-EHV-1-gD stimulated strong cellular immune responses, characterized by the upregulation of both interferon- and tumor necrosis factor-positive CD4 + T cells and CD8 + T cells. Overall, the data suggest that the HSV-1 VC2 vaccine strain may be used as a viral vector for the vaccination of horses as well as, potentially, for the vaccination of other economically important animals. IMPORTANCE A novel virus-vectored VC2-EHV-1-gD vaccine was constructed using the live-attenuated HSV-1 VC2 vaccine strain. This vaccine stimulated strong humoral

  18. Expression of dehydrin gene from Arctic Cerastium arcticum increases abiotic stress tolerance and enhances the fermentation capacity of a genetically engineered Saccharomyces cerevisiae laboratory strain.

    Science.gov (United States)

    Kim, Il-Sup; Kim, Hyun-Young; Kim, Young-Saeng; Choi, Han-Gu; Kang, Sung-Ho; Yoon, Ho-Sung

    2013-10-01

    We investigated Arctic plants to determine if they have a specific mechanism enabling them to adapt to extreme environments because they are subject to such conditions throughout their life cycles. Among the cell defense systems of the Arctic mouse-ear chickweed Cerastium arcticum, we identified a stress-responsive dehydrin gene CaDHN that belongs to the SK5 subclass and contains conserved regions with one S segment at the N-terminus and five K segments from the N-terminus to the C-terminus. To investigate the molecular properties of CaDHN, the yeast Saccharomyces was transformed with CaDHN. CaDHN-expressing transgenic yeast (TG) cells recovered more rapidly from challenge with exogenous stimuli, including oxidants (hydrogen peroxide, menadione, and tert-butyl hydroperoxide), high salinity, freezing and thawing, and metal (Zn(2+)), than wild-type (WT) cells. TG cells were sensitive to copper, cobalt, and sodium dodecyl sulfate. In addition, the cell survival of TG cells was higher than that of WT cells when cells at the mid-log and stationary stages were exposed to increased ethanol concentrations. There was a significant difference in cultures that have an ethanol content >16 %. During glucose-based batch fermentation at generally used (30 °C) and low (18 °C) temperatures, TG cells produced a higher alcohol concentration through improved cell survival. Specifically, the final alcohol concentrations were 13.3 and 13.2 % in TG cells during fermentation at 30 and 18 °C, respectively, whereas they were 10.2 and 9.4 %, respectively, in WT cells under the same fermentation conditions. An in vitro assay revealed that purified CaDHN acted as a reactive oxygen species scavenger by neutralizing H2O2 and a chaperone by preventing high temperature-mediated catalase inactivation. Taken together, our results show that CaDHN expression in transgenic yeast confers tolerance to various abiotic stresses by improving redox homeostasis and enhances fermentation capacity

  19. IGFR-I expression and structural analysis of the hard palatine mucosa in an ethanol-drinking rat strain (UChA and UChB).

    Science.gov (United States)

    Martinez, M; De Oliveira, S A; Pinheiro, P F F; Almeida-Francia, C; Pereira, S; Martins, O A; Mello-Júnior, W; Mendes, L O; Chuffa, L G A; Tirapelli, L F; Fávaro, W J; Cagnon, V H A; Martinez, F E

    2011-04-01

    The study analyzed the effects of chronic alcohol ingestion on the ultrastructure of the lining epithelium of the hard palatine mucosa of rats UChA and UChB (lines with voluntary alcohol consumption) in order to contribute to the understanding of the consequences of alcohol abuse for the morphology of the digestive system. Thirty female adult animals aged 120 days were divided into three experimental groups. (1) Ten UChA rats (genetically low ethanol consumer) with voluntary intake of 10% v/v (5.45 g/kg/day) ethanol solution and water. (2) Ten UChB (genetically high ethanol consumer) rats with voluntary intake of 10% v/v (7.16 g/kg/day) ethanol solution and water. (3) Ten Wistar rats with voluntary ad libitum water intake (control group). Both groups received Nuvital pellets ad libitum. The IGFR-I expression was intense in both experimental groups. The epithelial cells of the alcoholic rats UChA and UChB showed many alterations such as the presence of lipid droplets, altered nuclei, nuclei in corneum layer and disrupted mitochondria. It was concluded that ethanol intake induces ultrastructural lesions in the hard palatine mucosa. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Cloning, expression and characterization of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767

    Directory of Open Access Journals (Sweden)

    Yang Dong-Dong

    2012-06-01

    Full Text Available Abstract Background The white-rot fungus Phanerochaete chrysosporium is among the small group of fungi that can degrade lignin to carbon dioxide while leaving the crystalline cellulose untouched. The efficient lignin oxidation system of this fungus requires cyclic redox reactions involving the reduction of aryl-aldehydes to the corresponding alcohols by aryl-alcohol dehydrogenase. However, the biochemical properties of this enzyme have not been extensively studied. These are of most interest for the design of metabolic engineering/synthetic biology strategies in the field of biotechnological applications of this enzyme. Results We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus Phanerochaete chrysosporium, its expression in Escherichia coli and the biochemical characterization of the encoded GST and His6 tagged protein. The purified recombinant enzyme showed optimal activity at 37°C and at pH 6.4 for the reduction of aryl- and linear aldehydes with NADPH as coenzyme. NADH could also be the electron donor, while having a higher Km (220 μM compared to that of NADPH (39 μM. The purified recombinant enzyme was found to be active in the reduction of more than 20 different aryl- and linear aldehydes showing highest specificity for mono- and dimethoxylated Benzaldehyde at positions 3, 4, 3,4 and 3,5. The enzyme was also capable of oxidizing aryl-alcohols with NADP + at 30°C and an optimum pH of 10.3 but with 15 to 100-fold lower catalytic efficiency than for the reduction reaction. Conclusions In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. We show that this enzyme functions in the reductive sense under physiological conditions and that it displays relatively large substrate specificity with highest activity towards the natural compound Veratraldehyde.

  1. Autochthonous fungal strains with high ligninolytic activities from ...

    African Journals Online (AJOL)

    These fungal strains were first screened for lignin-modifying enzymes on solid media containing Poly R-478 or ABTS. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity, expressed within the first week of incubation and only 18 strains decolourised the Poly R-478. These positive strains were further ...

  2. A phase 1 study of a meningococcal native outer membrane vesicle vaccine made from a group B strain with deleted lpxL1 and synX, over-expressed factor H binding protein, two PorAs and stabilized OpcA expression.

    Science.gov (United States)

    Keiser, P B; Biggs-Cicatelli, S; Moran, E E; Schmiel, D H; Pinto, V B; Burden, R E; Miller, L B; Moon, J E; Bowden, R A; Cummings, J F; Zollinger, W D

    2011-02-04

    This phase I clinical trial assessed the safety and immunogenicity of a native outer membrane vesicle (NOMV) vaccine prepared from an lpxL1(-) synX(-) mutant of strain 8570(B:4:P1.19,15:L8-5) of Neisseria meningitidis. Additional mutations enhance the expression of factor H binding protein variant 1 (fHbp v.1), stabilize expression of OpcA and introduce a second PorA (P1.22,14). Thirty-six volunteers were assigned to one of four dose groups (10, 25, 50 and 75 mcg, based on protein content) to receive three intramuscular injections at six week intervals with aluminum hydroxide adjuvant. Specific local and systemic adverse events were solicited by diary and at visits on days 2, 7, and 14 after each vaccination. Blood chemistries, complete blood count, and coagulation studies were measured on each vaccination day and again 2 and 14 days later. Blood for ELISA and serum bactericidal assays was drawn two and six weeks after each vaccination. The proportion of volunteers who developed a fourfold or greater increase in bactericidal activity to the wild type parent of the vaccine strain at two weeks after the third dose was 27 out of 34 (0.79, 95% C.I. 0.65-0.93). Against four other group B strains the response rate ranged from 41% to 82% indicating a good cross reactive antibody response. Depletion assays show contributions to bactericidal activity from antibodies to lipooligosaccharide (LOS), fHbp v.1 and OpcA. Published by Elsevier Ltd.

  3. Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens.

    Science.gov (United States)

    Wang, Jichun; Ge, Aimin; Xu, Mengwei; Wang, Zhisheng; Qiao, Yongfeng; Gu, Yiqi; Liu, Chang; Liu, Yamei; Hou, Jibo

    2015-08-13

    Highly pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry. Duck enteritis virus (DEV)-vectored vaccines expressing AIV H5N1 hemagglutinin (HA) may be viable AIV and DEV vaccine candidates. To facilitate the generation and further improvement of DEV-vectored HA(H5) vaccines, we first constructed an infectious clone of DEV Chinese vaccine strain C-KCE (DEV(C-KCE)). Then, we generated a DEV-vectored HA(H5) vaccine (DEV-H5(UL55)) based on the bacterial artificial chromosome (BAC) by inserting a synthesized HA(H5) expression cassette with a pMCMV IE promoter and a consensus HA sequence into the noncoding area between UL55 and LORF11. The immunogenicity and protective efficacy of the resulting recombinant vaccine against DEV and AIV H5N1 were evaluated in both ducks and chickens. The successful construction of DEV BAC and DEV-H5(UL55) was verified by restriction fragment length polymorphism analysis. Recovered virus from the BAC or mutants showed similar growth kinetics to their parental viruses. The robust expression of HA in chicken embryo fibroblasts infected with the DEV-vectored vaccine was confirmed by indirect immunofluorescence and western blotting analyses. A single dose of 10(6) TCID50 DEV-vectored vaccine provided 100 % protection against duck viral enteritis in ducks, and the hemagglutination inhibition (HI) antibody titer of AIV H5N1 with a peak of 8.2 log2 was detected in 3-week-old layer chickens. In contrast, only very weak HI titers were observed in ducks immunized with 10(7) TCID50 DEV-vectored vaccine. A mortality rate of 60 % (6/10) was observed in 1-week-old specific pathogen free chickens inoculated with 10(6) TCID50 DEV-vectored vaccine. We demonstrate the following in this study. (i) The constructed BAC is a whole genome clone of DEV(C-KCE). (ii) The insertion of an HA expression cassette sequence into the noncoding area between UL55 and LORF11 of DEV(C-KCE) affects neither the growth kinetics of the virus nor its

  4. Escherichia coli MTC, a human NADPH P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450 isoforms 1A1, 1A2, 2A6, 3A4, or 3A5: catalytic activities and mutagenicity studies.

    NARCIS (Netherlands)

    Kranendonk, M.; Carreira, F.; Theisen, P.; Laires, A.; Fischer, C.W.; Rueff, J.; Estabrook, R.W.; Vermeulen, N.P.E.; Roda, R.

    1999-01-01

    We report here on the genetic engineering of four new Escherichia coli tester bacteria, coexpressing human CYP1A1, CYP2A6, CYP3A4 or CYP3A5 with human NADPH cytochrome P450 reductase (RED) by a biplasmid coexpression system, recently developed to express human CYP1A2 in the tester strain MTC. The

  5. Homologous expression of the nrdF gene of Corynebacterium ammoniagenes strain ATCC 6872 generates a manganese-metallocofactor (R2F) and a stable tyrosyl radical (Y˙) involved in ribonucleotide reduction.

    Science.gov (United States)

    Stolle, Patrick; Barckhausen, Olaf; Oehlmann, Wulf; Knobbe, Nadine; Vogt, Carla; Pierik, Antonio J; Cox, Nicholas; Schmidt, Peter P; Reijerse, Edward J; Lubitz, Wolfgang; Auling, Georg

    2010-12-01

    Ribonucleotide reduction, the unique step in the pathway to DNA synthesis, is catalyzed by enzymes via radical-dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene (nrdF) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac-promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron (30:1), high activity (69 μmol 2'-deoxyribonucleotide·mg⁻¹ ·min⁻¹) and distinct absorption at 408 nm, characteristic of a tyrosyl radical (Y˙), which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of Y˙ in ribonucleotide reduction because 0.2 nmol 2'-deoxyribonucleotide was formed, driven by 0.4 nmol Y˙ located on R2F. X-band electron paramagnetic resonance spectroscopy demonstrated a tyrosyl radical at an effective g-value of 2.004. Temperature dependent X/Q-band EPR studies revealed that this radical is coupled to a metallocofactor. Similarities of the native C. ammoniagenes ribonucleotide reductase to the in vitro activated Escherichia coli class Ib enzyme containing a dimanganese(III)-tyrosyl metallocofactor are discussed. © 2010 The Authors Journal compilation © 2010 FEBS.

  6. Attenuated Escherichia coli strains expressing the colonization factor antigen I (CFA/I) and a detoxified heat-labile enterotoxin (LThK63) enhance clearance of ETEC from the lungs of mice and protect mice from intestinal ETEC colonization and LT-induced fluid accumulation.

    Science.gov (United States)

    Byrd, Wyatt; Boedeker, Edgar C

    2013-03-15

    Although enterotoxigenic Escherichia coli (ETEC) infections are important causes of infantile and traveler's diarrhea there is no licensed vaccine available for those at-risk. Our goal is to develop a safe, live attenuated ETEC vaccine. We used an attenuated E. coli strain (O157:H7, Δ-intimin, Stx1-neg, Stx2-neg) as a vector (ZCR533) to prepare two vaccine strains, one strain expressing colonization factor antigen I (ZCR533-CFA/I) and one strain expressing CFA/I and a detoxified heat-labile enterotoxin (ZCR533-CFA/I+LThK63) to deliver ETEC antigens to mucosal sites in BALB/c mice. Following intranasal and intragastric immunization with the vaccine strains, serum IgG and IgA antibodies were measured to the CFA/I antigen, however, only serum IgG antibodies were detected to the heat-labile enterotoxin. Intranasal administration of the vaccine strains induced respiratory and intestinal antibody responses to the CFA/I and LT antigens, while intragastric administration induced only intestinal antibody responses with no respiratory antibodies detected to the CFA/I and LT antigens. Mice immunized intranasally with the vaccine strains showed enhanced clearance of wild-type (wt) ETEC bacteria from the lungs. Mice immunized intranasally and intragastrically with the vaccine strains were protected from intestinal colonization following oral challenge with ETEC wt bacteria. Mice immunized intragastrically with the ZCR533-CFA/I+LThK63 vaccine strain had less fluid accumulate in their intestine following challenge with ETEC wt bacteria or with purified LT as compared to the sham mice indicating that the immunized mice were protected from LT-induced intestinal fluid accumulation. Thus, mice intragastrically immunized with the ZCR533-CFA/I+LThK63 vaccine strain were able to effectively neutralize the activity of the LT enterotoxin. However, no difference in intestinal fluid accumulation was detected in the mice immunized intranasally with the vaccine strain as compared to the sham

  7. Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

    Directory of Open Access Journals (Sweden)

    Luisina Martorelli

    Full Text Available Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS(BLS-Stx2B, in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB, 3 antigens (IntiminC280, EspB, BLS-Stx2B, BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

  8. Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

    Science.gov (United States)

    Martorelli, Luisina; Garbaccio, Sergio; Vilte, Daniel A; Albanese, Adriana A; Mejías, María P; Palermo, Marina S; Mercado, Elsa C; Ibarra, Cristina E; Cataldi, Angel A

    2017-01-01

    Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

  9. Ehrlichia canis (Jaboticabal strain induces the expression of TNF-α in leukocytes and splenocytes of experimentally infected dogs Amostra Ehrlichia canis (Jaboticabal induz a expressão de TNF-α em leucócitos e esplenócitos de cães experimentalmente infectados

    Directory of Open Access Journals (Sweden)

    Joice Lara Maia Faria

    2011-03-01

    Full Text Available Canine ehrlichiosis is caused by the bacterium Ehrlichia canis and is characterized by a systemic febrile disease of unknown pathogenesis. This study evaluated the expression of cytokines TNF-α, IL-10, IFN-γ, in splenic cells and blood leukocytes during the acute phase of ehrlichiosis and after treatment with doxycycline hyclate in dogs experimentally infected with the E. canis Jaboticabal strain. The study results showed a significant expression of TNF-α 18 days post-inoculation, reducing by approximately 70% after treatment. There was a unique peak of expression of IL-10 and IFN-γ 18 and 30 days post-inoculation, respectively. This study suggests that TNF-α plays a role in the pathogenesis of the acute phase of canine ehrlichiosis and that treatment with doxycycline hyclate reduces the systemic effects of this cytokine, possibly by reducing or eliminating parasitemia.A erliquiose canina é causada pela bactéria Ehrlichia canis, que desencadeia no hospedeiro uma doença febril e sistêmica, de patogênese pouco conhecida. O presente estudo avaliou a expressão das citocinas TNF-α, IL-10, IFN-γ, em células esplênicas e em leucócitos sanguíneos, durante a fase aguda da erliquiose e após o tratamento com hiclato de doxiciclina, em cães experimentalmente infectados com a amostra E. canis Jaboticabal. Os resultados mostraram expressão significativa de TNF-α 18 dias após a inoculação, reduzindo aproximadante 70% após o tratamento. Houve um único pico de expressão de IL-10 e de IFN-γ entre 18 e 30 dias após a inoculação, respectivamente. Este estudo sugere que o TNF-α participa da patogenia da fase aguda da erliquiose canina, e que o tratamento com hiclato de doxiciclina reduz os efeitos sistêmicos dessa citocina, possivelmente por reduzir ou eliminar a parasitemia.

  10. Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains

    NARCIS (Netherlands)

    Maassen, C.B.M.; Holten-Neelen, C. van; Balk, F.; Bak-Glashouwer, M.-J.H. den; Leer, R.J.; Laman, J.D.; Boersma, W.J.A.; Claassen, E.

    2000-01-01

    Different Lactobacillus strains are frequently used in consumer food products. In addition, recombinant lactobacilli which contain novel expression vectors can now be used in immunotherapeutic applications such as oral vaccination strategies and in T cell tolerance induction approaches for

  11. TL transgenic mouse strains

    International Nuclear Information System (INIS)

    Obata, Y.; Matsudaira, Y.; Hasegawa, H.; Tamaki, H.; Takahashi, T.; Morita, A.; Kasai, K.

    1993-01-01

    As a result of abnormal development of the thymus of these mice, TCR αβ lineage of the T cell differentiation is disturbed and cells belonging to the TCR γδ CD4 - CD8 - double negative (DN) lineage become preponderant. The γδ DN cells migrate into peripheral lymphoid organs and constitute nearly 50% of peripheral T cells. Immune function of the transgenic mice is severely impaired, indicating that the γδ cells are incapable of participating in these reactions. Molecular and serological analyses of T-cell lymphomas reveal that they belong to the γδ lineage. Tg.Tla a -3-1 mice should be useful in defining the role of TL in normal and abnormal T cell differentiation as well as in the development of T-cell lymphomas, and further they should facilitate studies on the differentiation and function of γδ T cells. We isolated T3 b -TL gene from B6 mice and constructed a chimeric gene in which T3 b -TL is driven by the promoter of H-2K b . With the chimeric gene, two transgenic mouse strains, Tg. Con.3-1 and -2 have been derived in C3H background. Both strains express TL antigen in various tissues including skin. The skin graft of transgenic mice on C3H and (B6 X C3H)F 1 mice were rejected. In the mice which rejected the grafts, CD8 + TCRαβ cytotoxic T cells (CTL) against TL antigens were recognized. The recognition of TL by CTL did not require the antigen presentation by H-2 molecules. The results indicated that TL antigen in the skin becomes a transplantation antigen and behaves like a typical allogeneic MHC class I antigen. The facts that (B6 X C3H)F 1 mice rejected the skin expressing T3 b -TL antigen and induced CTL that killed TL + lymphomas of B6 origin revealed that TL antigen encoded by T3 b -TL is recognized as non-self in B6 mice. Experiments are now extended to analyze immune responses to TL antigen expressed on autochthonous T cell lymphomas. (J.P.N.)

  12. SarA is a repressor of hla (alpha-hemolysin) transcription in Staphylococcus aureus: its apparent role as an activator of hla in the prototype strain NCTC 8325 depends on reduced expression of sarS.

