Functional significance of the intermediate conductance Ca2+-activated K+ channel for the short-term survival of injured erythrocytes.

PubMed

Föller, Michael; Bobbala, Diwakar; Koka, Saisudha; Boini, Krishna M; Mahmud, Hasan; Kasinathan, Ravi S; Shumilina, Ekaterina; Amann, Kerstin; Beranek, Golo; Sausbier, Ulrike; Ruth, Peter; Sausbier, Matthias; Lang, Florian; Huber, Stephan M

2010-11-01

Increased cytosolic Ca(2+) concentrations activate Gardos K(+) channels in human erythrocytes with membrane hyperpolarization, efflux of K(+), Cl⁻, and osmotically obliged H₂O resulting in cell shrinkage, a phenomenon referred to as Gardos effect. We tested whether the Gardos effect delays colloid osmotic hemolysis of injured erythrocytes from mice lacking the Ca(2+)-activated K(+) channel K(Ca)3.1. To this end, we applied patch clamp and flow cytometry and determined in vitro as well as in vivo hemolysis. As a result, erythrocytes from K(Ca)3.1-deficient (K(Ca)3.1(-/-)) mice lacked Gardos channel activity and the Gardos effect. Blood parameters, reticulocyte count, or osmotic erythrocyte resistance, however, did not differ between K(Ca)3.1(-/-) mice and their wild-type littermates, suggesting low or absent Gardos channel activity in unstressed erythrocytes. Oxidative stress-induced Ca(2+) entry and phospholipid scrambling were significantly less pronounced in K(Ca)3.1(-/-) than in wild-type erythrocytes. Moreover, in vitro treatment with α-toxin from Staphylococcus aureus, which forms pores in the cellular membrane, resulted in significantly stronger hemolysis of K(Ca)3.1(-/-) than of wild-type erythrocytes. Intravenous injection of α-toxin induced more profound hemolysis in K(Ca)3.1(-/-) than in wild-type mice. Similarly, intra-peritoneal application of the redox-active substance phenylhydrazine, an agent for the induction of hemolytic anemia, was followed by a significantly stronger decrease of hematocrit in K(Ca)3.1(-/-) than in wild-type mice. Finally, malaria infection triggered the activation of K(Ca)3.1 and transient shrinkage of the infected erythrocytes. In conclusion, K(Ca)3.1 channel activity and Gardos effect counteract hemolysis of injured erythrocytes, thus decreasing hemoglobin release into circulating blood.

Synergy against drug-resistant HIV-1 with the microbicide antiretrovirals, dapivirine and tenofovir, in combination.

PubMed

Schader, Susan M; Colby-Germinario, Susan P; Schachter, Jordana R; Xu, Hongtao; Wainberg, Mark A

2011-08-24

To evaluate the candidate antiretroviral microbicide compounds, dapivirine (DAP) and tenofovir (TFV), alone and in combination against the transmission of wild-type and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 from different subtypes. We determined single-drug efficacy of the RTIs, DAP and TFV, against subtype B and non-B wild-type and NNRTI-resistant HIV-1 in vitro. To assess breadth of activity, compounds were tested alone and in combination against wild-type and NNRTI-resistant subtype C primary HIV-1 isolates and complimentary clonal HIV-1 from subtypes B, C and CRF02_AG to control for viral variation. Early infection was quantified by counting light units emitted from TZM-bl cells less than 48-h postinfection. Combination ratios were based on drug inhibitory concentrations (IC(50)s) and combined effects were determined by calculating combination indices. Both candidate microbicide antiretrovirals demonstrated potent anti-NNRTI-resistant HIV-1 activity in vitro, albeit the combination protected better than the single-drug treatments. Of particular interest, the DAP with TFV combination exhibited synergy (50% combination index, CI(50) = 0.567) against subtype C NNRTI-resistant HIV-1, whereas additivity (CI(50) = 0.987) was observed against the wild-type counterpart from the same patient. The effect was not compounded by the presence of subdominant viral fractions, as experiments using complimentary clonal subtype C wild-type (CI(50) = 0.968) and NNRTI-resistant (CI(50) = 0.672) HIV-1, in lieu of the patient quasispecies, gave similar results. This study supports the notion that antiretroviral drug combinations may retain antiviral activity against some drug-resistant HIV-1 despite subtype classification and quasispecies diversity.