    Science.gov (United States)

    Oscarsson, Jan; Kanth, Anna; Tegmark-Wisell, Karin; Arvidson, Staffan

    2006-12-01

    In most Staphylococcus aureus strains, inactivation of sarA increases hla transcription, indicating that sarA is a repressor. However, in S. aureus NCTC 8325 and its derivatives, used for most studies of hla regulation, inactivation of sarA resulted in decreased hla transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its rsbU mutation, which leads to sigma(B) deficiency. This has now been verified by the demonstration that sarA repressed hla transcription in an rsbU+ derivative of strain 8325-4 (SH1000). That sarA could act as a repressor of hla in an 8325-4 background was confirmed by the observation that inactivation of sarA in an agr sarS rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sarA as an activator of hla in 8325-4 was not a result of the rsbU mutation alone, as inactivation of sarA in another rsbU mutant, strain V8, led to increased hla transcription. Northern blot analysis revealed much higher levels of sarS mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the sarS activator, tcaR, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in sarS transcription upon the inactivation of sarA was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of sarA in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of sarA means a net decrease in repressor activity and, therefore, enhanced hla transcription.

  13. Hamstring strain - aftercare

    Science.gov (United States)

    Pulled hamstring muscle; Sprain - hamstring ... There are 3 levels of hamstring strains: Grade 1 -- mild muscle strain or pull Grade 2 -- partial muscle tear Grade 3 -- complete muscle tear Recovery time depends ...

  14. Obturator internus muscle strains

    Directory of Open Access Journals (Sweden)

    Caoimhe Byrne, MB BCh, BAO

    2017-03-01

    Full Text Available We report 2 cases of obturator internus muscle strains. The injuries occurred in young male athletes involved in kicking sports. Case 1 details an acute obturator internus muscle strain with associated adductor longus strain. Case 2 details an overuse injury of the bilateral obturator internus muscles. In each case, magnetic resonance imaging played a crucial role in accurate diagnosis.

  15. A strain gauge

    DEFF Research Database (Denmark)

    2016-01-01

    The invention relates to a strain gauge of a carrier layer and a meandering measurement grid positioned on the carrier layer, wherein the strain gauge comprises two reinforcement members positioned on the carrier layer at opposite ends of the measurement grid in the axial direction....... The reinforcement members are each placed within a certain axial distance to the measurement grid with the axial distance being equal to or smaller than a factor times the grid spacing. The invention further relates to a multi-axial strain gauge such as a bi-axial strain gauge or a strain gauge rosette where each...... of the strain gauges comprises reinforcement members. The invention further relates to a method for manufacturing a strain gauge as mentioned above....

  16. EFFECTS OF THE ANTIMUTAGENS VANILLIN AND CINNAMALDEHYDE ON SPONTANEOUS MUTATION IN E. COLI LACL STRAINS AND ON GLOBAL GENE EXPRESSION IN SALMONELLA TA104 AND HUMAN HEPG2 CELLS

    Science.gov (United States)

    Effects of the Antimutagens Vanillin and Cinnamaldehyde on Spontaneous Mutation in E. coli lacI Strains and on Global Gene Epression in Salmonella TAlO4 and Human HepG2 Cells In previous work we have shown that vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutag...

  17. In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin

    DEFF Research Database (Denmark)

    Damgaard, C.; Reinholdt, J.; Palarasah, Y.

    2017-01-01

    BACKGROUND AND OBJECTIVE: The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans wi...

  18. Generation and evaluation of a LaSota strain-based recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of avian metapneumovirus subgroup C (aMPV-C) as a bivalent vaccine

    Science.gov (United States)

    To develop a bivalent vaccine, a recombinant Newcastle disease virus was generated by using the NDV LaSota strain with insertion of the G gene of aMPV-C. The biological assessments of the recombinant virus, rLS/aMPV-CG, by conducting the mean death time, intracerebral pathogenicity index, and growth...

  19. Development of intra-strain self-cloning procedure for breeding baker's yeast strains.

    Science.gov (United States)

    Nakagawa, Youji; Ogihara, Hiroyuki; Mochizuki, Chisato; Yamamura, Hideki; Iimura, Yuzuru; Hayakawa, Masayuki

    2017-03-01

    Previously reported self-cloning procedures for breeding of industrial yeast strains require DNA from other strains, plasmid DNA, or mutagenesis. Therefore, we aimed to construct a self-cloning baker's yeast strain that exhibits freeze tolerance via an improved self-cloning procedure. We first disrupted the URA3 gene of a prototrophic baker's yeast strain without the use of any marker gene, resulting in a Δura3 homozygous disruptant. Then, the URA3 gene of the parental baker's yeast strain was used as a selection marker to introduce the constitutive TDH3 promoter upstream of the PDE2 gene encoding high-affinity cyclic AMP phosphodiesterase. This self-cloning procedure was performed without using DNA from other Saccharomyces cerevisiae strains, plasmid DNA, or mutagenesis and was therefore designated an intra-strain self-cloning procedure. Using this self-cloning procedure, we succeeded in producing self-cloning baker's yeast strains that harbor the TDH3p-PDE2 gene heterozygously and homozygously, designated TDH3p-PDE2 hetero and TDH3p-PDE2 homo strains, respectively. These self-cloning strains expressed much higher levels of PDE2 mRNA than the parental strain and exhibited higher viability after freeze stress, as well as higher fermentation ability in frozen dough, when compared with the parental strain. The TDH3p-PDE2 homo strain was genetically more stable than the TDH3p-PDE2 hetero strain. These results indicate that both heterozygous and homozygous strains of self-cloning PDE2-overexpressing freeze-tolerant strains of industrial baker's yeast can be prepared using the intra-strain self-cloning procedure, and, from a practical viewpoint, the TDH3p-PDE2 homo strain constructed in this study is preferable to the TDH3p-PDE2 hetero strain for frozen dough baking. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Differences in 5-HT2A and mGlu2 Receptor Expression Levels and Repressive Epigenetic Modifications at the 5-HT2A Promoter Region in the Roman Low- (RLA-I) and High- (RHA-I) Avoidance Rat Strains.

    Science.gov (United States)

    Fomsgaard, Luna; Moreno, Jose L; de la Fuente Revenga, Mario; Brudek, Tomasz; Adamsen, Dea; Rio-Alamos, Cristobal; Saunders, Justin; Klein, Anders Bue; Oliveras, Ignasi; Cañete, Toni; Blazquez, Gloria; Tobeña, Adolf; Fernandez-Teruel, Albert; Gonzalez-Maeso, Javier; Aznar, Susana

    2018-03-01

    The serotonin 2A (5-HT 2A ) and metabotropic glutamate 2 (mGlu2) receptors regulate each other and are associated with schizophrenia. The Roman high- (RHA-I) and the Roman low- (RLA-I) avoidance rat strains present well-differentiated behavioral profiles, with the RHA-I strain emerging as a putative genetic rat model of schizophrenia-related features. The RHA-I strain shows increased 5-HT 2A and decreased mGlu2 receptor binding levels in prefrontal cortex (PFC). Here, we looked for differences in gene expression and transcriptional regulation of these receptors. The striatum (STR) was included in the analysis. 5-HT 2A , 5-HT 1A , and mGlu2 mRNA and [ 3 H]ketanserin binding levels were measured in brain homogenates. As expected, 5-HT 2A binding was significantly increased in PFC in the RHA-I rats, while no difference in binding was observed in STR. Surprisingly, 5-HT 2A gene expression was unchanged in PFC but significantly decreased in STR. mGlu2 receptor gene expression was significantly decreased in both PFC and STR. No differences were observed for the 5-HT 1A receptor. Chromatin immunoprecipitation assay revealed increased trimethylation of histone 3 at lysine 27 (H3K27me3) at the promoter region of the HTR2A gene in the STR. We further looked at the Akt/GSK3 signaling pathway, a downstream point of convergence of the serotonin and glutamate system, and found increased phosphorylation levels of GSK3β at tyrosine 216 and increased β-catenin levels in the PFC of the RHA-I rats. These results reveal region-specific regulation of the 5-HT 2A receptor in the RHA-I rats probably due to absence of mGlu2 receptor that may result in differential regulation of downstream pathways.

  1. Interactions of Burkholderia terrae with soil fungi: Mechanisms, gene expression patterns and ecological behavior of Burkholderia terrae BS001 during its interaction with Lyophyllum sp. strain Karsten and Trichoderma asperellum 302 in soil

    OpenAIRE

    Haq, Irshad U.

    2016-01-01

    Even though soil is a nutrient-limited environment, there are zones of high microbial activity. Mainly zones that are influenced by plants, fungi, or a combination of both, are of interest. The mycosphere (niche under the influence of fungi) is the zone where, in particular, bacterial-fungal interactions take place. In my thesis, I studied the interaction of the soil bacterium Burkholderia terrae with Lyophyllum sp. strain studied Karsten and Trichoderma asperellum 302. In particular, I studi...

  2. Transcriptomics and adaptive genomics of the asymptomatic bacteriuria Escherichia coli strain 83972

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Seshasayee, Aswin S.; Ussery, David

    2008-01-01

    Escherichia coli strains are the major cause of urinary tract infections in humans. Such strains can be divided into virulent, UPEC strains causing symptomatic infections, and asymptomatic, commensal-like strains causing asymptomatic bacteriuria, ABU. The best-characterized ABU strain is strain...... factors for the human urinary tract could be identified. Also, presence/absence data of the gene expression was used as an adaptive genomics tool to model the gene pool of 83972 using primarily UPEC strain CFT073 as a scaffold. In our analysis, 96% of the transcripts filtered present in strain 83972 can...

  3. Variable expression of the ssb-1 allele in different strains of Escherichia coli K12 and B: Differential suppression of its effects on DNA replication, DNA repair and ultraviolet mutagenesis

    International Nuclear Information System (INIS)

    Lieberman, H.B.; Witkin, E.M.

    1981-01-01

    We have transduced the mutant allele ssb-1, which encodes a temperature-sensitive single-strand DNA binding protein (SSB), into several Escherichia coli strains, and have examined colony-forming ability, DNA replication, sensitivity to ultraviolet light (UV) and UV-induced mutability at the nonpermissive temperature. We have found: 1) that the degree of ssb-1-mediated temperature-sensitivity of colony-forming ability and of DNA replication is strain-dependent, resulting in plating efficiencies at 42 0 C (relative to 30 0 C) ranging from 100% to 0.002%; 2) that complete suppression of the temperature-sensitivity caused by ssb-1 occurs only on nutrient agar, and not in any other medium tested; 3) that strains in which ssb-1-mediated temperature-sensitivity is completely suppressed show moderate UV sensitivity and normal UV mutability at 30 0 C, but much more extreme UV sensitivity and drastically reduced UV mutability at 42 0 C; and 4) that defects in excision repair or in other Uvr + -dependent processes are not responsible for most of the UV sensitivity promoted by ssb-1. We discuss our results in relation to the known properties of SSB and its possible role in the induction of DNA damage-inducible (SOS) functions. (orig.) [de

  4. Strain-engineered MOSFETs

    CERN Document Server

    Maiti, CK

    2012-01-01

    Currently strain engineering is the main technique used to enhance the performance of advanced silicon-based metal-oxide-semiconductor field-effect transistors (MOSFETs). Written from an engineering application standpoint, Strain-Engineered MOSFETs introduces promising strain techniques to fabricate strain-engineered MOSFETs and to methods to assess the applications of these techniques. The book provides the background and physical insight needed to understand new and future developments in the modeling and design of n- and p-MOSFETs at nanoscale. This book focuses on recent developments in st

  5. Engineering and analysis of a Saccharomyces cerevisiae strain that uses formaldehyde as an auxiliary substrate

    NARCIS (Netherlands)

    Baerends, Richard J. S.; de Hulster, Erik; Geertman, Jan-Maarten A.; Daran, Jean-Marc; van Maris, Antonius J. A.; Veenhuis, Marten; van der Klei, Ida J.; Pronk, Jack T.

    We demonstrated that formaldehyde can be efficiently coutilized by an engineered Saccharomyces cerevisiae strain that expresses Hansenula polymorpha genes encoding formaldehyde dehydrogenase (FLD1) and formate dehydrogenase (FMD), in contrast to wild-type strains. Initial chemostat experiments

  6. The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Ulett, G.C.; Schembri, M.A.

    2006-01-01

    Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract....... The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory...... to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human...

  7. A strain gauge

    DEFF Research Database (Denmark)

    2017-01-01

    The invention relates to a strain gauge of a carrier layer and a meandering measurement grid (101) positioned on the carrier layer, wherein the measurement grid comprises a number of measurement grid sections placed side by side with gaps in between, and a number of end loops (106) interconnecting...... relates to a method for manufacturing a strain gauge as mentioned above....

  8. Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains

    DEFF Research Database (Denmark)

    Greppi, Anna; Krych, Lukasz; Costantini, Antonella

    2015-01-01

    Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces...... marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best...... growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase...

  9. An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains.

    Science.gov (United States)

    Fleetwood, Filippa; Andersson, Ken G; Ståhl, Stefan; Löfblom, John

    2014-12-30

    Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection

  10. Identification of Bacillus Strains for Biological Control of Catfish Pathogens

    Science.gov (United States)

    Ran, Chao; Carrias, Abel; Williams, Malachi A.; Capps, Nancy; Dan, Bui C. T.; Newton, Joseph C.; Kloepper, Joseph W.; Ooi, Ei L.; Browdy, Craig L.; Terhune, Jeffery S.; Liles, Mark R.

    2012-01-01

    Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×107 CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (PBacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture. PMID:23029244

  11. Identification of Bacillus strains for biological control of catfish pathogens.

    Science.gov (United States)

    Ran, Chao; Carrias, Abel; Williams, Malachi A; Capps, Nancy; Dan, Bui C T; Newton, Joseph C; Kloepper, Joseph W; Ooi, Ei L; Browdy, Craig L; Terhune, Jeffery S; Liles, Mark R

    2012-01-01

    Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7) CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (PBacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.

  12. Photothermal strain imaging

    Science.gov (United States)

    Choi, Changhoon; Ahn, Joongho; Jeon, Seungwan; Kim, Chulhong

    2017-07-01

    Vulnerable plaques are the major cause of cardiovascular disease, but they are difficult to detect with conventional intravascular imaging techniques. Techniques are needed to identify plaque vulnerability based on the presence of lipids in plaque. Thermal strain imaging (TSI) is an imaging technique based on ultrasound (US) wave propagation speed, which varies with the medium temperature. In TSI, the strain that occurs during tissue temperature change can be used for lipid detection because it has a different tendency depending on the type of tissue. Here, we demonstrate photothermal strain imaging (pTSI) using an intravascular ultrasound catheter. pTSI is performed by slightly and selectively heating lipid using a relatively inexpensive continuous laser source. We applied a speckle-tracking algorithm to US B-mode images for strain calculations. As a result, the strain produced in porcine fat was different from the strain produced in water-bearing gelatin phantom, which made it possible to distinguish the two. This suggests that pTSI could potentially be a way of differentiating lipids in coronary artery.

  13. Strain: Fact or Fiction?

    Science.gov (United States)

    Heilbronner, Renée

    2017-04-01

    2017 marks the 50th anniversary of the publication of John Ramsay's well known textbook "Folding and Fracturing of Rocks" - ... and the 30th anniversary of the rejection of a rather less well known paper entitled "Strain: Fact or Fiction?" submitted by Renée Panozzo to the Journal of Structural Geology. The gist of the paper was simple and straight forward: it was argued that not every fabric that can be observed in deformed rocks is necessarily a measure of the amount of strain the rock incurred. A distinction was made between a general "fabric", i.e., the traceable geometry of grain boundaries, for example, and a so-called "strain fabric", i.e., the model geometry that would result from homogeneously straining an initially isotropic fabric and that would exhibit at least orthorhombic symmetry. To verify if a given fabric was indeed a strain fabric it was therefore suggested to use the SURFOR method (published by Panozzo) and to carry out a so-called strain test, i.e., a check of symmetry, before interpreting the results of a fabric analysis in terms of strain. The problem with the paper was that it was very obviously written out of frustration. The frustration came form having reviewed a number of manuscripts which tried to use the then novel SURFOR method for strain analysis without first checking if the the fabric was a indeed a "strain fabric" or not, and then blaming the SURFOR method for producing ambiguous results. As a result, the paper was not exactly well balanced and carefully thought out. It was considered "interesting but not scholarly" by one of the reviewers and down-right offensive by the second. To tell the truth, however, the paper was not formally rejected. The editor Sue Treagus strongly encouraged Panozzo to revise the paper, ... and 30 years later, I will follow her advise and offer a revised paper as a tribute to John Ramsay. To quote from the original manuscript: "We should be a little more impressed that strain works so well, and less

  14. Genetic Diversity in the Lactose Operons of Lactobacillus helveticus Strains and Its Relationship to the Role of These Strains as Commercial Starter Cultures

    OpenAIRE

    Callanan, M. J.; Beresford, T. P.; Ross, R. P.

    2005-01-01

    Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the β-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced β-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed β-galactosidase in the absence of lactose.

  15. Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene.

    Directory of Open Access Journals (Sweden)

    Cristina W Cunha

    Full Text Available Herpes simplex virus 1 (HSV-1 ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture.

  16. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Teramura, Takeshi; Takehara, Toshiyuki; Onodera, Yuta; Nakagawa, Koichi; Hamanishi, Chiaki; Fukuda, Kanji

    2012-01-01

    Highlights: ► Mechanical stimulation is an important factor for regulation of stem cell fate. ► Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. ► Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. ► This reaction could be reproduced only by transfection of dominant active Rho. ► Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

  17. Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene.

    Science.gov (United States)

    Cunha, Cristina W; Taylor, Kathryne E; Pritchard, Suzanne M; Delboy, Mark G; Komala Sari, Tri; Aguilar, Hector C; Mossman, Karen L; Nicola, Anthony V

    2015-01-01

    Herpes simplex virus 1 (HSV-1) ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture.

  18. Strain superlattices in graphene

    Science.gov (United States)

    Zhang, Yingjie; Kim, Youngseok; Lyding, Joseph; Gilbert, Matthew; Mason, Nadya

    Superlattices have been widely explored to tailor the electronic properties of two-dimensional electron systems. Previous approaches to create superlattices have been limited to periodic potential modulations, either in the form of electrostatic gating or moiré heterostructures. Here we present a new strategy to generate superlattices in 2D materials. We deposit these 2D membranes on a periodic array of dielectric nanospheres, and achieve superlattices with periodic strain modulations. We studied the electronic and magneto-transport properties of strained graphene superlattices, and observed salient features of Dirac point cloning and Hofstadter's butterfly. Furthermore, we were able to tune the transport properties by changing the magnitude of strain in the graphene superlattice. This new degree of freedom provides a novel platform both for fundamental studies of 2D electron correlations and for prospective applications in 2D electronic devices. Y.Z. and N.M. acknowledge support from the National Science Foundation under Grant No. ENG-1434147.

  19. Strain and temperature measurement

    International Nuclear Information System (INIS)

    Stewart, P.A.E.; Fowler, P.H.

    1988-01-01

    A method of non-invasively measuring strain and temperature of an object, substantially simultaneously, using neutrons of selected energy levels is described. A pulsed neutron source is made to emit thermal and epithermal neutrons in a collimated beam directed at the object. Temperature is monitored by observing the thermal Doppler broadening of resonances in the neutron transmission characteristic for the epithermal neutrons and strain is measured from observations made of changes to the thermal neutron diffraction pattern. The object may be a gas turbine blade or a thrust bearing. (author)

  20. Effects of a 3 strain -based direct-fed microbial and dietary fiber concentration on growth performance and expression of genes related to absorption and metabolism of volatile fatty acids in weanling pigs.