Control of peroxisome proliferator-activated receptor gamma2 stability and activity by SUMOylation.

PubMed

Floyd, Z Elizabeth; Stephens, Jacqueline M

2004-06-01

To determine whether small ubiquitin-related modifier (SUMO)ylation of lysine 107 plays a role in regulating the activity of peroxisome proliferator-activated receptor gamma (PPARgamma). Transient expression of wild-type and K107R-PPARgamma2 in the NIH 3T3 fibroblast cell line was carried out in conjunction with half-life studies, luciferase activity assays, and indirect immunofluorescence localization studies. Additional in vitro analysis was carried out using recombinant SUMOylation pathway proteins along with in vitro transcribed and translated wild-type or K107R-PPARgamma2 to examine the SUMO-1 modification state of wild-type and SUMO-deficient K107R-PPARgamma2. While examining PPARgamma2 for potential ubiquitylation sites, we identified a strong consensus site for SUMO modification that contains lysine 107. In vitro, SUMOylation studies showed that lysine 107 of PPARgamma2 is a major SUMOylation site and that at least one other SUMOylation site is present in PPARgamma. In addition, our results demonstrated that SUMO-1 affects PPARgamma stability and transcriptional activity but not the nuclear localization of PPARgamma. These results indicated that SUMOylation plays a role in regulating PPARgamma, both indirectly and directly by modification of lysine 107. Because PPARgamma is regulated in numerous animal models of obesity, understanding the covalent modifications of PPARgamma may enhance our understanding of the metabolic syndrome.

Mycobacterium tuberculosis embB codon 306 mutations confer moderately increased resistance to ethambutol in vitro and in vivo.

PubMed

Plinke, Claudia; Walter, Kerstin; Aly, Sahar; Ehlers, Stefan; Niemann, Stefan

2011-06-01

Ethambutol (EMB) is a major component of the first-line therapy of tuberculosis. Mutations in codon 306 of embB (embB306) were suggested as a major resistance mechanism in clinical isolates. To directly analyze the impact of individual embB306 mutations on EMB resistance, we used allelic exchange experiments to generate embB306 mutants of M. tuberculosis H37Rv. The level of EMB resistance conferred by particular mutations was measured in vitro and in vivo after EMB therapy by daily gavage in a mouse model of aerogenic tuberculosis. The wild-type embB306 ATG codon was replaced by embB306 ATC, ATA, or GTG, respectively. All of the obtained embB306 mutants exhibited a 2- to 4-fold increase in EMB MIC compared to the wild-type H37Rv. In vivo, the one selected embB306 GTG mutant required a higher dose of ethambutol to restrict its growth in the lung compared to wild-type H37Rv. These experiments demonstrate that embB306 point mutations enhance the EMB MIC in vitro to a moderate, but significant extent, and reduce the efficacy of EMB treatment in the animal model. We propose that conventional EMB susceptibility testing, in combination with embB306 genotyping, may guide dose adjustment to avoid clinical treatment failure in these low-level resistant strains.

In Vitro Transcripts of Wild-Type and Fluorescent Protein-Tagged Triticum mosaic virus (Family Potyviridae) are Biologically Active in Wheat.

PubMed

Tatineni, Satyanarayana; McMechan, Anthony J; Bartels, Melissa; Hein, Gary L; Graybosch, Robert A

2015-11-01

Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat. Wheat seedlings inoculated with in vitro transcripts elicited mosaic and mottling symptoms similar to the wild-type virus, and the progeny virus was efficiently transmitted by wheat curl mites, indicating that the cloned virus retained pathogenicity, movement, and wheat curl mite transmission characteristics. A series of TriMV-based expression vectors was constructed by engineering a green fluorescent protein (GFP) or red fluorescent protein (RFP) open reading frame with homologous NIa-Pro cleavage peptides between the P1 and HC-Pro cistrons. We found that GFP-tagged TriMV with seven or nine amino acid cleavage peptides efficiently processed GFP from HC-Pro. TriMV-GFP vectors were stable in wheat for more than 120 days and for six serial passages at 14-day intervals by mechanical inoculation and were transmitted by wheat curl mites similarly to the wild-type virus. Fluorescent protein-tagged TriMV was observed in wheat leaves, stems, and crowns. The availability of fluorescent protein-tagged TriMV will facilitate the examination of virus movement and distribution in cereal hosts and the mechanisms of cross protection and synergistic interactions between TriMV and Wheat streak mosaic virus.

In vitro re-expression of the aryl hydrocarbon receptor (Ahr) in cultured Ahr-deficient mouse antral follicles partially restores the phenotype to that of cultured wild-type mouse follicles.

PubMed

Ziv-Gal, A; Gao, L; Karman, B N; Flaws, J A

2015-03-01

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of various endocrine disrupting chemicals. In female mice, global deletion of the Ahr (AhrKO) results in slow growth of ovarian antral follicles. No studies, however, have examined whether injection of the Ahr restores the phenotypes of cultured AhrKO ovarian antral follicles to wild-type levels. We developed a system to construct a recombinant adenovirus containing the Ahr to re-express the Ahr in AhrKO granulosa cells and whole antral follicles. We then compared follicle growth and levels of factors in the AHR signaling pathway (Ahr, Ahrr, Cyp1a1, and Cyp1b1) in wild-type, AhrKO, and Ahr re-expressed follicles. Further, we compared the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild-type, AhrKO, and Ahr re-expressed follicles. Ahr injection into AhrKO follicles partially restored their growth pattern to wild-type levels. Further, Ahr re-expressed follicles had significantly higher levels of Ahr, Ahrr, Cyp1a1, and Cyp1b1 compared to wild-type follicles. Upon TCDD treatment, only Cyp1a1 levels were significantly higher in Ahr re-expressed follicles compared to the levels in wild-type follicles. Our system of re-expression of the Ahr partially restores follicle growth and transcript levels of factors in the AHR signaling pathway to wild-type levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

Retinal ganglion cell responses to voltage and current stimulation in wild-type and rd1 mouse retinas

NASA Astrophysics Data System (ADS)

Goo, Yong Sook; Ye, Jang Hee; Lee, Seokyoung; Nam, Yoonkey; Ryu, Sang Baek; Kim, Kyung Hwan

2011-06-01

Retinal prostheses are being developed to restore vision for those with retinal diseases such as retinitis pigmentosa or age-related macular degeneration. Since neural prostheses depend upon electrical stimulation to control neural activity, optimal stimulation parameters for successful encoding of visual information are one of the most important requirements to enable visual perception. In this paper, we focused on retinal ganglion cell (RGC) responses to different stimulation parameters and compared threshold charge densities in wild-type and rd1 mice. For this purpose, we used in vitro retinal preparations of wild-type and rd1 mice. When the neural network was stimulated with voltage- and current-controlled pulses, RGCs from both wild-type and rd1 mice responded; however the temporal pattern of RGC response is very different. In wild-type RGCs, a single peak within 100 ms appears, while multiple peaks (approximately four peaks) with ~10 Hz rhythm within 400 ms appear in RGCs in the degenerated retina of rd1 mice. We find that an anodic phase-first biphasic voltage-controlled pulse is more efficient for stimulation than a biphasic current-controlled pulse based on lower threshold charge density. The threshold charge densities for activation of RGCs both with voltage- and current-controlled pulses are overall more elevated for the rd1 mouse than the wild-type mouse. Here, we propose the stimulus range for wild-type and rd1 retinas when the optimal modulation of a RGC response is possible.

In vitro transcripts of wild-type and fluorescent protein-tagged triticum mosaic virus (family potyviridae) are biologically active in wheat

USDA-ARS?s Scientific Manuscript database

Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat, and the progeny virus was efficiently transmitted by wheat curl m...

A defect in the inflammation-primed macrophage-activation cascade in osteopetrotic rats.

PubMed

Yamamoto, N; Lindsay, D D; Naraparaju, V R; Ireland, R A; Popoff, S N

1994-05-15

Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages.

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

PubMed

Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

2013-12-01

The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA. © 2013.

Viral evolution in response to the broad-based retroviral protease inhibitor TL-3.