    Science.gov (United States)

    Jaworski, N W; Owusu-Asiedu, A; Walsh, M C; McCann, J C; Loor, J J; Stein, H H

    2017-01-01

    Effects of a -based direct-fed microbial (DFM) on growth performance, plasma tumor necrosis factor ɑ (TNFɑ), relative gene expression, and intestinal VFA concentrations in weanling pigs fed low- or high-fiber diets were evaluated. Two hundred pigs (initial BW: 6.31 ± 0.73 kg) were allotted to 1 of 4 dietary treatments (5 pigs per pen and 10 pens per treatment). Treatments were arranged in a 2 × 2 factorial design with 2 diet types [low-fiber (LF) or high-fiber (HF)] and 2 concentrations of DFM (0 or 60 g DFM/t of feed). The DFM contained 1.5 × 10 cfu/g and was obtained from Danisco Animal Nutrition-DuPont Industrial Biosciences, Marlborough, UK. Phase 1 diets were fed for 2 wk post-weaning and phase 2 diets were fed over the following 29 d. Low fiber diets contained corn and soybean meal as main ingredients and HF diets contained corn, soybean meal, corn distillers dried grains with solubles (7.5 and 15.0% in phase 1 and 2, respectively), and wheat middlings (10.0%). Pigs and feed were weighed at the start and at the end of each phase, and ADG, ADFI, and G:F were calculated. At the conclusion of phase 2, blood was collected from 1 pig per pen and 1 pig per pen was sacrificed. Cecum and rectum contents were analyzed for VFA, and tissue samples were collected from the ileum, cecum, rectum, and liver to determine expression of genes related to absorption and metabolism of VFA using quantitative reverse transcription-PCR. Results indicated that feeding HF diets reduced ( ≤ 0.05) ADFI and ADG of pigs compared with feeding LF diets. Pigs fed DFM diets had improved ( ≤ 0.05) G:F compared with pigs fed non-DFM diets. Pigs fed LF diets had greater ( ≤ 0.05) BW at the end of phase 2 compared with pigs fed HF diets. The concentration of VFA in rectum contents was greater ( ≤ 0.05) in pigs fed LF diets than in pigs fed HF diets. The expression of in the rectum of pigs fed HF diets was greater ( ≤ 0.05) than for pigs fed LF diets, and pigs fed DFM

  1. Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae.

    Directory of Open Access Journals (Sweden)

    Sirjana Devi Shrestha

    Full Text Available The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076 with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains.

  2. Expression of aggregative adherence to hela cells by Escherichia coli strains isolated from sick horses Expressão de aderência agregativa em células HeLa por amostras de E. coli isoladas de eqüinos doentes

    Directory of Open Access Journals (Sweden)

    Ana Maria Alvim Liberatore

    2007-03-01

    Full Text Available The virulence attributes of 56 Escherichia coli strains isolated from sick horses (secretions of uterine cervices; gastrointestinal and lung fragments of necropsy; diarrheic feces, and tracheal washings was examined by determining their adherence pattern to HeLa cells and searching for the presence of virulence genes of the various E. coli pathotypes. Two non-adherent strains presented astA, which encodes the enteroaggregative E. coli heat-stable toxin. Twenty-seven strains (48.2% adhered to HeLa cells, 21 (77.8% of which presented the aggregative adherence pattern (AA that characterize the Enteroaggregative E. coli pathotype (EAEC. Nine of the strains presenting AA were isolated from secretions of uterine cervix, including one carrying virulence genes of the EAEC pathotype (aggR,aap,irp2, and pic. This is the first description of the AA phenotype amongst E. coli strains from sick horses. Such strains should be further evaluated regarding their potential role in the pathogenesis of diverse equine diseases and as reservoirs of human infections.Características de virulência de 56 amostras de Escherichia coli isoladas de eqüinos doentes (secreção de colo uterino, fragmentos de necrópsia do trato gastrointestinal e de pulmões, fezes diarréicas e lavado traqueal foram examinadas para determinar o padrão de aderência em células HeLa e pesquisar a presença de genes de virulência de vários patotipos de E. coli. Duas amostras não aderentes apresentaram astA, gene que codifica a toxina termo-estável de E. coli enteroagregativa. Das vinte e sete amostras (48,2% que aderiram a células HeLa, 21 (77,8% apresentaram o padrão de aderência agregativa (AA que caracteriza o patotipo de E. coli Enteroagregativa (EAEC. Nove destas amostras que apresentaram AA foram isoladas de secreção de colo uterino, incluindo uma que apresentava genes de virulência de patotipos de EAEC (aggR,aap,irp2 e pic. Esta é a primeira descrição do fenótipo AA em

  3. Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions.

    Science.gov (United States)

    Ditu, Lia-Mara; Chifiriuc, Mariana Carmen; Bezirtzoglou, Eugenia; Voltsi, Chrysa; Bleotu, Coralia; Pelinescu, Diana; Mihaescu, Grigore; Lazar, Veronica

    2011-12-01

    The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains. The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. LAOS: The strain softening/strain hardening paradox

    NARCIS (Netherlands)

    Mermet-Guyennet, M.R.B.; de Castro, J.G.; Habibi, M.; Martzel, N.; Denn, M.M.; Bonn, D.

    2015-01-01

    Numerous materials, from biopolymers to filled rubbers, exhibit strain softening at high strain amplitudes during a strain sweep in oscillatory rheology: The modulus decreases with increasing deformation. On the other hand, if the nonlinear elastic response is analyzed within a single oscillation

  5. Ratchetting strain prediction

    International Nuclear Information System (INIS)

    Noban, Mohammad; Jahed, Hamid

    2007-01-01

    A time-efficient method for predicting ratchetting strain is proposed. The ratchetting strain at any cycle is determined by finding the ratchetting rate at only a few cycles. This determination is done by first defining the trajectory of the origin of stress in the deviatoric stress space and then incorporating this moving origin into a cyclic plasticity model. It is shown that at the beginning of the loading, the starting point of this trajectory coincides with the initial stress origin and approaches the mean stress, displaying a power-law relationship with the number of loading cycles. The method of obtaining this trajectory from a standard uniaxial asymmetric cyclic loading is presented. Ratchetting rates are calculated with the help of this trajectory and through the use of a constitutive cyclic plasticity model which incorporates deviatoric stresses and back stresses that are measured with respect to this moving frame. The proposed model is used to predict the ratchetting strain of two types of steels under single- and multi-step loadings. Results obtained agree well with the available experimental measurements

  6. Macro-residual strains due to cyclic loading of composites

    CERN Document Server

    Hashin, Z

    1999-01-01

    Macro-residual strains produced by load cycles on elastic-brittle composites are analytically expressed in terms of the effective thermal expansion coefficients of the composite as affected by the damage states developing during the $9 cycling. Limiting values of residual strain are evaluated for unidirectional fiber composites and cross-ply laminates. Frictional losses due to internal sliding are not considered. (17 refs).

  7. Strain measurement based battery testing

    Science.gov (United States)

    Xu, Jeff Qiang; Steiber, Joe; Wall, Craig M.; Smith, Robert; Ng, Cheuk

    2017-05-23

    A method and system for strain-based estimation of the state of health of a battery, from an initial state to an aged state, is provided. A strain gauge is applied to the battery. A first strain measurement is performed on the battery, using the strain gauge, at a selected charge capacity of the battery and at the initial state of the battery. A second strain measurement is performed on the battery, using the strain gauge, at the selected charge capacity of the battery and at the aged state of the battery. The capacity degradation of the battery is estimated as the difference between the first and second strain measurements divided by the first strain measurement.

  8. Studies on Drosophila radiosensitive strains

    International Nuclear Information System (INIS)

    Varentsova, E.P.; Zakharov, I.A.

    1976-01-01

    45 of radiosensitive strains of Drosophila melanogaster were isolated by Curly/Lobe technique after EMS treatment of Livadia population males. The lethality of non-Curly late larvae after gamma-irradiation (4000r) characterized radiosensitivity strains. Most of them exhibited higher frequency of the spontaneous dominant lethals (up to 69%). The males of 6 strains were semi-sterile. 5 of these strains exhibited higher frequency of X-chromosome non-disjunction

  9. Health-promoting properties exhibited by Lactobacillus helveticus strains.

    Science.gov (United States)

    Skrzypczak, Katarzyna; Gustaw, Waldemar; Waśko, Adam

    2015-01-01

    Many strains belonging to lactobacilli exert a variety of beneficial health effects in humans and some of the bacteria are regarded as probiotic microorganisms. Adherence and capabilities of colonization by Lactobacillus strains of the intestinal tract is a prerequisite for probiotic strains to exhibit desired functional properties. The analysis conducted here aimed at screening strains of Lactobacillus helveticus possessing a health-promoting potential. The molecular analysis performed, revealed the presence of a slpA gene encoding the surface S-layer protein SlpA (contributing to the immunostimulatory activity of L. helveticus M 92 probiotic strain) in all B734, DSM, T80, and T105 strains. The product of gene amplification was also identified in a Bifidobacterium animalis ssp. lactis BB12 probiotic strain. SDS-PAGE of a surface protein extract demonstrated the presence of a protein with a mass of about 50 kDa in all strains, which refers to the mass of the S-layer proteins. These results are confirmed by observations carried with transmission electron microscopy, where a clearly visible S-layer was registered in all the strains analyzed. The in vitro study results obtained indicate that the strongest adhesion capacity to epithelial cells (HT-29) was demonstrated by L. helveticus B734, while coaggregation with pathogens was highly diverse among the tested strains. The percentage degree of coaggregation was increasing with the incubation time. After 5 h of incubation, the strongest ability to coaggregate with Escherichia coli was expressed by T104. The T80 strain demonstrated a significant ability to co-aggregate with Staphylococcus aureus, while DSM with Bacillus subtilis. For B734, the highest values of co-aggregation coefficient was noted in samples with Salmonella. The capability of autoaggregation, antibiotic susceptibility, resistance to increasing salt concentrations, and strain survival in simulated small intestinal juice were also analyzed.

  10. Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae

    Directory of Open Access Journals (Sweden)

    Klein Cátia S

    2009-12-01

    Full Text Available Abstract Background Mycoplasma hyopneumoniae is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP. Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422. Results In 2DE comparisons, we were able to identify differences in expression levels for 67 proteins, including the overexpression of some cytoadherence-related proteins only in the pathogenic strains. 2DE immunoblot analyses allowed the identification of differential proteolytic cleavage patterns of the P97 adhesin in the three strains. For more comprehensive protein profiling, an LC-MS/MS strategy was used. Overall, 35% of the M. hyopneumoniae genome coding capacity was covered. Partially overlapping profiles of identified proteins were observed in the strains with 81 proteins identified only in one strain and 54 proteins identified in two strains. Abundance analysis of proteins detected in more than one strain demonstrates the relative overexpression of 64 proteins, including the P97 adhesin in the pathogenic strains. Conclusions Our results indicate the physiological differences between the non-pathogenic strain, with its non-infective proliferate lifestyle, and the pathogenic strains, with its constitutive expression of adhesins, which would render the bacterium competent for adhesion and infection prior to host contact.

  11. Thermal Strain Analysis of Optic Fiber Sensors

    Directory of Open Access Journals (Sweden)

    Chih-Ying Huang

    2013-01-01

    Full Text Available An optical fiber sensor surface bonded onto a host structure and subjected to a temperature change is analytically studied in this work. The analysis is developed in order to assess the thermal behavior of an optical fiber sensor designed for measuring the strain in the host structure. For a surface bonded optical fiber sensor, the measuring sensitivity is strongly dependent on the bonding characteristics which include the protective coating, adhesive layer and the bonding length. Thermal stresses can be generated due to a mismatch of thermal expansion coefficients between the optical fiber and host structure. The optical fiber thermal strain induced by the host structure is transferred via the adhesive layer and protective coating. In this investigation, an analytical expression of the thermal strain and stress in the optical fiber is presented. The theoretical predictions are validated using the finite element method. Numerical results show that the thermal strain and stress are linearly dependent on the difference in thermal expansion coefficients between the optical fiber and host structure and independent of the thermal expansion coefficients of the adhesive and coating.

  12. Angiogenesis is induced by airway smooth muscle strain.

    Science.gov (United States)

    Hasaneen, Nadia A; Zucker, Stanley; Lin, Richard Z; Vaday, Gayle G; Panettieri, Reynold A; Foda, Hussein D

    2007-10-01

    Angiogenesis is an important feature of airway remodeling in both chronic asthma and chronic obstructive pulmonary disease (COPD). Airways in those conditions are exposed to excessive mechanical strain during periods of acute exacerbations. We recently reported that mechanical strain of human airway smooth muscle (HASM) led to an increase in their proliferation and migration. Sustained growth in airway smooth muscle in vivo requires an increase in the nutritional supply to these muscles, hence angiogenesis. In this study, we examined the hypothesis that cyclic mechanical strain of HASM produces factors promoting angiogenic events in the surrounding vascular endothelial cells. Our results show: 1) a significant increase in human lung microvascular endothelial cell (HMVEC-L) proliferation, migration, and tube formation following incubation in conditioned media (CM) from HASM cells exposed to mechanical strain; 2) mechanical strain of HASM cells induced VEGF expression and release; 3) VEGF neutralizing antibodies inhibited the proliferation, migration, and tube formations of HMVEC-L induced by the strained airway smooth muscle CM; 4) mechanical strain of HASM induced a significant increase in hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA and protein, a transcription factor required for VEGF gene transcription; and 5) mechanical strain of HASM induced HIF-1alpha/VEGF through dual phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and ERK pathways. In conclusion, exposing HASM cells to mechanical strain induces signal transduction pathway through PI3K/Akt/mTOR and ERK pathways that lead to an increase in HIF-1alpha, a transcription factor required for VEGF expression. VEGF release by mechanical strain of HASM may contribute to the angiogenesis seen with repeated exacerbation of asthma and COPD.

  13. Differential stress transcriptome landscape of historic and recently emerged hypervirulent strains of Clostridium difficile strains determined using RNA-seq.

    Directory of Open Access Journals (Sweden)

    Joy Scaria

    Full Text Available C. difficile is the most common cause of nosocomial diarrhea in North America and Europe. Genomes of individual strains of C. difficile are highly divergent. To determine how divergent strains respond to environmental changes, the transcriptomes of two historic and two recently isolated hypervirulent strains were analyzed following nutrient shift and osmotic shock. Illumina based RNA-seq was used to sequence these transcriptomes. Our results reveal that although C. difficile strains contain a large number of shared and strain specific genes, the majority of the differentially expressed genes were core genes. We also detected a number of transcriptionally active regions that were not part of the primary genome annotation. Some of these are likely to be small regulatory RNAs.

  14. Developmentally regulated expression of reporter gene in adult ...

    Indian Academy of Sciences (India)

    Figure 2. GAL4 expression pattern in the larval central nervous system of representative adult brain specific. GAL4 enhancer trap strains. Transgenic strains with P-GAL4 insertion were crossed to UAS-Nuc LacZ strain and the F1 larval ganglion at different instar was stained for β-galactosidase activity. (a & b) the brain lobes.

  15. Physical nature of strain rate sensitivity of metals and alloys at high strain rates

    Science.gov (United States)

    Borodin, E. N.; Gruzdkov, A. A.; Mayer, A. E.; Selyutina, N. S.

    2018-04-01

    The role of instabilities of plastic flow at plastic deformation of various materials is one of the important cross-disciplinary problems which is equally important in physics, mechanics and material science. The strain rate sensitivities under slow and high strain rate conditions of loading have different physical nature. In the case of low strain rate, the sensitivity arising from the inertness of the defect structures evolution can be expressed by a single parameter characterizing the plasticity mechanism. In our approach, this is the value of the characteristic relaxation time. In the dynamic case, there are additional effects of “high-speed sensitivity” associated with the micro-localization of the plastic flow near the stress concentrators. In the frames of mechanical description, this requires to introduce additional strain rate sensitivity parameters, which is realized in numerous modifications of Johnson–Cook and Zerilli–Armstrong models. The consideration of both these factors is fundamental for an adequate description of the problems of dynamic deformation of highly inhomogeneous metallic materials such as steels and alloys. The measurement of the dispersion of particle velocities on the free surface of a shock-loaded material can be regarded as an experimental expression of the effect of micro-localization. This is also confirmed by our results of numerical simulation of the propagation of shock waves in a two-dimensional formulation and analytical estimations.

  16. [Echinococcus and strain concepts].

    Science.gov (United States)

    Utük, Armağan Erdem; Simsek, Sami

    2008-01-01

    Hydatid disease (echinococcosis) is one of the most important parasitic zoonoses and remains a public health and economic problem all over the world. Echinococcus granulosus includes a number of genetic variants and, up to date, analyses of mitochondrial DNA sequences have identified ten distinct genetic types (genotypes G1-10). This categorization follows closely the pattern of strain variation emerging based on biological characteristics. The extensive variation in E. granulosus may influence life-cycle patterns, host specificity, development rate, antigenicity, transmission dynamics, sensitivity to chemotherapeutic agents, and pathology. In this review, the recent genetic characterizations of Echinococcus genus have been summarized.

  17. Improved Eco-Friendly Recombinant Anabaena sp. Strain PCC7120 with Enhanced Nitrogen Biofertilizer Potential▿

    Science.gov (United States)

    Chaurasia, Akhilesh Kumar; Apte, Shree Kumar

    2011-01-01

    Photosynthetic, nitrogen-fixing Anabaena strains are native to tropical paddy fields and contribute to the carbon and nitrogen economy of such soils. Genetic engineering was employed to improve the nitrogen biofertilizer potential of Anabaena sp. strain PCC7120. Constitutive enhanced expression of an additional integrated copy of the hetR gene from a light-inducible promoter elevated HetR protein expression and enhanced functional heterocyst frequency in the recombinant strain. The recombinant strain displayed consistently higher nitrogenase activity than the wild-type strain and appeared to be in homeostasis with compatible modulation of photosynthesis and respiration. The enhanced combined nitrogen availability from the recombinant strain positively catered to the nitrogen demand of rice seedlings in short-term hydroponic experiments and supported better growth. The engineered strain is stable, eco-friendly, and useful for environmental application as nitrogen biofertilizer in paddy fields. PMID:21057013

  18. Improved eco-friendly recombinant Anabaena sp. strain PCC7120 with enhanced nitrogen biofertilizer potential.

    Science.gov (United States)

    Chaurasia, Akhilesh Kumar; Apte, Shree Kumar

    2011-01-01

    Photosynthetic, nitrogen-fixing Anabaena strains are native to tropical paddy fields and contribute to the carbon and nitrogen economy of such soils. Genetic engineering was employed to improve the nitrogen biofertilizer potential of Anabaena sp. strain PCC7120. Constitutive enhanced expression of an additional integrated copy of the hetR gene from a light-inducible promoter elevated HetR protein expression and enhanced functional heterocyst frequency in the recombinant strain. The recombinant strain displayed consistently higher nitrogenase activity than the wild-type strain and appeared to be in homeostasis with compatible modulation of photosynthesis and respiration. The enhanced combined nitrogen availability from the recombinant strain positively catered to the nitrogen demand of rice seedlings in short-term hydroponic experiments and supported better growth. The engineered strain is stable, eco-friendly, and useful for environmental application as nitrogen biofertilizer in paddy fields.

  19. Reconstruction of axisymmetric strain distributions via neutron strain tomography

    Science.gov (United States)

    Abbey, Brian; Zhang, Shu Yan; Vorster, Wim; Korsunsky, Alexander M.

    2012-01-01

    Predicting the behaviour of structural components under a particular set of loading conditions requires knowledge of the residual elastic strain distribution throughout the bulk of these components. Characterising the 3D strain state at any particular point involves the measurement of six independent components which make up the second order strain tensor. Mapping the complete strain distribution throughout large volumes thus presents significant practical challenges. One possible solution to this problem is to reconstruct the 3D variation of strain components using tomographic techniques. The basic principle underpinning this idea is that the multi-component strain tensor can be reconstructed from a redundant set of lower order projection data. Here we demonstrate this fundamental concept for two samples: a shrink fit 'ring-and-plug' sample, and a spray-quenched circular cylinder, both possessing axially symmetric internal strain distribution. We present and contrast different approaches to the strain tomography problem. The methods described here can also be readily applied to high-energy X-ray diffraction measurements and represent an important step toward developing the tomographic reconstruction framework for strain tensor distributions of arbitrary complexity. The major benefit of neutron strain tomography is that the incident beam flux is utilised more fully, greatly reducing the data collection times. Using micro-channel plate (MCP) neutron detectors, a spatial resolution of the order of 0.1 mm can be achieved [1].