PubMed

Bühler, B; Lin, Y C; Morris, G; Olson, A J; Wong, C H; Richman, D D; Elder, J H; Torbett, B E

2001-10-01

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

Viral Evolution in Response to the Broad-Based Retroviral Protease Inhibitor TL-3†

PubMed Central

Bühler, Bernd; Lin, Ying-Chuan; Morris, Garrett; Olson, Arthur J.; Wong, Chi-Huey; Richman, Douglas D.; Elder, John H.; Torbett, Bruce E.

2001-01-01

TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity. PMID:11533212

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

PubMed

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

1993-08-05

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

Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

PubMed Central

Yu, Xia; Wang, Guirong; Chen, Suting; Wei, Guomei; Shang, Yuanyuan; Dong, Lingling; Schön, Thomas; Moradigaravand, Danesh; Peacock, Sharon J.

2016-01-01

Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective. PMID:27324769

Muskmelon embryo rescue techniques using in vitro embryo culture.

PubMed

Nuñez-Palenius, Hector Gordon; Ramírez-Malagón, Rafael; Ochoa-Alejo, Neftalí

2011-01-01

Among the major cucurbit vegetables, melon (Cucumis melo) has one of the greatest polymorphic fruit types and botanical varieties. Some melon fruits have excellent aroma, variety of flesh colors, deeper flavor, and more juice compared to other cucurbits. Despite numerous available melon cultivars, some of them are exceedingly susceptible to several diseases. The genetic background carrying the genes for tolerance and/or resistance for those diseases is found in wild melon landraces. Unfortunately, the commercial melon varieties are not able to produce viable hybrids when crossed with their wild melon counterparts. Plant tissue culture techniques are needed to surpass those genetic barriers. In vitro melon embryo rescue has played a main role to obtain viable hybrids originated from commercial versus wild melon crosses. In this chapter, an efficient and simple embryo rescue melon protocol is thoroughly described.

Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

NASA Technical Reports Server (NTRS)

Tsang, Joyce; Joyce, Gerald F.

1996-01-01

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

Myostatin Attenuation In Vivo Reduces Adiposity, but Activates Adipogenesis

PubMed Central

Li, Naisi; Yang, Qiyuan; Walker, Ryan G.; Thompson, Thomas B.; Du, Min

2016-01-01

A potentially novel approach for treating obesity includes attenuating myostatin as this increases muscle mass and decreases fat mass. Notwithstanding, conflicting studies report that myostatin stimulates or inhibits adipogenesis and it is unknown whether reduced adiposity with myostatin attenuation results from changes in fat deposition or adipogenesis. We therefore quantified changes in the stem, transit amplifying and progenitor cell pool in white adipose tissue (WAT) and brown adipose tissue (BAT) using label-retaining wild-type and mstn−/− (Jekyll) mice. Muscle mass was larger in Jekyll mice, WAT and BAT mass was smaller and label induction was equal in all tissues from both wild-type and Jekyll mice. The number of label-retaining cells, however, dissipated quicker in WAT and BAT of Jekyll mice and was only 25% and 17%, respectively, of wild-type cell counts 1 month after induction. Adipose cell density was significantly higher in Jekyll mice and increased over time concomitant with label-retaining cell disappearance, which is consistent with enhanced expansion and differentiation of the stem, transit amplifying and progenitor pool. Stromal vascular cells from Jekyll WAT and BAT differentiated into mature adipocytes at a faster rate than wild-type cells and although Jekyll WAT cells also proliferated quicker in vitro, those from BAT did not. Differentiation marker expression in vitro, however, suggests that mstn−/− BAT preadipocytes are far more sensitive to the suppressive effects of myostatin. These results suggest that myostatin attenuation stimulates adipogenesis in vivo and that the reduced adiposity in mstn−/− animals results from nutrient partitioning away from fat and in support of muscle. PMID:26580671

Myostatin Attenuation In Vivo Reduces Adiposity, but Activates Adipogenesis.