  20. Sadovskii vortex in strain

    Science.gov (United States)

    Freilich, Daniel; Llewellyn Smith, Stefan

    2014-11-01

    A Sadovskii vortex is a patch of fluid with uniform vorticity surrounded by a vortex sheet. Using a boundary element type method, we investigate the steady states of this flow in an incompressible, inviscid straining flow. Outside the vortex, the fluid is irrotational. In the limiting case where the entire circulation is due to the vortex patch, this is a patch vortex (Moore & Saffman, Aircraft wake turbulence and its detection 1971). In the other limiting case, where all the circulation is due to the vortex sheet, this is a hollow vortex (Llewellyn Smith and Crowdy, J. Fluid Mech. 691, 2012). This flow has two governing nondimensional parameters, relating the strengths of the straining field, vortex sheet, and patch vorticity. We study the relationship between these two parameters, and examine the shape of the resulting vortices. We also work towards a bifurcation diagram of the steady states of the Sadovskii vortex in an attempt to understand the connection between vortex sheet and vortex patch desingularizations of the point vortex. Support from NSF-CMMI-0970113.

  1. Cloning, sequencing, expression, and characterization of the tsh gene from an avian pathogenic Escherichia coli strain/ Clonagem, sequenciamento, expressão e caracterização do gene tsh de uma cepa de Escherichia coli patogênica de aves

    Directory of Open Access Journals (Sweden)

    Marilda C. Vidotto

    2006-06-01

    Full Text Available A hemaglutinina temperatura sensível (Tsh pertence à família das serino-proteases autotransporte de Enterobacteriacea (SPATE, as quais são capazes de clivar diferentes substratos. Nós isolamos e caracterizamos o gene de Escherichia coli patogênica aviária (APEC amostra APEC 13, sorotipo O2:H9,clonado em pET101. A região de 4.2 kb do DNA clonado codifificou uma proteína de aproximadamente 140 kDa (r-Tsh. O plasmídio recombinante pET101-tsh conferiu um fenótipo de hemaglutinação positivo para a linhagem BL21 (tsh- para eritrócitos de galinha. A proteína r-Tsh foi purificada em coluna de níquel e utilizada na produção de anticorpos anti-Tsh. Um fragmento de 1.6 kb foi amplificado e subclonado em pCR4, e a seqüência parcial mostrou alta homologia com outras seqüências analisadas. O anti-Tsh reagiu com as proteínas r-Tsh e Tsh nativa da amostra APEC13, como demonstrado pela técnica de Western blot, mostrando que a r-Tsh tem epitopos conservados e que sua antigenicidade foi preservada. O anti-Tsh também inibiu a atividade hemaglutinante das amostras APEC13 e BL21/pET 101-tsh.The temperature-sensitive hemagglutinin (Tsh belongs to a family of high-molecular-weight serine protease autotransporters of Enterobacteriaceae (SPATEs, which can cleave different substrates. We isolated and characterised the tsh gene from an avian pathogenic Escherichia coli (APEC strain, APEC13 serotype O2:H9, which was cloned in pET101. The 4.2 kb region of cloned DNA coded one protein of approximately 140 kDa (r-Tsh. The recombinant plasmid pET101-tsh conferred to E. coli BL21 strain (tsh the hemagglutination-positive phenotype against chicken erythrocytes. The r-Tsh was purified by Ni-NTA column and used to produce antibody anti-Tsh. A 1.6 kb fragment of the tsh sequence was also amplified and cloned in pCR4, and a partial sequence showed high homology with other sequence analysed. The anti-Tsh reacted with the protein r-Tsh and native Tsh of APEC13

  2. Microarray estimation of genomic inter-strain variability in the genus Ectocarpus (Phaeophyceae

    Directory of Open Access Journals (Sweden)

    Peters Akira F

    2011-01-01

    Full Text Available Background Brown algae of the genus Ectocarpus exhibit high levels of genetic diversity and variability in morphological and physiological characteristics. With the establishment of E. siliculosus as a model and the availability of a complete genome sequence, it is now of interest to analyze variability among different species, ecotypes, and strains of the genus Ectocarpus both at the genome and the transcriptome level. Results We used an E. siliculosus gene expression microarray based on EST sequences from the genome-sequenced strain (reference strain to carry out comparative genome hybridizations for five Ectocarpus strains: four E. siliculosus isolates (the male genome strain, a female strain used for outcrosses with the genome strain, a strain isolated from freshwater, and a highly copper-tolerant strain, as well as one strain of the sister species E. fasciculatus. Our results revealed significant genomic differences between ecotypes of the same species, and enable the selection of conserved probes for future microarray experiments with these strains. In the two closely related strains (a male and a female strain used for crosses, genomic differences were also detected, but concentrated in two smaller genomic regions, one of which corresponds to a viral insertion site. Conclusion The high variability between strains supports the concept of E. siliculosus as a complex of cryptic species. Moreover, our data suggest that several parts of the Ectocarpus genome may have evolved at different rates: high variability was detected particularly in transposable elements and fucoxanthin chlorophyll a/c binding proteins.

  3. Colony Dimorphism in Bradyrhizobium Strains

    Science.gov (United States)

    Sylvester-Bradley, Rosemary; Thornton, Philip; Jones, Peter

    1988-01-01

    Ten isolates of Bradyrhizobium spp. which form two colony types were studied; the isolates originated from a range of legume species. The two colony types differed in the amount of gum formed or size or both, depending on the strain. Whole 7-day-old colonies of each type were subcultured to determine the proportion of cells which had changed to the other type. An iterative computerized procedure was used to determine the rate of switching per generation between the two types and to predict proportions reached at equilibrium for each strain. The predicted proportions of the wetter (more gummy) or larger colony type at equilibrium differed significantly between strains, ranging from 0.9999 (strain CIAT 2383) to 0.0216 (strain CIAT 2469), because some strains switched faster from dry to wet (or small to large) and others switched faster from wet to dry (or large to small). Predicted equilibrium was reached after about 140 generations in strain USDA 76. In all but one strain (CIAT 3030) the growth rate of the wetter colony type was greater than or similar to that of the drier type. The mean difference in generation time between the two colony types was 0.37 h. Doubling times calculated for either colony type after 7 days of growth on the agar surface ranged from 6.0 to 7.3 h. The formation of two persistent colony types by one strain (clonal or colony dimorphism) may be a common phenomenon among Bradyrhizobium strains. Images PMID:16347599

  4. GAL4 enhancer trap strains with reporter gene expression during ...

    Indian Academy of Sciences (India)

    Understanding the molecular regulation of adult brain development has remained difficult even in a genetically tractable system like Drosophila. An important reason for this is that the same genes are deployed repeatedly several times at different stages to carry out different functions in a developmental programme.

  5. A recombinant lactobacillus strain expressing genes coding for ...

    African Journals Online (AJOL)

    SERVER

    2007-08-06

    Aug 6, 2007 ... ... however, overshadowed by the foresight of immunologic rejection and unpredictable safety (Web cutter Version 2.0. http://rna.lund- berg.gu.se/cutter2/; Perelson et al., 1996; Perelson et al.,. 1997; Miller et al., 1993; Jolly, 1994; Weissman et al.,. 1995). We have hypothesized that through ex-vivo deli- ...

  6. A gene expression study on strains of Nostoc (Cyanobacteria ...

    African Journals Online (AJOL)

    Cyanobacteria are well known for their production of a multitude of highly allelopathic compounds. These products have features such as incorporation of non-proteinogenic amino acids which are characteristics of peptides biosynthesized by non-ribosomal peptide synthetases (NRPSs). Some of these peptides have ...

  7. Hydrogen production by recombinant Escherichia coli strains

    Science.gov (United States)

    Maeda, Toshinari; Sanchez‐Torres, Viviana; Wood, Thomas K.

    2012-01-01

    Summary The production of hydrogen via microbial biotechnology is an active field of research. Given its ease of manipulation, the best‐studied bacterium Escherichia coli has become a workhorse for enhanced hydrogen production through metabolic engineering, heterologous gene expression, adaptive evolution, and protein engineering. Herein, the utility of E. coli strains to produce hydrogen, via native hydrogenases or heterologous ones, is reviewed. In addition, potential strategies for increasing hydrogen production are outlined and whole‐cell systems and cell‐free systems are compared. PMID:21895995

  8. Asymptomatic bacteriuria Escherichia coli strains

    DEFF Research Database (Denmark)

    Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

    2006-01-01

    Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast...... to uropathogenic E. coli (UPEC) that cause symptomatic UTI, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the growth characteristics in human urine as well as adhesin repertoire of nine ABU strains; the ability of ABU strains to compete...

  9. Hydrogen production from microbial strains

    Science.gov (United States)

    Harwood, Caroline S; Rey, Federico E

    2012-09-18

    The present invention is directed to a method of screening microbe strains capable of generating hydrogen. This method involves inoculating one or more microbes in a sample containing cell culture medium to form an inoculated culture medium. The inoculated culture medium is then incubated under hydrogen producing conditions. Once incubating causes the inoculated culture medium to produce hydrogen, microbes in the culture medium are identified as candidate microbe strains capable of generating hydrogen. Methods of producing hydrogen using one or more of the microbial strains identified as well as the hydrogen producing strains themselves are also disclosed.

  10. Design and Construction of Vibrio cholerae Strains That Harbor Various CTX Prophage Arrays

    Directory of Open Access Journals (Sweden)

    Hyun J. Yu

    2018-03-01

    Full Text Available Toxigenic Vibrio cholerae strains arise upon infection and integration of the lysogenic cholera toxin phage, the CTX phage, into bacterial chromosomes. The V. cholerae serogroup O1 strains identified to date can be broadly categorized into three main groups: the classical biotype strains, which harbor CTX-cla; the prototype El Tor strains (Wave 1 strains, which harbor CTX-1; and the atypical El Tor strains, which harbor CTX-2 (Wave 2 strains or CTX-3~6 (Wave 3 strains. The efficiencies of replication and transmission of CTX phages are similar, suggesting the possibility of existence of more diverse bacterial strains harboring various CTX phages and their arrays in nature. In this study, a set of V. cholerae strains was constructed by the chromosomal integration of CTX phages into strains that already harbored CTX phages or those that did not harbor any CTX phage or RS1 element. Strains containing repeats of the same kind of CTX phage, strains containing the same kind of CTX phage in each chromosome, strains containing alternative CTX phages in one chromosome, or containing different CTX phages in each chromosome have been constructed. Thus, strains with any CTX array can be designed and constructed. Moreover, the strains described in this study contained the toxT-139F allele, which enhances the expression of TcpA and cholera toxin. These characteristics are considered to be important for cholera vaccine development. Once their capacity to provoke immunity in human against V. cholerae infection is evaluated, some of the generated strains could be developed further to yield cholera vaccine strains.

  11. Live Brucella abortus rough vaccine strain RB51 stimulates enhanced innate immune response in vitro compared to rough vaccine strain RB51SOD and virulent smooth strain 2308 in murine bone marrow-derived dendritic cells.

    Science.gov (United States)

    Surendran, Naveen; Hiltbold, Elizabeth M; Heid, Bettina; Sriranganathan, Nammalwar; Boyle, Stephen M; Zimmerman, Kurt L; Makris, Melissa R; Witonsky, Sharon G

    2011-01-10

    Brucella spp. are Gram-negative, coccobacillary, facultative intracellular pathogens. B. abortus strain 2308 is a pathogenic strain affecting cattle and humans. Rough B. abortus strain RB51, which lacks the O-side chain of lipopolysaccharide (LPS), is the live attenuated USDA approved vaccine for cattle in the United States. Strain RB51SOD, which overexpresses Cu-Zn superoxide dismutase (SOD), has been shown to confer better protection than strain RB51 in a murine model. Protection against brucellosis is mediated by a strong CD4+ Th(1) and CD8+ Tc(1) adaptive immune response. In order to stimulate a robust adaptive response, a solid innate immune response, including that mediated by dendritic cells, is essential. As dendritic cells (DCs) are highly susceptible to Brucella infection, it is possible that pathogenic strains could limit the innate and thereby adaptive immune response. By contrast, vaccine strains could limit or bolster the innate and subsequent adaptive immune response. Identifying how Brucella vaccines stimulate innate and adaptive immunity is critical for enhancing vaccine efficacy. The ability of rough vaccine strains RB51 and RB51SOD to stimulate DC function has not been characterized. We report that live rough vaccine strain RB51 induced significantly better (p ≤ 0.05) DC maturation and function compared to either strain RB51SOD or smooth virulent strain 2308, based on costimulatory marker expression and cytokine production. Copyright © 2010. Published by Elsevier B.V.

  12. Burial stress and elastic strain of carbonate rocks

    DEFF Research Database (Denmark)

    Fabricius, Ida Lykke

    2014-01-01

    strain multiplied by the rock frame modulus. We cannot measure the strain directly in the subsurface, but from the data on bulk density and P‐wave velocity, we can estimate the rock frame modulus and Biot's coefficient and then calculate the “effective vertical stress” as the total vertical stress minus......Burial stress on a sediment or sedimentary rock is relevant for predicting compaction or failure caused by changes in, e.g., pore pressure in the subsurface. For this purpose, the stress is conventionally expressed in terms of its effect: “the effective stress” defined as the consequent elastic...... the product of pore pressure and Biot's coefficient. We can now calculate the elastic strain by dividing “effective stress” with the rock frame modulus. By this procedure, the degree of elastic deformation at a given time and depth can be directly expressed. This facilitates the discussion of the deformation...

  13. Quantum magnetotransport properties of topological insulators under strain

    KAUST Repository

    Tahir, M.

    2012-08-15

    We present a detailed theoretical investigation of the quantum magnetotransport properties of topological insulators under strain. We consider an external magnetic field perpendicular to the surface of the topological insulator in the presence of strain induced by the substrate. The strain effects mix the lower and upper surface states of neighboring Landau levels into two unequally spaced energy branches. Analytical expressions are derived for the collisional conductivity for elastic impurity scattering in the first Born approximation. We also calculate the Hall conductivity using the Kubo formalism. Evidence for the beating of Shubnikov–de Haas oscillations is found from the temperature and magnetic field dependence of the collisional and Hall conductivities. In the regime of a strong magnetic field, the beating pattern is replaced by a splitting of the magnetoresistance peaks due to finite strain energy. These results are in excellent agreement with recent HgTe transport experiments.

  14. A NURBS approximation of experimental stress-strain curves

    International Nuclear Information System (INIS)

    Fedorov, Timofey V.; Morrev, Pavel G.

    2016-01-01

    A compact universal representation of monotonic experimental stress-strain curves of metals and alloys is proposed. It is based on the nonuniform rational Bezier splines (NURBS) of second order and may be used in a computer library of materials. Only six parameters per curve are needed; this is equivalent to a specification of only three points in a stress-strain plane. NURBS-functions of higher order prove to be surplus. Explicit expressions for both yield stress and hardening modulus are given. Two types of curves are considered: at a finite interval of strain and at infinite one. A broad class of metals and alloys of various chemical compositions subjected to various types of preliminary thermo-mechanical working is selected from a comprehensive data base in order to test the methodology proposed. The results demonstrate excellent correspondence to the experimental data. Keywords: work hardening, stress-strain curve, spline approximation, nonuniform rational B-spline, NURBS.

  15. Enterobacter Strains Might Promote Colon Cancer.

    Science.gov (United States)

    Yurdakul, Dilşad; Yazgan-Karataş, Ayten; Şahin, Fikrettin

    2015-09-01

    Many studies have been performed to determine the interaction between bacterial species and cancer. However, there has been no attempts to demonstrate a possible relationship between Enterobacter spp. and colon cancer so far. Therefore, in the present study, it is aimed to investigate the effects of Enterobacter strains on colon cancer. Bacterial proteins were isolated from 11 Enterobacter spp., one Morganella morganii, and one Escherichia coli strains, and applied onto NCM460 (Incell) and CRL1790 (ATCC) cell lines. Cell viability and proliferation were determined in MTS assay. Flow Cytometry was used to detect CD24 level and apoptosis. Real-Time PCR studies were performed to determine NFKB and Bcl2 expression. Graphpad Software was used for statistical analysis. The results showed that proteins, isolated from the Enterobacter spp., have significantly increased cell viability and proliferation, while decreasing the apoptosis of the cell lines tested. The data in the present study indicated that Enterobacter strains might promote colon cancer. Moreover, Enterobacter spp. could be a clinically important factor for colon cancer initiation and progression. Studies can be extended on animal models in order to develop new strategies for treatment.

  16. Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system.

    Science.gov (United States)

    Cheng, L; Chen, G; Ding, G; Zhao, Z; Dong, T; Hu, Z

    2015-04-27

    The Rhodobacter sphaeroides system has been used to express membrane proteins. However, its low yield has substantially limited its application. In order to promote the protein expression capability of this system, the pucC gene, which plays a crucial role in assembling the R. sphaeroides light-harvesting 2 complex (LH2), was overexpressed. To build a pucC overexpression strain, a pucC overexpression vector was constructed and transformed into R. sphaeroides CQU68. The overexpression efficiency was evaluated by quantitative real-time polymerase chain reaction. A well-used reporter β-glucuronidase (GUS) was fusion-expressed with LH2 to evaluate the heterologous protein expression level. As a result, the cell culture and protein in the pucC overexpression strain showed much higher typical spectral absorption peaks at 800 and 850 nm compared with the non-overexpression strain, suggesting a higher expression level of LH2-GUS fusion protein in the pucC overexpression strain. This result was further confirmed by Western blot, which also showed a much higher level of heterologous protein expression in the pucC overexpression strain. We further compared GUS activity in pucC overexpression and non-overexpression strains, the results of which showed that GUS activity in the pucC overexpression strain was approximately ten-fold that in the non-overexpression strain. These results demonstrate that overexpressed pucC can promote heterologous protein expression levels in R. sphaeroides.

  17. Nutritionally fastidious Ruminococct $ flovefociens : strains

    African Journals Online (AJOL)

    than those obtained in a medium containing rumen fluid. Growth of all strains was remarkably uniform. Where the same inoculum was used, differences in the qualitative com- position of the medium usually had little effect on growth. In contrast, the R. f/avefaciens strains were much more variable in their growth responses.

  18. Haldane model under nonuniform strain

    Science.gov (United States)

    Ho, Yen-Hung; Castro, Eduardo V.; Cazalilla, Miguel A.

    2017-10-01

    We study the Haldane model under strain using a tight-binding approach, and compare the obtained results with the continuum-limit approximation. As in graphene, nonuniform strain leads to a time-reversal preserving pseudomagnetic field that induces (pseudo-)Landau levels. Unlike a real magnetic field, strain lifts the degeneracy of the zeroth pseudo-Landau levels at different valleys. Moreover, for the zigzag edge under uniaxial strain, strain removes the degeneracy within the pseudo-Landau levels by inducing a tilt in their energy dispersion. The latter arises from next-to-leading order corrections to the continuum-limit Hamiltonian, which are absent for a real magnetic field. We show that, for the lowest pseudo-Landau levels in the Haldane model, the dominant contribution to the tilt is different from graphene. In addition, although strain does not strongly modify the dispersion of the edge states, their interplay with the pseudo-Landau levels is different for the armchair and zigzag ribbons. Finally, we study the effect of strain in the band structure of the Haldane model at the critical point of the topological transition, thus shedding light on the interplay between nontrivial topology and strain in quantum anomalous Hall systems.

  19. Characterization of putative virulence factors of Serratia marcescens strain SEN for pathogenesis in Spodoptera litura.