PubMed

Li, Naisi; Yang, Qiyuan; Walker, Ryan G; Thompson, Thomas B; Du, Min; Rodgers, Buel D

2016-01-01

A potentially novel approach for treating obesity includes attenuating myostatin as this increases muscle mass and decreases fat mass. Notwithstanding, conflicting studies report that myostatin stimulates or inhibits adipogenesis and it is unknown whether reduced adiposity with myostatin attenuation results from changes in fat deposition or adipogenesis. We therefore quantified changes in the stem, transit amplifying and progenitor cell pool in white adipose tissue (WAT) and brown adipose tissue (BAT) using label-retaining wild-type and mstn(-/-) (Jekyll) mice. Muscle mass was larger in Jekyll mice, WAT and BAT mass was smaller and label induction was equal in all tissues from both wild-type and Jekyll mice. The number of label-retaining cells, however, dissipated quicker in WAT and BAT of Jekyll mice and was only 25% and 17%, respectively, of wild-type cell counts 1 month after induction. Adipose cell density was significantly higher in Jekyll mice and increased over time concomitant with label-retaining cell disappearance, which is consistent with enhanced expansion and differentiation of the stem, transit amplifying and progenitor pool. Stromal vascular cells from Jekyll WAT and BAT differentiated into mature adipocytes at a faster rate than wild-type cells and although Jekyll WAT cells also proliferated quicker in vitro, those from BAT did not. Differentiation marker expression in vitro, however, suggests that mstn(-/-) BAT preadipocytes are far more sensitive to the suppressive effects of myostatin. These results suggest that myostatin attenuation stimulates adipogenesis in vivo and that the reduced adiposity in mstn(-/-) animals results from nutrient partitioning away from fat and in support of muscle.

PECTIN METHYLESTERASE48 Is Involved in Arabidopsis Pollen Grain Germination1[OPEN

PubMed Central

Leroux, Christelle; Bouton, Sophie; Kiefer-Meyer, Marie-Christine; Fabrice, Tohnyui Ndinyanka; Mareck, Alain; Guénin, Stéphanie; Fournet, Françoise; Ringli, Christoph; Pelloux, Jérôme; Driouich, Azeddine; Lerouge, Patrice; Lehner, Arnaud; Mollet, Jean-Claude

2015-01-01

Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme48−/− pollen grains. In contrast, the PME activity was lower in pme48−/−, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48−/− with the optimum germination medium supplemented with 2.5 mm calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca2+ necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination. PMID:25524442

Curcumin Affects Phase II Disposition of Resveratrol Through Inhibiting Efflux Transporters MRP2 and BCRP

PubMed Central

Ge, Shufan; Yin, Taijun; Xu, Beibei; Gao, Song; Hu, Ming

2015-01-01

Purpose To evaluate the impact of curcumin on the disposition of resveratrol phase II metabolites in vivo, and explain the observations by performing in vitro studies in transporter-overexpressed cells. Methods Pharmacokinetic studies of resveratrol with and without the co-administration of curcumin were performed in both FVB wild-type and Bcrp1 (−/−) mice. Human UGT1A9-overexpressing HeLa cells and human MRP2-overexpressing MDCK II-UGT1A1 cells were used as in vitro tools to further determine the impact of curcumin as a transporter inhibitor on resveratrol metabolites. Results We observed higher exposure of resveratrol conjugates in Bcrp1 (−/−) mice compared to wild-type mice. In wild-type mice, curcumin increased the AUC of resveratrol glucuronide by 4-fold compared to the mice treated without curcumin. The plasma levels of resveratrol and its sulfate conjugate also increased moderately. In Bcrp1 (−/−) mice, there was a further increase (6-fold increase) in AUC of resveratrol glucuronide observed when curcumin was co-administered compared to AUC values obtained in wild-type mice without curcumin treatment. In the presence of 50nM curcumin, the clearance of resveratrol-3-O-glucuronide and resveratrol-3-O-sulfate reduced in both MRP2-overexpressing MDCKII-UGT1A1 cells and Human UGT1A9-overexpressing HeLa cells. Conclusions These results suggest that curcumin alters the phase II distribution of resveratrol through inhibiting efflux transporters including MRP2 and BCRP. PMID:26502886

Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon.