    Science.gov (United States)

    Aggarwal, Chetana; Paul, Sangeeta; Tripathi, Vishwas; Paul, Bishwajeet; Khan, Md Aslam

    2017-02-01

    Two Serratia marcescens strains, SEN and ICC-4, isolated from diseased insect cadavers were observed to differ considerably in their virulence towards Spodoptera litura. The present study was aimed to characterize the possible virulence factors present in the virulent Serratia marcescens strain SEN. Both the S. marcescens strains were evaluated for the presence of various lytic enzymes such as chitinase, lipase, protease and phospholipase. The virulent S. marcescens strain SEN was observed to possess considerably higher activity of chitinase and protease enzymes; activity of phospholipase enzyme was also higher. Although, all the three toxin genes shlA, phlA and swr could be detected in both the S. marcescens strains, there was a higher expression of these genes in the virulent strain SEN. S. marcescens strain ICC-4 showed greater reduction in overall growth yield in the post-exponential phase in the presence of midgut juice and hemolymph of S. litura larvae, as compared to S. marcescens strain SEN. Proliferation of the S. marcescens strain SEN was also considerably higher in foregut, midgut and hemolymph of S. litura larvae, as compared to strain ICC-4. Peritrophic membrane treated with broth culture of the S. marcescens strain SEN showed higher damage as compared to strain ICC-4. The peritrophic membrane of larvae fed on diet treated with the virulent strain showed considerable damage while the peritrophic membrane of larvae fed on diet treated with the non-virulent strain showed no damage. This is the first report documenting the fate of ingested S. marcescens in S. litura gut and the relative expression of toxin genes from two S. marcescens strains differing in their virulence towards S. litura. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Strain gradients in epitaxial ferroelectrics

    International Nuclear Information System (INIS)

    Catalan, G.; Noheda, B.; McAneney, J.; Sinnamon, L.J.; Gregg, J.M.

    2005-01-01

    X-ray analysis of ferroelectric thin layers of Ba 1/2 Sr 1/2 TiO 3 with different thicknesses reveals the presence of strain gradients across the films and allows us to propose a functional form for the internal strain profile. We use this to calculate the influence of strain gradient, through flexoelectric coupling, on the degradation of the ferroelectric properties of films with decreasing thickness, in excellent agreement with the observed behavior. This paper shows that strain relaxation can lead to smooth, continuous gradients across hundreds of nanometers, and it highlights the pressing need to avoid such strain gradients in order to obtain ferroelectric films with bulklike properties

  1. Comparative Proteomics Analysis of Mice Lymphocytes in Early Stages of Infection by Different Strains of Rabies Virus

    OpenAIRE

    Vaziri, Behrouz; Torkashvand, Fatemeh; Eslami, Naser; Fayaz, Ahmad

    2012-01-01

    The CNS immune response to rabies virus has been shown to be influenced by virulence of the virus strains. There is no comprehensive report of the peripheral immune response against different strains of rabies virus. In this report we used a comparative proteome analysis to find the early events in the spleen lymphocytes of mice infected by a street strain and an attenuated strain of the rabies virus. Differentially expressed proteins were identified which play important biological roles such...

  2. Local and Nonlocal Strain Rate Fields and Vorticity Alignment in Turbulent Flows

    OpenAIRE

    Hamlington, Peter E.; Schumacher, Jörg; Dahm, Werner J. A.

    2008-01-01

    Local and nonlocal contributions to the total strain rate tensor at any point in a flow are formulated from an expansion of the vorticity field in a local spherical neighborhood of radius R centered on x. The resulting exact expression allows the nonlocal (background) strain rate tensor to be obtained from the total strain rate tensor. In turbulent flows, where the vorticity naturally concentrates into relatively compact structures, this allows the local alignment of vorticity with the most e...

  3. A cell strain system for small homogeneous strain applications.

    Science.gov (United States)

    Bottlang, M; Simnacher, M; Schmitt, H; Brand, R A; Claes, L

    1997-11-01

    A cell culture system has been developed that enables application of well characterized, homogeneously distributed cyclic strains to monolayer cell cultures. Optically clear silicone culture dishes atop Plexiglas base plates are deformed by four-point bending of flexible silicone culture wells driven in user specified strain cycle patterns using computer controlled electromagnetic linear actuators. Cyclic mechano-transduction can be induced in amplitudes of 0 to 3000 mustrain, in frequencies of 0 to 30 Hz and in any specified strain cycle pattern. The cell culture system, which contains six simultaneously driven culture wells, has been mechanically characterized by holographic interferometry, laser displacement sensor recordings of the dish surfaces, strain gauge monitoring of the base plates, and finite element modeling of the dishes on the base plates. The standard deviation of the strain amplitudes among the six simultaneously stimulated culture wells is less than 5%. The cell culture system allows accurate generation of small magnitudes of well characterized, homogeneous strain, easy handling of the culture wells, flexible setting of cyclic strain pattern parameters, simultaneous stimulation of 6 culture wells, and light microscopic observation of the cell cultures.

  4. Strain expansion-reduction approach

    Science.gov (United States)

    Baqersad, Javad; Bharadwaj, Kedar

    2018-02-01

    Validating numerical models are one of the main aspects of engineering design. However, correlating million degrees of freedom of numerical models to the few degrees of freedom of test models is challenging. Reduction/expansion approaches have been traditionally used to match these degrees of freedom. However, the conventional reduction/expansion approaches are only limited to displacement, velocity or acceleration data. While in many cases only strain data are accessible (e.g. when a structure is monitored using strain-gages), the conventional approaches are not capable of expanding strain data. To bridge this gap, the current paper outlines a reduction/expansion technique to reduce/expand strain data. In the proposed approach, strain mode shapes of a structure are extracted using the finite element method or the digital image correlation technique. The strain mode shapes are used to generate a transformation matrix that can expand the limited set of measurement data. The proposed approach can be used to correlate experimental and analytical strain data. Furthermore, the proposed technique can be used to expand real-time operating data for structural health monitoring (SHM). In order to verify the accuracy of the approach, the proposed technique was used to expand the limited set of real-time operating data in a numerical model of a cantilever beam subjected to various types of excitations. The proposed technique was also applied to expand real-time operating data measured using a few strain gages mounted to an aluminum beam. It was shown that the proposed approach can effectively expand the strain data at limited locations to accurately predict the strain at locations where no sensors were placed.

  5. Differences in 5-HT2A and mGlu2 Receptor Expression Levels and Repressive Epigenetic Modifications at the 5-HT2A Promoter Region in the Roman Low- (RLA-I) and High- (RHA-I) Avoidance Rat Strains

    DEFF Research Database (Denmark)

    Fomsgaard, Luna; Moreno, Jose L; de la Fuente Revenga, Mario

    2018-01-01

    The serotonin 2A (5-HT2A) and metabotropic glutamate 2 (mGlu2) receptors regulate each other and are associated with schizophrenia. The Roman high- (RHA-I) and the Roman low- (RLA-I) avoidance rat strains present well-differentiated behavioral profiles, with the RHA-I strain emerging as a putativ...

  6. Comparative Transcriptomic Analysis Reveals Similarities and Dissimilarities in Saccharomyces cerevisiae Wine Strains Response to Nitrogen Availability

    Science.gov (United States)

    Barbosa, Catarina; García-Martínez, José; Pérez-Ortín, José E.; Mendes-Ferreira, Ana

    2015-01-01

    Nitrogen levels in grape-juices are of major importance in winemaking ensuring adequate yeast growth and fermentation performance. Here we used a comparative transcriptome analysis to uncover wine yeasts responses to nitrogen availability during fermentation. Gene expression was assessed in three genetically and phenotypically divergent commercial wine strains (CEG, VL1 and QA23), under low (67 mg/L) and high nitrogen (670 mg/L) regimes, at three time points during fermentation (12h, 24h and 96h). Two-way ANOVA analysis of each fermentation condition led to the identification of genes whose expression was dependent on strain, fermentation stage and on the interaction of both factors. The high fermenter yeast strain QA23 was more clearly distinct from the other two strains, by differential expression of genes involved in flocculation, mitochondrial functions, energy generation and protein folding and stabilization. For all strains, higher transcriptional variability due to fermentation stage was seen in the high nitrogen fermentations. A positive correlation between maximum fermentation rate and the expression of genes involved in stress response was observed. The finding of common genes correlated with both fermentation activity and nitrogen up-take underlies the role of nitrogen on yeast fermentative fitness. The comparative analysis of genes differentially expressed between both fermentation conditions at 12h, where the main difference was the level of nitrogen available, showed the highest variability amongst strains revealing strain-specific responses. Nevertheless, we were able to identify a small set of genes whose expression profiles can quantitatively assess the common response of the yeast strains to varying nitrogen conditions. The use of three contrasting yeast strains in gene expression analysis prompts the identification of more reliable, accurate and reproducible biomarkers that will facilitate the diagnosis of deficiency of this nutrient in the grape

  7. Comparative transcriptomic analysis reveals similarities and dissimilarities in Saccharomyces cerevisiae wine strains response to nitrogen availability.

    Directory of Open Access Journals (Sweden)

    Catarina Barbosa

    Full Text Available Nitrogen levels in grape-juices are of major importance in winemaking ensuring adequate yeast growth and fermentation performance. Here we used a comparative transcriptome analysis to uncover wine yeasts responses to nitrogen availability during fermentation. Gene expression was assessed in three genetically and phenotypically divergent commercial wine strains (CEG, VL1 and QA23, under low (67 mg/L and high nitrogen (670 mg/L regimes, at three time points during fermentation (12 h, 24 h and 96 h. Two-way ANOVA analysis of each fermentation condition led to the identification of genes whose expression was dependent on strain, fermentation stage and on the interaction of both factors. The high fermenter yeast strain QA23 was more clearly distinct from the other two strains, by differential expression of genes involved in flocculation, mitochondrial functions, energy generation and protein folding and stabilization. For all strains, higher transcriptional variability due to fermentation stage was seen in the high nitrogen fermentations. A positive correlation between maximum fermentation rate and the expression of genes involved in stress response was observed. The finding of common genes correlated with both fermentation activity and nitrogen up-take underlies the role of nitrogen on yeast fermentative fitness. The comparative analysis of genes differentially expressed between both fermentation conditions at 12 h, where the main difference was the level of nitrogen available, showed the highest variability amongst strains revealing strain-specific responses. Nevertheless, we were able to identify a small set of genes whose expression profiles can quantitatively assess the common response of the yeast strains to varying nitrogen conditions. The use of three contrasting yeast strains in gene expression analysis prompts the identification of more reliable, accurate and reproducible biomarkers that will facilitate the diagnosis of deficiency of this

  8. A comparison of four major antigens in five human and several animal strains of ureaplasmas.

    Science.gov (United States)

    Thirkell, D; Myles, A D; Taylor-Robinson, D

    1990-07-01

    A comparison of the antigens of single representatives of five serotypes of Ureaplasma urealyticum, of three strains of U. diversum and of single ureaplasmal strains from four other animal hosts was performed by immunoblotting with monoclonal antibodies and a urease 'enzyme-catch test'. The U. urealyticum serotype 8-specific, surface-expressed, 96-Kda antigen was not found in any of the strains of non-human origin. Differences in the distribution of 16- and 17-Kda antigens were also seen, not only between seroclusters A and B of U. urealyticum, but also with respect to animal strains. Five distinct epitopes were expressed on the urease from U. urealyticum and from chimpanzee ureaplasmal strains, but between one and three of these epitopes were either poorly expressed or not detected on the urease from the other animal strains. Apart from lacking the 96-Kda antigen of U. urealyticum serotype 8, chimpanzee strains gave results similar to those obtained with serocluster A of U. urealyticum. The results with the marmoset strain differed from those of all other non-human strains.

  9. Shape recovery and irrecoverable strain control in polyurethane shape-memory polymer

    International Nuclear Information System (INIS)

    Tobushi, Hisaaki; Ejiri, Yoshihiro; Hayashi, Syunichi; Hoshio, Kazumasa

    2008-01-01

    In shape-memory polymers, large strain can be fixed at a low temperature and thereafter recovered at a high temperature. If the shape-memory polymer is held at a high temperature for a long time, the irrecoverable strain can attain a new intermediate shape between the shape under the maximum stress and the primary shape. Irrecoverable strain control can be applied to the fabrication of a shape-memory polymer element with a complex shape in a simple method. In the present study, the influence of the strain-holding conditions on the shape recovery and the irrecoverable strain control in polyurethane shape-memory polymer is investigated by tension test of a film and three-point bending test of a sheet. The higher the shape-holding temperature and the longer the shape-holding time, the higher the irrecoverable strain rate. The equation that expresses the characteristics of the irrecoverable strain control is formulated

  10. Shape recovery and irrecoverable strain control in polyurethane shape-memory polymer

    Directory of Open Access Journals (Sweden)

    Hisaaki Tobushi et al

    2008-01-01

    Full Text Available In shape-memory polymers, large strain can be fixed at a low temperature and thereafter recovered at a high temperature. If the shape-memory polymer is held at a high temperature for a long time, the irrecoverable strain can attain a new intermediate shape between the shape under the maximum stress and the primary shape. Irrecoverable strain control can be applied to the fabrication of a shape-memory polymer element with a complex shape in a simple method. In the present study, the influence of the strain-holding conditions on the shape recovery and the irrecoverable strain control in polyurethane shape-memory polymer is investigated by tension test of a film and three-point bending test of a sheet. The higher the shape-holding temperature and the longer the shape-holding time, the higher the irrecoverable strain rate. The equation that expresses the characteristics of the irrecoverable strain control is formulated.

  11. Comparative Proteomic Profiling of Mycobacterium bovis and BCG Vaccine Strains

    KAUST Repository

    Gao, Ge

    2013-09-01

    BCG is the only licensed human vaccine currently available against TB. Derived from a virulent strain of M. bovis, the vaccine was thought to have struck a balance between reduced virulence and preserved immunogenicity. Nowadays, BCG vaccine strains used in different countries and vaccination programs show clear variations in their genomes and immune protective properties. The aim of this study was to characterize the proteomic profile on Mycobacterium bovis and five BCG strains Pasteur, Tokyo, Danish, Phipps and Birkhaug by Tandem Mass Tag® (TMT®)-labeling quantitative proteomic approach. In total, 420 proteins were identified and 377 of them were quantitated for their relative abundance. We reported the number and relationship of differential expressed proteins in BCG strains compared to M. bovis and investigated their functions by bioinformatics analysis. Several interesting up-regulated and down-regulated protein targets were found. The identified proteins and their quantitative expression profiles provide a basis for further understanding of the cellular biology of M. bovis and BCG vaccine strains, and hopefully would assist in the design of better anti-TB vaccine and drugs.

  12. Strain fluctuations and elastic constants

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, M.; Rahman, A.

    1982-03-01

    It is shown that the elastic strain fluctuations are a direct measure of elastic compliances in a general anisotropic medium; depending on the ensemble in which the fluctuation is measured either the isothermal or the adiabatic compliances are obtained. These fluctuations can now be calculated in a constant enthalpy and pressure, and hence, constant entropy, ensemble due to recent develpments in the molecular dynamics techniques. A calculation for a Ni single crystal under uniform uniaxial 100 tensile or compressive load is presented as an illustration of the relationships derived between various strain fluctuations and the elastic modulii. The Born stability criteria and the behavior of strain fluctuations are shown to be related.

  13. Strain accumulation in quasicrystalline solids

    International Nuclear Information System (INIS)

    Nori, F.; Ronchetti, M.; Elser, V.

    1988-01-01

    We study the relaxation of 2D quasicrystalline elastic networks when their constituent bonds are perturbed homogeneously. Whereas ideal, quasiperiodic networks are stable against such perturbations, we find significant accumulations of strain in a class of disordered networks generated by a growth process. The grown networks are characterized by root mean square phason fluctuations which grow linearly with system size. The strain accumulation we observe in these networks also grows linearly with system size. Finally, we find a dependence of strain accumulation on cooling rate

  14. Roll bonding of strained aluminium

    DEFF Research Database (Denmark)

    Staun, Jakob M.

    2003-01-01

    This report investigates roll bonding of pre-strained (å ~ 4) aluminium sheets to produce high strain material from high purity aluminium (99.996%) and commercial pure aluminium (99.6%). The degree of bonding is investigated by optical microscopy and ultrasonic scanning. Under the right...... of the cross rolled volume fraction is found. To further asses this effect, and the anisotropy, it is necessary to acquire knowledge about both texture and microstructure, e.g. by TEM. Roll bonding of pre-strained aluminium is found to be a possible alternative to ARB in the quest for ultra-fine grained...

  15. Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics

    Science.gov (United States)

    Douillard, François P.; Ribbera, Angela; Järvinen, Hanna M.; Kant, Ravi; Pietilä, Taija E.; Randazzo, Cinzia; Paulin, Lars; Laine, Pia K.; Caggia, Cinzia; von Ossowski, Ingemar; Reunanen, Justus; Satokari, Reetta; Salminen, Seppo; Palva, Airi

    2013-01-01

    Four Lactobacillus strains were isolated from marketed probiotic products, including L. rhamnosus strains from Vifit (Friesland Campina) and Idoform (Ferrosan) and L. casei strains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail with L. casei strain BL23 and L. rhamnosus strain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization between L. casei and L. rhamnosus strains, which could be linked to their genotypes. The two isolated L. rhamnosus strains had genomes that were virtually identical to that of L. rhamnosus GG, testifying to their genomic stability and integrity in food products. The L. casei strains showed much greater genomic heterogeneity. Remarkably, all strains contained an intact spaCBA pilus gene cluster. However, only the L. rhamnosus strains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of an iso-IS30 element upstream of the pilus gene cluster in L. rhamnosus strains but absent in L. casei strains had constituted a functional promoter driving pilus gene expression. All L. rhamnosus strains triggered an NF-κB response via Toll-like receptor 2 (TLR2) in a reporter cell line, whereas the L. casei strains did not or did so to a much lesser extent. This study demonstrates that the two L. rhamnosus strains isolated from probiotic products are virtually identical to L. rhamnosus GG and further highlights the differences between these and L. casei strains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities. PMID:23315726

  16. Strain differences in intermale aggression and possible factors regulating increased aggression in Japanese quail.

    Science.gov (United States)

    Maekawa, Fumihiko; Nagino, Koki; Yang, Jiaxin; Htike, Nang T T; Tsukahara, Shinji; Ubuka, Takayoshi; Tsutsui, Kazuyoshi; Kawashima, Takaharu

    2018-01-15

    The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail (Coturnix japonica) known as NIES-L by rotation breeding in a closed colony for over 35years; accordingly, the strain has highly inbred-like characteristics. Another strain called NIES-Brn has been maintained by randomized breeding in a closed colony to produce outbred-like characteristics. The current study aimed to characterize intermale aggressive behaviors in both strains and to identify possible factors regulating higher aggression in the hypothalamus, such as sex hormone and neuropeptide expression. Both strains displayed a common set of intermale aggressive behaviors that included pecking, grabbing, mounting, and cloacal contact behavior, although NIES-Brn quail showed significantly more grabbing, mounting, and cloacal contact behavior than did NIES-L quail. We examined sex hormone levels in the blood and diencephalon in both strains. Testosterone concentrations were significantly higher in the blood and diencephalon of NIES-Brn quail compared to NIES-L quail. We next examined gene expression in the hypothalamus of both strains using an Agilent gene expression microarray and real-time RT-PCR and found that gene expression of mesotocin (an oxytocin homologue) was significantly higher in the hypothalamus of NIES-Brn quail compared to NIES-L quail. Immunohistochemistry of the hypothalamus revealed that numbers of large cells (cell area>500μm 2 ) expressing mesotocin were significantly higher in the NIES-Brn strain compared to the NIES-L strain. Taken together, our findings suggest that higher testosterone and mesotocin levels in the hypothalamus may be responsible for higher aggression in the NIES-Brn quail strain. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Stress–strain relations for hydrogels under multiaxial deformation

    DEFF Research Database (Denmark)

    Drozdov, Aleksey; Christiansen, Jesper de Claville

    2013-01-01

    Constitutive equations are derived for the elastic response of swollen elastomers and hydrogels under an arbitrary deformation with finite strains. An expression is developed for the free energy density of a polymer network based on the Flory concept of flexible chains with constrained junctions...... and solvent-dependent reference configuration. The importance of introduction of a reference configuration evolving under swelling is confirmed by the analysis of experimental data on nanocomposite hydrogels subjected to swelling and drying. Adjustable parameters in the stress–strain relations are found...

  18. Strain localisation in granular media

    OpenAIRE

    Desrues , Jacques

    1984-01-01

    This study is devoted to strain localisation in Granular materials. Both experimental and theoretical results have been obtained.The first part of the thesis is a review of the methods and theories about rupture in sols mechanics and more generally, in solid mechanics. The classical framework of Shear Band analysis is presented, and the main results available for different classes of materials are discussed.The second part describes an experimental study of strain localisation in sand specime...

  19. Different transcriptional profiles of human monocyte-derived dendritic cells infected with distinct strains of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin.

    Science.gov (United States)

    Sanarico, Nunzia; Colone, Alessia; Grassi, Manuela; Speranza, Viviana; Giovannini, Daniela; Ciaramella, Antonio; Colizzi, Vittorio; Mariani, Francesca

    2011-01-01

    In order to analyze dendritic cells (DCs) activation following infection with different mycobacterial strains, we studied the expression profiles of 165 genes of human monocyte-derived DCs infected with H37Rv, a virulent Mycobacterium tuberculosis (MTB) laboratory strain, CMT97, a clinical MTB isolate, Mycobacterium bovis bacillus Calmette-Guérin (BCG), Aventis Pasteur, and BCG Japan, both employed as vaccine against tuberculosis. The analysis of the gene expression reveals that, despite a set of genes similarly modulated, DCs response resulted strain dependent. In particular, H37Rv significantly upregulated EBI3 expression compared with BCG Japan, while it was the only strain that failed to release a significant IL-10 amount. Of note, BCG Japan showed a marked increase in CCR7 and TNF-α expression regarding both MTB strains and it resulted the only strain failing in exponential intracellular growth. Our results suggest that DCs display the ability to elicit a tailored strain-specific immune response.

  20. The Effect of Grain Size and Strain on the Tensile Flow Stress of Aluminium at Room Temperature

    DEFF Research Database (Denmark)

    Hansen, Niels

    1977-01-01

    Tensile-stress-strain data over a strain range from 0.2 to 30% were obtained at room temperature for 99.999 and 99.5% aluminium as a function of grain size. The yield stress-grain size relationship can be expressed by a Petch-Hall relation with approximately the same slope for the two materials....... The flow stress-grain size relationship can adequately be expressed by a modified Petch-Hall relation; for 99.999% aluminium material the slope increases with strain through a maximum around 15–20%, whereas for 99.5% aluminium the slope decreases with the strain to zero at strains about 10%. The flow...... stress-grain size relationship was analyzed in terms of matrix strengthening and grain boundary strengthening according to the dislocation concept of Ashby. At intermediate strains this approach gives a good description of the effect of strain, grain size and purity on the flow stress....

  1. The Strain and Grain Size Dependence of the Flow Stress of Copper

    DEFF Research Database (Denmark)

    Hansen, Niels; Ralph, B.

    1982-01-01

    in terms of a Hall-Petch relationship. At low strains an inhomogeneous distribution of dislocations is seen whilst at higher strains (0.1–0.2) a more regular cell structure begins to develop. This tends to have a minimum size near to grain boundaries. These microstructural observations are correlated......Tensile stress strain data for 99.999% copper at room and liquid nitrogen temperature as a function of grain size are presented together with some microstructural observations made by transmission electron microscopy. It is shown that the flow stress data, at constant strain may be expressed...

  2. [Production of pertussis toxin by Bordetella pertussis strains isolated from patients with whooping cough].

    Science.gov (United States)

    Zaĭtsev, E M; Mertsalova, N U; Shinkarev, A S; Mazurova, I K; Zakharova, N S

    2011-01-01

    To assess level of pertussin toxin (PT) production by vaccine strains of Bordetella pertussis and strains isolated from patients with whooping cough. Concentration of PT in supernatants of microbial cultures of 3 vaccine strains and 25 strains of B. pertussis isolated from patients with pertussis in 2001 - 2005 was measured with enzyme immunoassay using gamma-globulin fractions of rabbit antiserum to PT as immunosorbent or included in peroxidase conjugates. Level of PT production by strains isolated from infected persons varied from 3 +/- 0.5 to 64.8 +/- 12.2 ng/MFU/ml: in 9 strains--from 3 +/- 0.5 to 9.4 +/- 2.1 ng/MFU/ml, in 7--10.5 +/- 1.8 to 18.4 +/- 2.6 ng/MFU/ml, and in 9--23.6 +/- 4.5 to 64.8 +/- 12.2 ng/MFU/ml. B. pertussis strains isolated from patients were heterogeneous on level of PT production. Difference in expression of PT between strains were as high as 20-fold. Conditionally low, moderate and high levels of PT production had 9 (36%), 7 (28%), and 9 (36%) of 25 studied strains. Three vaccine strains had levels of toxin production similar to recently isolated strains with moderate level of its production.

  3. Effects of genome duplication on phenotypes and industrial applications of Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Zhang, Ke; Fang, Ya-Hong; Gao, Ke-Hui; Sui, Yang; Zheng, Dao-Qiong; Wu, Xue-Chang

    2017-07-01

    Polyploidy is common in Saccharomyces cerevisiae strains, but the physiological and phenotypic effects of ploidy changes have not been fully clarified. Here, isogenic diploid, triploid, and tetraploid S. cerevisiae strains were constructed from a haploid strain, CEN.PK2-1C. Stress tolerance and ethanol fermentation performance of the four euploid strains were compared. Each euploid strain had strengths and weaknesses in tolerance to certain stressors, and no single strain was tolerant of all stressors. The diploid had higher ethanol production than the other strains in normal fermentation medium, while the triploid strain showed the fastest fermentation rate in the presence of inhibitors found in lignocellulosic hydrolysate. Physiological determination revealed diverse physiological attributes, such as trehalose, ergosterol, glutathione, and anti-oxidative enzymes among the strains. Our analyses suggest that both ploidy parity and number of chromosome sets contribute to changes in physiological status. Using qRT-PCR, different expression patterns of genes involved in the regulation of cell morphology and the biosynthesis of key physiological attributes among strains were determined. Our data provide novel insights into the multiple effects of genome duplication on yeast cells and are a useful reference for breeding excellent strains used in specific industrial applications.

  4. Effects of equiaxial strain on the differentiation of dental pulp stem cells without using biochemical reagents.

    Science.gov (United States)

    Tabatabaei, F S; Jazayeri, M; Ghahari, P; Haghighipour, N

    2014-09-01

    During orthodontic treatments, applied mechanical forces create strain and result in tooth movement through the alveolar bone. This response to mechanical strain is a fundamental biological reaction. The present study evaluated the effect of equiaxial strain within the range of orthodontic forces on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). Following isolation and culture of hDPSCs, 3rd passage cells were transferred on a silicone membrane covered with collagen. Cell adhesion to the membrane was evaluated under scanning electron microscope (SEM). Cells were divided into three groups: the first group was placed in a conventional culture medium, transferred to an equiaxial stretching device (3% strain for 2 weeks). The positive control was placed in an osteogenic medium with no mechanical strain. The negative control group was placed in the conventional culture medium with no mechanical strain either. Study groups were evaluated for expression ofosteogenic markers (Alkaline phosphatase and Osteopontin) with immunofluorescence and real time PCR. SEM images revealed optimal adhesion of cells to the silicone membrane. Immunofluorescence study demonstrated that osteocalcin expression occurred after 2 weeks in the two groups under mechanical and chemical signals. After application of equiaxial strain, level of expression of osteogenic markers was significantly higher than in the negative and positive control groups. Based on the study results, static equiaxial strain which mimics the types of orthodontic forces can result in differentiation of hDPSCs to osteoblasts. The results obtained may be used in cell therapy and tissue engineering.

  5. Germinant receptor diversity and germination responses of four strains of the Bacillus cereus group.

    Science.gov (United States)

    van der Voort, Menno; García, Diego; Moezelaar, Roy; Abee, Tjakko

    2010-04-30

    Four strains of the Bacillus cereus group were compared for their germinant receptor composition and spore germination capacity. Phylogenetic analysis of the germinant receptor encoding operons of the enterotoxic strains B. cereus ATCC 14579 and ATCC 10987, the emetic strain AH187, and the psychrotolerant strain Bacillus weihenstephanensis KBAB4, indicated a core group of five germinant receptor operons to be present in the four strains, with each strain containing one to three additional receptors. Using quantitative PCR, induction of expression during sporulation was confirmed for all identified germinant receptor operons in these strains. Despite the large overlap in receptors, diversity in amino acid-induced germination capacity was observed, with six out of 20 amino acids, serving as germinants for spores of all four strains. Each strain showed unique features: efficient germination of strain KBAB4 spores required non-inducing amounts of inosine as the co-germinant, strain ATCC 10987 spores germinated only efficiently after heat activation. Furthermore, strain ATCC 14579 and AH187 spores germinated without heat activation or inosine, with strain ATCC 14579 spores being triggered by all amino acids except phenylalanine and strain AH187 spores being specifically triggered efficiently only by phenylalanine. Analysis of all germination data did not reveal strict linkages between specific germinants and germinant receptors. Finally, the diversity in nutrient-induced germination capacity was also reflected in the diverse germination responses of heat-activated spores of the four B. cereus strains in food matrices, such as milk, rice water and meat bouillon, indicating that amino acid composition and/or availability of inosine are important germination determinants in foods. Copyright 2010 Elsevier B.V. All rights reserved.

  6. Short-period strain (0.1-105 s): Near-source strain field for an earthquake (M L 3.2) near San Juan Bautista, California

    Science.gov (United States)

    Johnston, M. J. S.; Borcherdt, R. D.; Linde, A. T.

    1986-10-01

    Measurements of dilational earth strain in the frequency band 25-10-5 Hz have been made on a deep borehole strainmeter installed near the San Andreas fault. These data are used to determine seismic radiation fields during nuclear explosions, teleseisms, local earthquakes, and ground noise during seismically quiet times. Strains of less than 10-10 on these instruments can be clearly resolved at short periods (< 10 s) and are recorded with wide dynamic range digital recorders. This permits measurement of the static and dynamic strain variations in the near field of local earthquakes. Noise spectra for earth strain referenced to 1 (strain)2/Hz show that strain resolution decreases at about 10 dB per decade of frequency from -150 dB at 10-4 Hz to -223 dB at 10 Hz. Exact expressions are derived to relate the volumetric strain and displacement field for a homogeneous P wave in a general viscoelastic solid as observed on colocated dilatometers and seismometers. A rare near-field recording of strain and seismic velocity was obtained on May 26, 1984, from an earthquake (ML 3.2) at a hypocentral distance of 3.2 km near the San Andreas fault at San Juan Bautista, California. While the data indicate no precursory strain release at the 5 × 10-11 strain level, a coseismic strain release of 1.86 nanostrain was observed. This change in strain is consistent with that calculated from a simple dislocation model of the event. Ground displacement spectra, determined from the downhole strain data and instrument-corrected surface seismic data, suggest that source parameters estimated from surface recordings may be contaminated by amplification effects in near-surface low-velocity materials.

  7. Sex Differences in Drosophila Somatic Gene Expression: Variation and Regulation by doublesex

    Directory of Open Access Journals (Sweden)

    Michelle N. Arbeitman

    2016-07-01

    Full Text Available Sex differences in gene expression have been widely studied in Drosophila melanogaster. Sex differences vary across strains, but many molecular studies focus on only a single strain, or on genes that show sexually dimorphic expression in many strains. How extensive variability is and whether this variability occurs among genes regulated by sex determination hierarchy terminal transcription factors is unknown. To address these questions, we examine differences in sexually dimorphic gene expression between two strains in Drosophila adult head tissues. We also examine gene expression in doublesex (dsx mutant strains to determine which sex-differentially expressed genes are regulated by DSX, and the mode by which DSX regulates expression. We find substantial variation in sex-differential expression. The sets of genes with sexually dimorphic expression in each strain show little overlap. The prevalence of different DSX regulatory modes also varies between the two strains. Neither the patterns of DSX DNA occupancy, nor mode of DSX regulation explain why some genes show consistent sex-differential expression across strains. We find that the genes identified as regulated by DSX in this study are enriched with known sites of DSX DNA occupancy. Finally, we find that sex-differentially expressed genes and genes regulated by DSX are highly enriched on the fourth chromosome. These results provide insights into a more complete pool of potential DSX targets, as well as revealing the molecular flexibility of DSX regulation.

  8. Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

    Directory of Open Access Journals (Sweden)

    Guocai Li

    Full Text Available Neisseria gonorrhoeae (N. gonorrhoeae outer membrane protein reduction modifiable protein (Rmp has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

  9. Experimental strain analysis of Clarens Sandstone colonised by endolithic lichens

    Directory of Open Access Journals (Sweden)

    D. Wessels

    1995-09-01

    Full Text Available Endolithic lichens occur commonly on Clarens Sandstone in South Africa, where they significantly contribute to the weathering of sandstone by means of mechanical and chemical weathering processes. This preliminary investigation reports on the success- ful use of strain gauges in detecting strain differences between sandstone without epilithic lichens and sandstone colonised by the euendolithic lichen Lecidea aff. sarcogynoides Korb. Mechanical weathering, expressed as strain changes, in Clarens Sandstone was studied during the transition from relatively dry winter to wet summer conditions. Daily weathering of sandstone due to thermal expansion and contraction of colonised and uncolonised sandstone could be shown. Our results show that liquid water in sandstone enhances the mechanical weathering of uncolonised Clarens Sandstone while water in the gaseous phase enhances mechanical weathering of sandstone by euendolithic lichens.

  10. Lactobacillus ruminis strains cluster according to their mammalian gut source.

    Science.gov (United States)

    O' Donnell, Michelle M; Harris, Hugh Michael B; Lynch, Denise B; Ross, Reynolds Paul; O'Toole, Paul W

    2015-04-01

    Lactobacillus ruminis is a motile Lactobacillus that is autochthonous to the human gut, and which may also be isolated from other mammals. Detailed characterization of L. ruminis has previously been restricted to strains of human and bovine origin. We therefore sought to expand our bio-bank of strains to identify and characterise isolates of porcine and equine origin by comparative genomics. We isolated five strains from the faeces of horses and two strains from pigs, and compared their motility, biochemistry and genetic relatedness to six human isolates and three bovine isolates including the type strain 27780(T). Multilocus sequence typing analysis based on concatenated sequence data for six individual loci separated the 16 L. ruminis strains into three clades concordant with human, bovine or porcine, and equine sources. Sequencing the genomes of four additional strains of human, bovine, equine and porcine origin revealed a high level of genome synteny, independent of the source animal. Analysis of carbohydrate utilization, stress survival and technological robustness in a combined panel of sixteen L. ruminis isolates identified strains with optimal survival characteristics suitable for future investigation as candidate probiotics. Under laboratory conditions, six human isolates of L. ruminis tested were aflagellate and non-motile, whereas all 10 strains of bovine, equine and porcine origin were motile. Interestingly the equine and porcine strains were hyper-flagellated compared to bovine isolates, and this hyper-flagellate phenotype correlated with the ability to swarm on solid medium containing up to 1.8% agar. Analysis by RNA sequencing and qRT-PCR identified genes for the biosynthesis of flagella, genes for carbohydrate metabolism and genes of unknown function that were differentially expressed in swarming cells of an equine isolate of L. ruminis. We suggest that Lactobacillus ruminis isolates have potential to be used in the functional food industry. We

  11. Intra-species Genomic and Physiological Variability Impact Stress Resistance in Strains of Probiotic Potential.

    Science.gov (United States)

    Arnold, Jason W; Simpson, Joshua B; Roach, Jeffrey; Kwintkiewicz, Jakub; Azcarate-Peril, M Andrea

    2018-01-01

    Large-scale microbiome studies have established that most of the diversity contained in the gastrointestinal tract is represented at the strain level; however, exhaustive genomic and physiological characterization of human isolates is still lacking. With increased use of probiotics as interventions for gastrointestinal disorders, genomic and functional characterization of novel microorganisms becomes essential. In this study, we explored the impact of strain-level genomic variability on bacterial physiology of two novel human Lactobacillus rhamnosus strains (AMC143 and AMC010) of probiotic potential in relation to stress resistance. The strains showed differences with known probiotic strains ( L. rhamnosus GG, Lc705, and HN001) at the genomic level, including nucleotide polymorphisms, mutations in non-coding regulatory regions, and rearrangements of genomic architecture. Transcriptomics analysis revealed that gene expression profiles differed between strains when exposed to simulated gastrointestinal stresses, suggesting the presence of unique regulatory systems in each strain. In vitro physiological assays to test resistance to conditions mimicking the gut environment (acid, alkali, and bile stress) showed that growth of L. rhamnosus AMC143 was inhibited upon exposure to alkaline pH, while AMC010 and control strain LGG were unaffected. AMC143 also showed a significant survival advantage compared to the other strains upon bile exposure. Reverse transcription qPCR targeting the bile salt hydrolase gene ( bsh ) revealed that AMC143 expressed bsh poorly (a consequence of a deletion in the bsh promoter and truncation of bsh gene in AMC143), while AMC010 had significantly higher expression levels than AMC143 or LGG. Insertional inactivation of the bsh gene in AMC010 suggested that bsh could be detrimental to bacterial survival during bile stress. Together, these findings show that coupling of classical microbiology with functional genomics methods for the

  12. Intra-species Genomic and Physiological Variability Impact Stress Resistance in Strains of Probiotic Potential

    Directory of Open Access Journals (Sweden)

    Jason W. Arnold

    2018-02-01

    Full Text Available Large-scale microbiome studies have established that most of the diversity contained in the gastrointestinal tract is represented at the strain level; however, exhaustive genomic and physiological characterization of human isolates is still lacking. With increased use of probiotics as interventions for gastrointestinal disorders, genomic and functional characterization of novel microorganisms becomes essential. In this study, we explored the impact of strain-level genomic variability on bacterial physiology of two novel human Lactobacillus rhamnosus strains (AMC143 and AMC010 of probiotic potential in relation to stress resistance. The strains showed differences with known probiotic strains (L. rhamnosus GG, Lc705, and HN001 at the genomic level, including nucleotide polymorphisms, mutations in non-coding regulatory regions, and rearrangements of genomic architecture. Transcriptomics analysis revealed that gene expression profiles differed between strains when exposed to simulated gastrointestinal stresses, suggesting the presence of unique regulatory systems in each strain. In vitro physiological assays to test resistance to conditions mimicking the gut environment (acid, alkali, and bile stress showed that growth of L. rhamnosus AMC143 was inhibited upon exposure to alkaline pH, while AMC010 and control strain LGG were unaffected. AMC143 also showed a significant survival advantage compared to the other strains upon bile exposure. Reverse transcription qPCR targeting the bile salt hydrolase gene (bsh revealed that AMC143 expressed bsh poorly (a consequence of a deletion in the bsh promoter and truncation of bsh gene in AMC143, while AMC010 had significantly higher expression levels than AMC143 or LGG. Insertional inactivation of the bsh gene in AMC010 suggested that bsh could be detrimental to bacterial survival during bile stress. Together, these findings show that coupling of classical microbiology with functional genomics methods for the

  13. Intra-species Genomic and Physiological Variability Impact Stress Resistance in Strains of Probiotic Potential

    Science.gov (United States)

    Arnold, Jason W.; Simpson, Joshua B.; Roach, Jeffrey; Kwintkiewicz, Jakub; Azcarate-Peril, M. Andrea

    2018-01-01

    Large-scale microbiome studies have established that most of the diversity contained in the gastrointestinal tract is represented at the strain level; however, exhaustive genomic and physiological characterization of human isolates is still lacking. With increased use of probiotics as interventions for gastrointestinal disorders, genomic and functional characterization of novel microorganisms becomes essential. In this study, we explored the impact of strain-level genomic variability on bacterial physiology of two novel human Lactobacillus rhamnosus strains (AMC143 and AMC010) of probiotic potential in relation to stress resistance. The strains showed differences with known probiotic strains (L. rhamnosus GG, Lc705, and HN001) at the genomic level, including nucleotide polymorphisms, mutations in non-coding regulatory regions, and rearrangements of genomic architecture. Transcriptomics analysis revealed that gene expression profiles differed between strains when exposed to simulated gastrointestinal stresses, suggesting the presence of unique regulatory systems in each strain. In vitro physiological assays to test resistance to conditions mimicking the gut environment (acid, alkali, and bile stress) showed that growth of L. rhamnosus AMC143 was inhibited upon exposure to alkaline pH, while AMC010 and control strain LGG were unaffected. AMC143 also showed a significant survival advantage compared to the other strains upon bile exposure. Reverse transcription qPCR targeting the bile salt hydrolase gene (bsh) revealed that AMC143 expressed bsh poorly (a consequence of a deletion in the bsh promoter and truncation of bsh gene in AMC143), while AMC010 had significantly higher expression levels than AMC143 or LGG. Insertional inactivation of the bsh gene in AMC010 suggested that bsh could be detrimental to bacterial survival during bile stress. Together, these findings show that coupling of classical microbiology with functional genomics methods for the characterization

  14. Antagonistic Pleiotropy and Fitness Trade-Offs Reveal Specialist and Generalist Traits in Strains of Canine Distemper Virus

    Science.gov (United States)

    Nikolin, Veljko M.; Osterrieder, Klaus; von Messling, Veronika; Hofer, Heribert; Anderson, Danielle; Dubovi, Edward; Brunner, Edgar; East, Marion L.