PubMed

Akashi, Kinya; Nishimura, Noriyuki; Ishida, Yoshinori; Yokota, Akiho

2004-10-08

Wild watermelon (Citrullus lanatus sp.) has the ability to tolerate severe drought/high light stress conditions despite carrying out normal C3-type photosynthesis. Here, mRNA differential display was employed to isolate drought-responsive genes in the leaves of wild watermelon. One of the isolated genes, CLMT2, shared significant homology with type-2 metallothionein (MT) sequences from other plants. The second-order rate constant for the reaction between a recombinant CLMT2 protein and hydroxyl radicals was estimated to be 1.2 x 10(11) M(-1) s(-1), demonstrating that CLMT2 had an extraordinary high activity for detoxifying hydroxyl radicals. Moreover, hydroxyl radical-catalyzed degradation of watermelon genomic DNA was effectively suppressed by CLMT2 in vitro. This is the first demonstration of a plant MT with antioxidant properties. The results suggest that CLMT2 induction contributes to the survival of wild watermelon under severe drought/high light stress conditions. Copyright 2004 Elsevier Inc.

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

PubMed

Stephen, Abish S; Millhouse, Emma; Sherry, Leighann; Aduse-Opoku, Joseph; Culshaw, Shauna; Ramage, Gordon; Bradshaw, David J; Burnett, Gary R; Allaker, Robert P

2016-01-01

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.

The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice

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

Ogawa, Eiichi; Hosokawa, Masaya; Faculty of Human Sciences, Tezukayama Gakuin University, Osaka

2011-01-07

Research highlights: {yields} Exogenous GIP inhibits intestinal motility through a somatostatin-mediated pathway. {yields} Exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility. {yields} The GIP-receptor-mediated action in intestine does not involve in GLP-1-mediated pathway. -- Abstract: Gastric inhibitory polypeptide (GIP) is released from the small intestine upon meal ingestion and increases insulin secretion from pancreatic {beta} cells. Although the GIP receptor is known to be expressed in small intestine, the effects of GIP in small intestine are not fully understood. This study was designed to clarify the effect of GIP on intestinal glucose absorption and intestinal motility. Intestinal glucosemore » absorption in vivo was measured by single-pass perfusion method. Incorporation of [{sup 14}C]-glucose into everted jejunal rings in vitro was used to evaluate the effect of GIP on sodium-glucose co-transporter (SGLT). Motility of small intestine was measured by intestinal transit after oral administration of a non-absorbed marker. Intraperitoneal administration of GIP inhibited glucose absorption in wild-type mice in a concentration-dependent manner, showing maximum decrease at the dosage of 50 nmol/kg body weight. In glucagon-like-peptide-1 (GLP-1) receptor-deficient mice, GIP inhibited glucose absorption as in wild-type mice. In vitro examination of [{sup 14}C]-glucose uptake revealed that 100 nM GIP did not change SGLT-dependent glucose uptake in wild-type mice. After intraperitoneal administration of GIP (50 nmol/kg body weight), small intestinal transit was inhibited to 40% in both wild-type and GLP-1 receptor-deficient mice. Furthermore, a somatostatin receptor antagonist, cyclosomatostatin, reduced the inhibitory effect of GIP on both intestinal transit and glucose absorption in wild-type mice. These results demonstrate that exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility through a somatostatin-mediated pathway rather than through a GLP-1-mediated pathway.« less

Revealing the drug-resistant mechanism for diarylpyrimidine analogue inhibitors of HIV-1 reverse transcriptase.

PubMed

Zhang, Hao; Qin, Fang; Ye, Wei; Li, Zeng; Ma, Songyao; Xia, Yan; Jiang, Yi; Zhu, Jiayi; Li, Yixue; Zhang, Jian; Chen, Hai-Feng

2011-09-01

Diaryltriazine (DATA) and diarylpyrimidine (DAPY) were two category inhibitors with highly potent activity for wild type (wt) and four principal mutant types (L100I, K103N, Y181C and Y188L) of HIV-1 reverse transcriptase (RT). We had revealed the drug-resistant mechanism of DATA analogue inhibitors with molecular dynamics simulation and three-dimensional quantitative structure-activity relationship (3D-QSAR) methods. In this work, we investigated the drug-resistant mechanism of DAPY analogue inhibitors. It was found that DAPY analogue inhibitors form more hydrogen bonds and hydrophobic contacts with wild type and mutants of HIV-1 RT than DATA inhibitors. This could explain that DAPY analogue inhibitors are more potent than DATA for the wild type and mutants of HIV-1 RT. Then, 3D-QSAR models were constructed for these inhibitors of wild type and four principal mutant types HIV-1 RT and evaluated by test set compounds. These combined models can be used to design new chemical entities and make quantitative prediction of the bioactivities for HIV-1 RT inhibitors before resorting to in vitro and in vivo experiment. © 2011 John Wiley & Sons A/S.