    2012-01-01

    Theoretically, homogeneous environments favor the evolution of specialists whereas heterogeneous environments favor generalists. Canine distemper is a multi-host carnivore disease caused by canine distemper virus (CDV). The described cell receptor of CDV is SLAM (CD150). Attachment of CDV hemagglutinin protein (CDV-H) to this receptor facilitates fusion and virus entry in cooperation with the fusion protein (CDV-F). We investigated whether CDV strains co-evolved in the large, homogeneous domestic dog population exhibited specialist traits, and strains adapted to the heterogeneous environment of smaller populations of different carnivores exhibited generalist traits. Comparison of amino acid sequences of the SLAM binding region revealed higher similarity between sequences from Canidae species than to sequences from other carnivore families. Using an in vitro assay, we quantified syncytia formation mediated by CDV-H proteins from dog and non-dog CDV strains in cells expressing dog, lion or cat SLAM. CDV-H proteins from dog strains produced significantly higher values with cells expressing dog SLAM than with cells expressing lion or cat SLAM. CDV-H proteins from strains of non-dog species produced similar values in all three cell types, but lower values in cells expressing dog SLAM than the values obtained for CDV-H proteins from dog strains. By experimentally changing one amino acid (Y549H) in the CDV-H protein of one dog strain we decreased expression of specialist traits and increased expression of generalist traits, thereby confirming its functional importance. A virus titer assay demonstrated that dog strains produced higher titers in cells expressing dog SLAM than cells expressing SLAM of non-dog hosts, which suggested possible fitness benefits of specialization post-cell entry. We provide in vitro evidence for the expression of specialist and generalist traits by CDV strains, and fitness trade-offs across carnivore host environments caused by antagonistic

  15. Antagonistic pleiotropy and fitness trade-offs reveal specialist and generalist traits in strains of canine distemper virus.

    Directory of Open Access Journals (Sweden)

    Veljko M Nikolin

    Full Text Available Theoretically, homogeneous environments favor the evolution of specialists whereas heterogeneous environments favor generalists. Canine distemper is a multi-host carnivore disease caused by canine distemper virus (CDV. The described cell receptor of CDV is SLAM (CD150. Attachment of CDV hemagglutinin protein (CDV-H to this receptor facilitates fusion and virus entry in cooperation with the fusion protein (CDV-F. We investigated whether CDV strains co-evolved in the large, homogeneous domestic dog population exhibited specialist traits, and strains adapted to the heterogeneous environment of smaller populations of different carnivores exhibited generalist traits. Comparison of amino acid sequences of the SLAM binding region revealed higher similarity between sequences from Canidae species than to sequences from other carnivore families. Using an in vitro assay, we quantified syncytia formation mediated by CDV-H proteins from dog and non-dog CDV strains in cells expressing dog, lion or cat SLAM. CDV-H proteins from dog strains produced significantly higher values with cells expressing dog SLAM than with cells expressing lion or cat SLAM. CDV-H proteins from strains of non-dog species produced similar values in all three cell types, but lower values in cells expressing dog SLAM than the values obtained for CDV-H proteins from dog strains. By experimentally changing one amino acid (Y549H in the CDV-H protein of one dog strain we decreased expression of specialist traits and increased expression of generalist traits, thereby confirming its functional importance. A virus titer assay demonstrated that dog strains produced higher titers in cells expressing dog SLAM than cells expressing SLAM of non-dog hosts, which suggested possible fitness benefits of specialization post-cell entry. We provide in vitro evidence for the expression of specialist and generalist traits by CDV strains, and fitness trade-offs across carnivore host environments caused by

  16. Modulation of gene expression made easy

    DEFF Research Database (Denmark)

    Solem, Christian; Jensen, Peter Ruhdal

    2002-01-01

    A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected...

  17. Acute and persistent infection by a transfected Mo7 strain of Babesia bovis

    Science.gov (United States)

    Stable transfection of the Mo7 strain of Babesia bovis and expression of an exogenous gene has been demonstrated in long term culture. However, the use of transfected parasites as marker vaccines or vehicles for expressing exogenous antigens in vivo requires demonstration of acute and persistent inf...

  18. Description of full-range strain hardening behavior of steels.

    Science.gov (United States)

    Li, Tao; Zheng, Jinyang; Chen, Zhiwei

    2016-01-01

    Mathematical expression describing plastic behavior of steels allows the execution of parametric studies for many purposes. Various formulas have been developed to characterize stress strain curves of steels. However, most of those formulas failed to describe accurately the strain hardening behavior of steels in the full range which shows various distinct stages. For this purpose, a new formula is developed based on the well-known Ramberg-Osgood formula to describe the full range strain hardening behavior of steels. Test results of all the six types of steels show a three-stage strain hardening behavior. The proposed formula can describe such behavior accurately in the full range using a single expression. The parameters of the formula can be obtained directly and easily through linear regression analysis. Excellent agreements with the test data are observed for all the steels tested. Furthermore, other formulas such as Ludwigson formula, Gardner formula, UGent formula are also applied for comparison. Finally, the proposed formula is considered to have wide suitability and high accuracy for all the steels tested.

  19. High level expression of human basic fibroblast growth factor in ...

    African Journals Online (AJOL)

    USER

    2010-04-19

    Apr 19, 2010 ... Meanwhile, wild-type gene remarkably expressed in all the strains especially in codon plus strain, rare codon substituted ... Key words: Translation initiation region mutagenesis, modified gene, codon optimization, GC content reduction. ..... Yeast-enhanced green fluorescent protein (yEGFP) a reporter of ...

  20. Gene expression

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

    1983-01-01

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  1. Yersinia enterocolitica YopH-Deficient Strain Activates Neutrophil Recruitment to Peyer's Patches and Promotes Clearance of the Virulent Strain.

    Science.gov (United States)

    Dave, Mabel N; Silva, Juan E; Eliçabe, Ricardo J; Jeréz, María B; Filippa, Verónica P; Gorlino, Carolina V; Autenrieth, Stella; Autenrieth, Ingo B; Di Genaro, María S

    2016-11-01

    Yersinia enterocolitica evades the immune response by injecting Yersinia outer proteins (Yops) into the cytosol of host cells. YopH is a tyrosine phosphatase critical for Yersinia virulence. However, the mucosal immune mechanisms subverted by YopH during in vivo orogastric infection with Y. enterocolitica remain elusive. The results of this study revealed neutrophil recruitment to Peyer's patches (PP) after infection with a YopH-deficient mutant strain (Y. enterocolitica ΔyopH). While the Y. enterocolitica wild-type (WT) strain in PP induced the major neutrophil chemoattractant CXCL1 mRNA and protein levels, infection with the Y. enterocolitica ΔyopH mutant strain exhibited a higher expression of the CXCL1 receptor, CXCR2, in blood neutrophils, leading to efficient neutrophil recruitment to the PP. In contrast, migration of neutrophils into PP was impaired upon infection with Y. enterocolitica WT strain. In vitro infection of blood neutrophils revealed the involvement of YopH in CXCR2 expression. Depletion of neutrophils during Y. enterocolitica ΔyopH infection raised the bacterial load in PP. Moreover, the clearance of WT Y. enterocolitica was improved when an equal mixture of Y. enterocolitica WT and Y. enterocolitica ΔyopH strains was used in infecting the mice. This study indicates that Y. enterocolitica prevents early neutrophil recruitment in the intestine and that the effector protein YopH plays an important role in the immune evasion mechanism. The findings highlight the potential use of the Y. enterocolitica YopH-deficient strain as an oral vaccine carrier. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Redox balancing in recombinant strains of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Anderlund, M.

    1998-09-01

    In metabolically engineered Saccharomyces cerevisiae expressing Pichia stipitis XYL1 and XYL2 genes, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, xylitol is excreted as the major product during anaerobic xylose fermentation and only low yields of ethanol are produced. This has been interpreted as a result of the dual cofactor dependence of XR and the exclusive use of NAD{sup +} by XDH. The excretion of xylitol was completely stopped and the formation of glycerol and acetic acid were reduced in xylose utilising S. cerevisiae strains cultivated in oxygen-limited conditions by expressing lower levels of XR than of XDH. The expression level of XYL1 and XYL2 were controlled by changing the promoters and transcription directions of the genes. A new functional metabolic pathway was established when Thermus thermophilus xylA gene was expressed in S. cerevisiae. The recombinant strain was able to ferment xylose to ethanol when cultivated on a minimal medium containing xylose as only carbon source. In order to create a channeled metabolic transfer in the two first steps of the xylose metabolism, XYL1 and XYL2 were fused in-frame and expressed in S. cerevisiae. When the fusion protein, containing a linker of three amino acids, was co expressed together with native XR and XDH monomers, enzyme complexes consisting of chimeric and native subunits were formed. The total activity of these complexes exhibited 10 and 9 times higher XR and XDH activity, respectively, than the original conjugates, consisting of only chimeric subunits. This strain produced less xylitol and the xylitol yield was lower than with strains only expressing native XR and XDH monomers. In addition, more ethanol and less acetic acid were formed. A new gene encoding the cytoplasmic transhydrogenase from Azotobacter vinelandii was cloned. The enzyme showed high similarity to the family of pyridine nucleotide-disulphide oxidoreductase. To analyse the physiological effect of

  3.  Prokaryotic expression systems

    Directory of Open Access Journals (Sweden)

    Dorota Porowińska

    2013-03-01

    Full Text Available For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host.The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized.Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities. 

  4. The core proteome and pan proteome of Salmonella Paratyphi A epidemic strains.

    Directory of Open Access Journals (Sweden)

    Li Zhang

    Full Text Available Comparative proteomics of the multiple strains within the same species can reveal the genetic variation and relationships among strains without the need to assess the genomic data. Similar to comparative genomics, core proteome and pan proteome can also be obtained within multiple strains under the same culture conditions. In this study we present the core proteome and pan proteome of four epidemic Salmonella Paratyphi A strains cultured under laboratory culture conditions. The proteomic information was obtained using a Two-dimensional gel electrophoresis (2-DE technique. The expression profiles of these strains were conservative, similar to the monomorphic genome of S. Paratyphi A. Few strain-specific proteins were found in these strains. Interestingly, non-core proteins were found in similar categories as core proteins. However, significant fluctuations in the abundance of some core proteins were also observed, suggesting that there is elaborate regulation of core proteins in the different strains even when they are cultured in the same environment. Therefore, core proteome and pan proteome analysis of the multiple strains can demonstrate the core pathways of metabolism of the species under specific culture conditions, and further the specific responses and adaptations of the strains to the growth environment.

  5. Photoacoustic spectroscopy of Entamoeba histolytica strains

    Science.gov (United States)

    Acosta-Avalos, D.; Alvarado-Gil, J. J.; Silva, E. F.; Orozco, E.; de Menezes, L. F.; Vargas, H.

    2005-06-01

    Pathogenic and non-pathogenic strains of E. histolytica are studied using photoacoustic spectroscopy. It is shown that the pathogenic strain presents a spectrum similar to that of iron sulfur proteins. The non-pathogenic strain does not show any relevant absorption at the studied wavelength range. The differences observed between the optical absorption spectra of both strains opens the possibility of using photoacoustic spectroscopy as a reliable and simple technique to identify different types of E. histolytica strains.

  6. Pseudomonas fluorescens induces strain-dependent and strain-independent host plant responses in defense networks, primary metabolism and photosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Pelletier, Dale A [ORNL; Morrell-Falvey, Jennifer L [ORNL; Karve, Abhijit A [ORNL; Lu, Tse-Yuan S [ORNL; Tschaplinski, Timothy J [ORNL; Tuskan, Gerald A [ORNL; Chen, Jay [ORNL; Martin, Madhavi Z [ORNL; Jawdy, Sara [ORNL; Weston, David [ORNL; Doktycz, Mitchel John [ORNL; Schadt, Christopher Warren [ORNL

    2012-01-01

    Colonization of plants by nonpathogenic Pseudomonas fluorescens strains can confer enhanced defense capacity against a broad spectrum of pathogens. Few studies, however, have linked defense pathway regulation to primary metabolism and physiology. In this study, physiological data, metabolites, and transcript profiles are integrated to elucidate how molecular networks initiated at the root-microbe interface influence shoot metabolism and whole-plant performance. Experiments with Arabidopsis thaliana were performed using the newly identified P. fluorescens GM30 or P. fluorescens Pf-5 strains. Co-expression networks indicated that Pf-5 and GM30 induced a subnetwork specific to roots enriched for genes participating in RNA regulation, protein degradation, and hormonal metabolism. In contrast, only GM30 induced a subnetwork enriched for calcium signaling, sugar and nutrient signaling, and auxin metabolism, suggesting strain dependence in network architecture. In addition, one subnetwork present in shoots was enriched for genes in secondary metabolism, photosynthetic light reactions, and hormone metabolism. Metabolite analysis indicated that this network initiated changes in carbohydrate and amino acid metabolism. Consistent with this, we observed strain-specific responses in tryptophan and phenylalanine abundance. Both strains reduced host plant carbon gain and fitness, yet provided a clear fitness benefit when plants were challenged with the pathogen P. syringae DC3000.

  7. Mobilomics in Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Menconi, Giulia; Battaglia, Giovanni; Grossi, Roberto; Pisanti, Nadia; Marangoni, Roberto

    2013-03-20

    Mobile Genetic Elements (MGEs) are selfish DNA integrated in the genomes. Their detection is mainly based on consensus-like searches by scanning the investigated genome against the sequence of an already identified MGE. Mobilomics aims at discovering all the MGEs in a genome and understanding their dynamic behavior: The data for this kind of investigation can be provided by comparative genomics of closely related organisms. The amount of data thus involved requires a strong computational effort, which should be alleviated. Our approach proposes to exploit the high similarity among homologous chromosomes of different strains of the same species, following a progressive comparative genomics philosophy. We introduce a software tool based on our new fast algorithm, called regender, which is able to identify the conserved regions between chromosomes. Our case study is represented by a unique recently available dataset of 39 different strains of S.cerevisiae, which regender is able to compare in few minutes. By exploring the non-conserved regions, where MGEs are mainly retrotransposons called Tys, and marking the candidate Tys based on their length, we are able to locate a priori and automatically all the already known Tys and map all the putative Tys in all the strains. The remaining putative mobile elements (PMEs) emerging from this intra-specific comparison are sharp markers of inter-specific evolution: indeed, many events of non-conservation among different yeast strains correspond to PMEs. A clustering based on the presence/absence of the candidate Tys in the strains suggests an evolutionary interconnection that is very similar to classic phylogenetic trees based on SNPs analysis, even though it is computed without using phylogenetic information. The case study indicates that the proposed methodology brings two major advantages: (a) it does not require any template sequence for the wanted MGEs and (b) it can be applied to infer MGEs also for low coverage genomes

  8. Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

    International Nuclear Information System (INIS)

    Sweeney, Nicholas von Offenberg; Cummins, Philip M.; Birney, Yvonne A.; Redmond, Eileen M.; Cahill, Paul A.

    2004-01-01

    Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38 ± 6%), an inhibitory effect which could be completely reversed by targeted siRNA 'knock-down' of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 μM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration

  9. In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum

    Directory of Open Access Journals (Sweden)

    Florent Ailloud

    2016-01-01

    Full Text Available Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana and NPB (not pathogenic to banana strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions.Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem.Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes.

  10. Contrasting transcriptional responses of a virulent and an attenuated strain of Mycobacterium tuberculosis infecting macrophages.

    Directory of Open Access Journals (Sweden)

    Alice H Li

    2010-06-01

    Full Text Available H37Rv and H37Ra are well-described laboratory strains of Mycobacterium tuberculosis derived from the same parental strain, H37, that show dramatically different pathogenic phenotypes.In this study, the transcriptomes of the two strains during axenic growth in broth and during intracellular growth within murine bone-marrow macrophages were compared by whole genome expression profiling. We identified and compared adaptations of either strain upon encountering an intracellular environment, and also contrasted the transcriptomes of the two strains while inside macrophages. In the former comparison, both strains induced genes that would facilitate intracellular survival including those involved in mycobactin synthesis and fatty acid metabolism. However, this response was stronger and more extensive for H37Rv than for H37Ra. This was manifested as the differential expression of a greater number of genes and an increased magnitude of expression for these genes in H37Rv. In comparing intracellular transcriptional signatures, fifty genes were found to be differentially expressed between the strains. Of these fifty, twelve were under control of the PhoPR regulon. Further differences between strains included genes whose products were members of the ESAT-6 family of proteins, or were associated with their secretion.Along with the recent identification of single nucleotide polymorphisms in H37Ra when compared to H37Rv, our demonstration of differential expression of PhoP-regulated and ESX-1 region-related genes during macrophage infection further highlights the significance of these genes in the attenuation of H37Ra.

  11. Coordinated Evolution of Transcriptional and Post-Transcriptional Regulation for Mitochondrial Functions in Yeast Strains.

    Directory of Open Access Journals (Sweden)

    Xuepeng Sun

    Full Text Available Evolution of gene regulation has been proposed to play an important role in environmental adaptation. Exploring mechanisms underlying coordinated evolutionary changes at various levels of gene regulation could shed new light on how organism adapt in nature. In this study, we focused on regulatory differences between a laboratory Saccharomyces cerevisiae strain BY4742 and a pathogenic S. cerevisiae strain, YJM789. The two strains diverge in many features, including growth rate, morphology, high temperature tolerance, and pathogenicity. Our RNA-Seq and ribosomal footprint profiling data showed that gene expression differences are pervasive, and genes functioning in mitochondria are mostly divergent between the two strains at both transcriptional and translational levels. Combining functional genomics data from other yeast strains, we further demonstrated that significant divergence of expression for genes functioning in the electron transport chain (ETC was likely caused by differential expression of a transcriptional factor, HAP4, and that post-transcriptional regulation mediated by an RNA-binding protein, PUF3, likely led to expression divergence for genes involved in mitochondrial translation. We also explored mito-nuclear interactions via mitochondrial DNA replacement between strains. Although the two mitochondrial genomes harbor substantial sequence divergence, neither growth nor gene expression were affected by mitochondrial DNA replacement in both fermentative and respiratory growth media, indicating compatible mitochondrial and nuclear genomes between these two strains in the tested conditions. Collectively, we used mitochondrial functions as an example to demonstrate for the first time that evolution at both transcriptional and post-transcriptional levels could lead to coordinated regulatory changes underlying strain specific functional variations.

  12. High Virulence and Antifungal Resistance in Clinical Strains of Candida albicans

    Directory of Open Access Journals (Sweden)

    Eric Monroy-Pérez

    2016-01-01

    Full Text Available Antifungal resistance and virulence properties of Candida albicans are a growing health problem worldwide. To study the expression of virulence and azole resistance genes in 39 clinical strains of C. albicans, we used a model of infection of human vaginal epithelial cells with C. albicans strains isolated from Mexican women with vulvovaginal candidiasis (VVC. The strains were identified by PCR amplification of the ITS1 and ITS2 regions of rRNA. The detection and expression of virulence genes and azole resistance genes MDR1 and CDR1 were performed using PCR and RT-PCR, respectively. All strains were sensitive to nystatin and 38 (97.4% and 37 (94.9% were resistant to ketoconazole and fluconazole, respectively. ALS1, SAP4–SAP6, LIP1, LIP2, LIP4, LIP6, LIP7, LIP9, LIP10, and PLB1-PLB2 were present in all strains; SAP1 was identified in 37 (94.8% isolates, HWP1 in 35 (89.7%, ALS3 in 14 (35.8%, and CDR1 in 26 (66.6%. In nearly all of the strains, ALS1, HWP1, SAP4–SAP6, LIP1–LIP10, PLB1, and PLB2 were expressed, whereas CDR1 was expressed in 20 (51.3% and ALS3 in 14 (35.8%. In our in vitro model of infection with C. albicans, the clinical strains showed different expression profiles of virulence genes in association with the azole resistance gene CDR1. The results indicate that the strains that infect Mexican patients suffering from VVC are highly virulent and virtually all are insensitive to azoles.

  13. Analysis of Spleen-Induced Fimbria Production in Recombinant Attenuated Salmonella enterica Serovar Typhimurium Vaccine Strains

    Directory of Open Access Journals (Sweden)

    Paweł Łaniewski

    2017-08-01

    Full Text Available Salmonella enterica serovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesized in vivo. We used an in vivo expression technology (IVET strategy to identify four fimbrial operons, agf, saf, sti, and stc that are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 105 CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 109 CFU of wild-type S. Typhimurium. We also examined the possible effect of these fimbriae on the ability of a Salmonella vaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express the Streptococcus pneumoniae antigen PspA. Strains that constitutively expressed saf or stc elicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carrying saf or stc deletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulent S. pneumoniae. Our results indicate that these fimbriae play important roles, as yet not understood, in Salmonella virulence and immunogenicity.

  14. Toxin production in a rare and genetically remote cluster of strains of the Bacillus cereus group

    Directory of Open Access Journals (Sweden)

    Granum Per

    2007-05-01

    Full Text Available Abstract Background Three enterotoxins are implicated in diarrhoeal food poisoning due to Bacillus cereus: Haemolysin BL (Hbl, Non-haemolytic enterotoxin (Nhe, and Cytotoxin K (CytK. Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of B. cereus group strains. B. cereus strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other B. cereus group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of cytK expression. To date, only three strains containing cytK-1 have been identified; B. cereus strains NVH 391/98, NVH 883/00, and INRA AF2. Results A novel gene variant encoding Nhe was identified in these three strains, which had an average of 80% identity in protein sequence with previously identified Nhe toxins. While culture supernatants containing CytK and Nhe from NVH 391/98 and INRA AF2 were highly cytotoxic, NVH 883/00 expressed little or no CytK and Nhe and was non-cytotoxic. Comparative sequence and expression studies indicated that neither the PlcR/PapR quorum sensing system, nor theYvrGH and YvfTU two-component systems, were responsible for the observed difference in toxin production. Additionally, phylogenetic analysis of 13 genes showed that NVH 391/98, NVH 883/00, and INRA AF2 comprise a novel cluster of strains genetically distant from other B. cereus group strains. Conclusion Due to its divergent sequence, the novel nhe operon had previously not been detected in NVH 391/98 using PCR and several monoclonal antibodies. Thus, toxigenic profiling based on the original nhe sequence will fail to detect the toxin in this group of strains. The observation that strain NVH 883/00 carries cytK-1 but is non-cytotoxic indicates that the detection of this gene

  15. A NEW STRAIN OF TRANSMISSIBLE LEUCEMIA IN FOWLS (STRAIN H).

    Science.gov (United States)

    Ellermann, V

    1921-03-31

    1. A new strain of fowl leucosis has been transmitted through twelve generations of fowls. 2. An increase in virulence was observed during its passage. This was shown in a shortening of the interval between inoculation and death. The increase in virulence does not affect the number of successful inoculations, which remains approximately constant in from 20 to 40 per cent of the birds employed. 3. As with former strains, the disease manifests itself in various forms; i.e., myeloid and intravascular lymphoid types. A single lymphatic case was observed. 4. In several intravascular cases a diminution in the hemolytic power of the serum was established. This phenomenon was absent in a number of myeloid cases. 5. Active immunization cannot be produced by means of the subcutaneous injection of virulent material. 6. The finding of previous experiments that the virus is filterable has been confirmed. 7. The inoculation of human leucemic material into fowls gave negative results.

  16. Natural plasmid transformation in a high-frequency-of transformation marine Vibrio strain

    International Nuclear Information System (INIS)

    Frischer, M.E.; Thurmond, J.M.; Paul, J.H.

    1990-01-01

    The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 x 10 -9 and 3.4 x 10 -7 transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42,857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 x 10 -8 to 1.3 x 10 -4 transformants per recipient with plasmid DNA and at an average frequency of 8.3 x 10 -5 transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [ 3 H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations

  17. Distinguishing highly-related outbreak-associated Clostridium botulinum type A(B) strains.

    Science.gov (United States)

    Raphael, Brian H; Shirey, Timothy B; Lúquez, Carolina; Maslanka, Susan E

    2014-07-16

    In the United States, most Clostridium botulinum type A strains isolated during laboratory investigations of human botulism demonstrate the presence of an expressed type A botulinum neurotoxin (BoNT/A) gene and an unexpressed BoNT/B gene. These strains are designated type A(B). The most common pulsed-field gel electrophoresis (PFGE) pattern in the C. botulinum PulseNet database is composed of A(B) strains. The purpose of this study was to evaluate the ability of genome sequencing and multi-loci variable number of tandem repeat analysis (MLVA) to differentiate such strains. The genome sequences of type A(B) strains evaluated in this study are closely related and cluster together compared to other available C. botulinum Group I genomes. In silico multilocus sequence typing (MLST) analysis (7-loci) was unable to differentiate any of the type A(B) strains isolated from seven different outbreak investigations evaluated in this study. A 15-locus MLVA scheme demonstrated an improved ability to differentiate these strains, however, repeat unit variation among the strains was restricted to only two loci. Reference-free single nucleotide polymorphism (SNP) analysis demonstrated the ability to differentiate strains from all of the outbreaks examined and a non-outbreak associated strain. This study confirms that type A(B) strains that share the same PFGE pattern also share closely-related genome sequences. The lack of a complete type A(B) strain representative genome sequence hinders the ability to assemble genomes by reference mapping and analysis of SNPs at pre-identified sites. However, compared to other methods evaluated in this study, a reference-free SNP analysis demonstrated optimal subtyping utility for type A(B) strains using de novo assembled genome sequences.

  18. The role of nitrogen uptake on the competition ability of three vineyard Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Vendramini, Chiara; Beltran, Gemma; Nadai, Chiara; Giacomini, Alessio; Mas, Albert; Corich, Viviana

    2017-10-03

    Three vineyard strains of Saccharomyces cerevisiae, P301.4, P304.4 and P254.12, were assayed in comparison with a commercial industrial strain, QA23. The aim was to understand if nitrogen availability could influence strain competition ability during must fermentation. Pairwise-strain fermentations and co-fermentations with the simultaneous presence of the four strains were performed in synthetic musts at two nitrogen levels: control nitrogen condition (CNC) that assured the suitable assimilable nitrogen amount required by the yeast strains to complete the fermentation and low nitrogen condition (LNC) where nitrogen is present at very low level. Results suggested a strong involvement of nitrogen availability, as the frequency in must of the vineyard strains, respect to QA23, in LNC was always higher than that found in CNC. Moreover, in CNC only strain P304.4 reached the same strain frequency as QA23. P304.4 competition ability increased during the fermentation, indicating better performance when nitrogen availability was dropping down. P301.4 was the only strain sensitive to QA23 killer toxin. In CNC, when it was co-inoculated with the industrial strain QA23, P301.4 was never detected. In LNC, P301.4 after 12h accounted for 10% of the total population. This percentage increased after 48h (20%). Single-strain fermentations were also run in both conditions and the nitrogen metabolism further analyzed. Fermentation kinetics, ammonium and amino-acid consumptions and the expression of genes under nitrogen catabolite repression evidenced that vineyard yeasts, and particularly strain P304.4, had higher nitrogen assimilation rate than the commercial control. In conclusion, the high nitrogen assimilation rate seems to be an additional strategy that allowed vineyard yeasts successful competition during the growth in grape musts. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Effect of starvation on survival and virulence expression of Aeromonas hydrophila from different sources.

    Science.gov (United States)

    Casabianca, Anna; Orlandi, Chiara; Barbieri, Federica; Sabatini, Luigia; Di Cesare, Andrea; Sisti, Davide; Pasquaroli, Sonia; Magnani, Mauro; Citterio, Barbara

    2015-04-01

    Aeromonas hydrophila is an aquatic bacterium responsible for several human illnesses. The aim of this work was to investigate the survival ability and virulence expression of two strains from different sources (fish, strain 87 and surface water, strain LS) maintained in a seawater microcosm. The strains were analyzed for the total and viable bacterial counts, adhesion ability to Hep-2 cells and aerA gene expression by qPCR throughout the experiment (35 days). Both strains reached a putative VBNC state and lost adhesive properties but exhibited a different behavior in the expression of aerA. This could be due to the different origin of the two strains; the former adapted to a habitat rich of nutrient and the latter already used to survive in a more hostile environment. Moreover, our results indicate that the quantitative determination of aerA mRNA can be a useful indicator of virulence expression under stress conditions.

  20. Differentiation of strains of varicella-zoster virus by changes in neutral lipid metabolism in infected cells.

    OpenAIRE

    Jerkofsky, M; De Siervo, A J

    1986-01-01

    Eleven isolates of varicella-zoster virus were tested for their effects on the incorporation of [14C]acetate into lipids in infected human embryonic lung cells. By relative percent, all virus isolates demonstrated a shift from polar lipid synthesis to neutral lipid, especially triglyceride, synthesis. By data expressed as counts per minute per microgram of protein, the VZV strains could be separated into two groups: those strains which depressed lipid synthesis and those strains which did not...

  1. Expression of recombinant Streptokinase from local Egyptian ...

    African Journals Online (AJOL)

    We reported for the first time the expression of a recombinant SK from a local Streptococcus strain. When produced on industrial scale this r-SK may substantially contribute to reducing the costs of thrombolytic therapy in developing countries. In this study, a highly purified r-SK from Streptococcus sp. isolated from Egyptian ...

  2. Multi-omics Quantification of Species Variation of Escherichia coli Links Molecular Features with Strain Phenotypes

    DEFF Research Database (Denmark)

    Monk, Jonathan M; Koza, Anna; Campodonico, Miguel A

    2016-01-01

    , metabolic engineering, and process design. Here, we quantified strain-specific differences in seven widely used strains of E. coli (BL21, C, Crooks, DH5a, K-12 MG1655, K-12 W3110, and W) using genomics, phenomics, transcriptomics, and genome-scale modeling. Metabolic physiology and gene expression varied...

  3. Monitoring the ethanol stress response of a sigM deletion strain of B. cereus ATCC 14579.

    NARCIS (Netherlands)

    Voort, van der M.

    2008-01-01

    Here, the role of σM and its regulon in stress response and survival of B. cereus ATCC 14579 was assessed by comparative transciptome and phenotypic analysis of this strain and its sigM deletion strain. Exposure of B. cereus ATCC 14579 to a wide range of stresses revealed expression of sigM,

  4. Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production

    DEFF Research Database (Denmark)

    Pedersen, K.; Gram, Lone; Austin, D.A.

    1997-01-01

    The virulence of 18 strains of Vibrio anguillarum serogroup 01 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only str...

  5. Beryllium strain under dynamic loading

    Directory of Open Access Journals (Sweden)

    Pushkov Victor

    2015-01-01

    Full Text Available There are some data (not much on dynamic characteristics of beryllium that are important, for example, when estimating construction performance at NPP emergencies. A number of data on stress-strain curves, spall strength, shear strength, fracture and structure responses of shock loaded beryllium have obtained in US and Russian laboratories. For today the model description of this complex metal behavior does not have a reasonable agreement with the experimental data, thus a wider spectrum of experimental data is required. This work presents data on dynamic compression-test diagrams of Russian beryllium. Experiments are performed using Hopkinson bar method (SHPB. Strain rates were ε ∼ 103 s−1.

  6. Fingerprints of a position-dependent Fermi velocity on scanning tunnelling spectra of strained graphene

    Science.gov (United States)

    Oliva-Leyva, M.; Barrios-Vargas, J. E.; Wang, Chumin

    2018-02-01

    Nonuniform strain in graphene induces a position dependence of the Fermi velocity, as recently demonstrated by scanning tunnelling spectroscopy experiments. In this work, we study the effects of a position-dependent Fermi velocity on the local density of states (LDOS) of strained graphene, with and without the presence of a uniform magnetic field. The variation of LDOS obtained from tight-binding calculations is successfully explained by analytical expressions derived within the Dirac approach. These expressions also rectify a rough Fermi velocity substitution used in the literature that neglects the strain-induced anisotropy. The reported analytical results could be useful for understanding the nonuniform strain effects on scanning tunnelling spectra of graphene, as well as when it is exposed to an external magnetic field.

  7. A virulent parent with probiotic progeny: comparative genomics of Escherichia coli strains CFT073, Nissle 1917 and ABU 83972

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk; Friis, Carsten; Hancock, Viktoria

    2010-01-01

    Escherichia coli is a highly versatile species encompassing a diverse spectrum of strains, i.e. from highly virulent isolates causing serious infectious diseases to commensals and probiotic strains. Although much is known about bacterial pathogenicity in E. coli, the understanding of which genetic...... determinants differentiates a virulent from an avirulent strain still remains limited. In this study we designed a new comparative genomic hybridization microarray based on 31 sequenced E. coli strains and used it to compare two E. coli strains used as prophylactic agents (i.e. Nissle 1917 and 83972...... to genotype and phenotype than 83972 (an asymptomatic bacteriuria strain). The study indicates that genetic variations (e.g. mutations) and expression differences, rather than genomic content per se, contribute to the divergence in disease-causing ability between these strains. This has implications...

  8. Transcriptional profiling of C57 and DBA strains of mice in the absence and presence of morphine

    Directory of Open Access Journals (Sweden)

    Golden Greg T

    2007-03-01

    Full Text Available Abstract Background The mouse C57BL/6 (C57 and DBA/2J (DBA inbred strains differ substantially in many aspects of their response to drugs of abuse. The development of microarray analyses represents a genome-wide method for measuring differences across strains, focusing on expression differences. In the current study, we carried out microarray analysis in C57 and DBA mice in the nucleus accumbens of drug-naïve and morphine-treated animals. Results We identified mRNAs with altered expression between the two strains. We validated the mRNA expression changes of several such mRNAs, including Gnb1, which has been observed to be regulated by several drugs of abuse. In addition, we validated alterations in the enzyme activity of one mRNA product, catechol-O-methyltransferase (Comt. Data mining of expression and behavioral data indicates that both Gnb1 and Comt expression correlate with aspects of drug response in C57/DBA recombinant inbred strains. Pathway analysis was carried out to identify pathways showing significant alterations as a result of treatment and/or due to strain differences. These analyses identified axon guidance genes, particularly the semaphorins, as showing altered expression in the presence of morphine, and plasticity genes as showing altered expression across strains. Pathway analysis of genes showing strain by treatment interaction suggest that the phosphatidylinositol signaling pathway may represent an important difference between the strains as related to morphine exposure. Conclusion mRNAs with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse. One such mRNA is Comt and we hypothesize that altered expression of Comt may represent a potential mechanism for regulating the effect of, and response to, multiple substances of abuse. Similarly, a role for Gnb1 in responses to multiple drugs of abuse is supported by expression data from our study and from other

  9. Hyper-inducible expression system for streptomycetes

    Science.gov (United States)

    Herai, Sachio; Hashimoto, Yoshiteru; Higashibata, Hiroki; Maseda, Hideaki; Ikeda, Haruo; Ōmura, Satoshi; Kobayashi, Michihiko

    2004-01-01

    Streptomycetes produce useful enzymes and a wide variety of secondary metabolites with potent biological activities (e.g., antibiotics, immunosuppressors, pesticides, etc.). Despite their importance in the pharmaceutical and agrochemical fields, there have been no reports for practical expression systems in streptomycetes. Here, we developed a “PnitA-NitR” system for regulatory gene expression in streptomycetes based on the expression mechanism of Rhodococcus rhodochrous J1 nitrilase, which is highly induced by an inexpensive and safe inducer, ε-caprolactam. Heterologous protein expression experiments demonstrated that the system allowed suppressed basal expression and hyper-inducible expression, yielding target protein levels of as high as ≈40% of all soluble protein. Furthermore, the system functioned in important streptomycete strains. Thus, the PnitA-NitR system should be a powerful tool for improving the productivity of various useful products in streptomycetes. PMID:15377796

  10. Production of d-lactate using a pyruvate-producing Escherichia coli strain.

    Science.gov (United States)

    Akita, Hironaga; Nakashima, Nobutaka; Hoshino, Tamotsu

    2017-07-01

    To generate an organism capable of producing d-lactate, NAD + -dependent d-lactate dehydrogenase was expressed in our pyruvate-producing strain, Escherichia coli strain LAFCPCPt-accBC-aceE. After determining the optimal culture conditions for d-lactate production, 18.4 mM d-lactate was produced from biomass-based medium without supplemental mineral or nitrogen sources. Our results show that d-lactate can be produced in simple batch fermentation processes.

  11. Improving sensitivity of the polyurethane/CNT laminate strain sensor by controlled mechanical preload

    International Nuclear Information System (INIS)

    Slobodian, Petr; Olejnik, Robert; Matyas, Jiri; Babar, Dipak Gorakh

    2016-01-01

    This article describes strain detection potential of polyurethane/CNT layered composite and further possible enhance of its sensitivity to strain, expressed by value of gauge factor, GF, employing its controlled mechanical preload. In course of its fabrication a non-woven polyurethane membrane made by electro spinning was used as filtering membrane for CNT aqueous dispersion. Final CNT polyurethane laminate composite is prepared by compression molding. Produced polyurethane/CNT composite laminate is electrically conductive and high elastic. Its elongation leads to change of its macroscopic electrical resistance. Changes in resistance are further reversible, reproducible and can monitor deformation in real time. Gauge factor reaches very high values around 8 for strain reaching 3.5% comparing with conventional metallic strain gauges. Finally, controlled mechanical preload significantly increases value of GF. For example for value of 8.1% of preload value of GF reaches 23.3 for strain 3.5%. (paper)

  12. Improving sensitivity of the polyurethane/CNT laminate strain sensor by controlled mechanical preload

    Science.gov (United States)

    Slobodian, Petr; Olejnik, Robert; Matyas, Jiri; Gorakh Babar, Dipak

    2016-03-01

    This article describes strain detection potential of polyurethane/CNT layered composite and further possible enhance of its sensitivity to strain, expressed by value of gauge factor, GF, employing its controlled mechanical preload. In course of its fabrication a non-woven polyurethane membrane made by electro spinning was used as filtering membrane for CNT aqueous dispersion. Final CNT polyurethane laminate composite is prepared by compression molding. Produced polyurethane/CNT composite laminate is electrically conductive and high elastic. Its elongation leads to change of its macroscopic electrical resistance. Changes in resistance are further reversible, reproducible and can monitor deformation in real time. Gauge factor reaches very high values around 8 for strain reaching 3.5% comparing with conventional metallic strain gauges. Finally, controlled mechanical preload significantly increases value of GF. For example for value of 8.1% of preload value of GF reaches 23.3 for strain 3.5%.

  13. Development of Industrial Yeast Platform Strains

    DEFF Research Database (Denmark)

    Bergdahl, Basti; Dato, Laura; Förster, Jochen

    2014-01-01

    Most of the current metabolic engineering projects are carried out using laboratory strains as the starting host. Although such strains are easily manipulated genetically, their robustness does not always meet the requirements set by industrial fermentation conditions. In such conditions, the cel...... screening of the 36 industrial and laboratory yeast strains. In addition, progress in the development of molecular biology methods for generating the new strains will be presented....

  14. Genetic diversity of FLO1 and FLO5 genes in wine flocculent Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Tofalo, Rosanna; Perpetuini, Giorgia; Di Gianvito, Paola; Schirone, Maria; Corsetti, Aldo; Suzzi, Giovanna

    2014-11-17

    Twenty-eight flocculent wine strains were tested for adhesion and flocculation phenotypic variability. Moreover, the ex