In Vitro Pharmacodynamics of AZD5206 against Staphylococcus aureus

PubMed Central

Chang, Kai-Tai; Yang, Zhen; Newman, Joseph; Hu, Ming

2013-01-01

AZD5206 is a novel antimicrobial agent with potent in vitro activity against Staphylococcus aureus. We evaluated the in vitro pharmacodynamics of AZD5206 against a standard wild-type methicillin-susceptible strain (ATCC 29213) and a clinical strain of methicillin-resistant S. aureus (SA62). Overall, bacterial killing against a low baseline inoculum was more remarkable. Low dosing exposures and/or high baseline inoculum resulted in early reduction in bacterial burden, followed by regrowth and selective amplification of the resistant population. PMID:23229481

Comparison of human and monkey cells for the ability to attenuate transcripts that begin at the adenovirus major late promoter

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

Seiberg, M.; Aloni, Y.; Levine, A.J.

1989-09-01

Late transcription from the adenovirus major late promoter can terminate prematurely at a site 182 to 188 nucleotides downstream. Experiments have been designed, with run-on transcription in nuclei in vitro or riboprobe protection of RNA obtained both in vivo and in vitro, that demonstrate that the ratio of attenuator RNA to readthrough RNA is greater in monkey cells (CV-1) than in human cells (HeLa). This may explain, in part, why the human adenoviruses replicate more poorly in CV-1 cells than in HeLa cells. A mutant adenovirus that replicates better than wild-type virus in monkey cells produces less of the attenuatormore » RNA than wild-type adenovirus does in monkey cells. Monkey cell extracts have been shown to contain a factor that, when added to human cell extracts transcribing adenovirus DNA in vitro, increases the production of attenuator RNA in these reactions. These observations help to explain a portion of the block to the production of infectious adenoviruses in monkey cells.« less

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor.

PubMed

Radke, Lars; Sandig, Grit; Lubitz, Annika; Schließer, Ulrike; von Horsten, Hans Henning; Blanchard, Veronique; Keil, Karolin; Sandig, Volker; Giese, Christoph; Hummel, Michael; Hinderlich, Stephan; Frohme, Marcus

2017-08-15

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN ® ). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.

Non-unity molecular heritability demonstrated by continuous evolution in vitro

NASA Technical Reports Server (NTRS)

Schmitt, T.; Lehman, N.

1999-01-01

INTRODUCTION: When catalytic RNA is evolved in vitro, the molecule's chemical reactivity is usually the desired selection target. Sometimes the phenotype of a particular RNA molecule cannot be unambiguously determined from its genotype, however. This can occur if a nucleotide sequence can adopt multiple folded states, an example of non-unity heritability (i.e. one genotype gives rise to more than one phenotype). In these cases, more rounds of selection are required to achieve a phenotypic shift. We tested the influence of non-unity heritability at the molecular level by selecting for variants of a ligase ribozyme via continuous evolution. RESULTS: During 20 bursts of continuous evolution of a 152-nucleotide ligase ribozyme in which the Mg2+ concentration was periodically lowered, a nine-error variant of the starting 'wild-type' molecule became dominant in the last eight bursts. This variant appears to be more active than the wild type. Kinetic analyses of the mutant suggest that it may not possess a higher first-order catalytic rate constant, however. Examination of the multiple RNA conformations present under the continuous evolution conditions suggests that the mutant is superior to the wild type because it is less likely to misfold into inactive conformers. CONCLUSIONS: The evolution of genotypes that are more likely to exhibit a particular phenotype is an epiphenomenon usually ascribed only to complex living systems. We show that this can occur at the molecular level, demonstrating that in vitro systems may have more life-like characteristics than previously thought, and providing additional support for an RNA world.

The LuxS/AI-2 Quorum-Sensing System of Streptococcus pneumoniae Is Required to Cause Disease, and to Regulate Virulence- and Metabolism-Related Genes in a Rat Model of Middle Ear Infection.

PubMed

Yadav, Mukesh K; Vidal, Jorge E; Go, Yoon Y; Kim, Shin H; Chae, Sung-Won; Song, Jae-Jun

2018-01-01

Objective: Streptococcus pneumoniae colonizes the nasopharynx of children, and from nasopharynx it could migrate to the middle ear and causes acute otitis media (AOM). During colonization and AOM, the pneumococcus forms biofilms. In vitro biofilm formation requires a functional LuxS/AI-2 quorum-sensing system. We investigated the role of LuxS/AI-2 signaling in pneumococcal middle ear infection, and identified the genes that are regulated by LuxS/AI-2 during pneumococcal biofilm formation. Methods: Streptococcus pneumoniae D39 wild-type and an isogenic D39Δ luxS strain were utilized to evaluate in vitro biofilm formation, and in vivo colonization and epithelial damage using a microtiter plate assay and a rat model of pneumococcal middle ear infection, respectively. Biofilm structures and colonization and epithelial damage were evaluated at the ultrastructural level by scanning electron microscopy and confocal microscopy. Microarrays were used to investigate the global genes that were regulated by LuxS/AI-2 during biofilm formation. Results: The biofilm biomass and density of D39Δ luxS were significantly ( p < 0.05) lower than those of D39 wild-type. SEM and confocal microscopy revealed that D39Δ luxS formed thin biofilms in vitro compared with D39 wild-type. The in vivo model of middle ear infection showed that D39Δ luxS resulted in ~60% less ( p < 0.05) bacterial colonization than the wild-type. SEM analysis of the rat middle ears revealed dense biofilm-like cell debris deposited on the cilia in wild-type D39-infected rats. However, little cell debris was deposited in the middle ears of the D39Δ luxS -inoculated rats, and the cilia were visible. cDNA-microarray analysis revealed 117 differentially expressed genes in D39Δ luxS compared with D39 wild-type. Among the 66 genes encoding putative proteins and previously characterized proteins, 60 were significantly downregulated, whereas 6 were upregulated. Functional annotation revealed that genes involved in DNA replication and repair, ATP synthesis, capsule biosynthesis, cell division, the cell cycle, signal transduction, transcription regulation, competence, virulence, and carbohydrate metabolism were downregulated in the absence of LuxS/AI-2. Conclusion: The S. pneumoniae LuxS/AI-2 quorum-sensing system is necessary for biofilm formation and the colonization of the ear epithelium, and caused middle ear infection in the rat model. LuxS/AI-2 regulates the expression of the genes involved in virulence and bacterial fitness during pneumococcal biofilm formation.

In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer

PubMed Central

Yasuda, Hiroyuki; Hamamoto, Junko; Oashi, Ayano; Ishioka, Kota; Arai, Daisuke; Nukaga, Shigenari; Miyawaki, Masayoshi; Kawada, Ichiro; Naoki, Katsuhiko; Costa, Daniel B.; Kobayashi, Susumu S.; Betsuyaku, Tomoko; Soejima, Kenzo

2015-01-01

EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the “therapeutic window” of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations. PMID:26515464

The Pea light-independent photomorphogenesis1 Mutant Results from Partial Duplication of COP1 Generating an Internal Promoter and Producing Two Distinct Transcripts

PubMed Central

Sullivan, James A.; Gray, John C.

2000-01-01

The pea lip1 (light-independent photomorphogenesis1) mutant shows many of the characteristics of light-grown development when grown in continuous darkness. To investigate the identity of LIP1, cDNAs encoding the pea homolog of COP1, a repressor of photomorphogenesis identified in Arabidopsis, were isolated from wild-type and lip1 pea seedlings. lip1 seedlings contained a wild-type COP1 transcript as well as a larger COP1′ transcript that contained an internal in-frame duplication of 894 bp. The COP1′ transcript segregated with the lip1 phenotype in F2 seedlings and could be translated in vitro to produce a protein of ∼100 kD. The COP1 gene in lip1 peas contained a 7.5-kb duplication, consisting of exons 1 to 7 of the wild-type sequence, located 2.5 kb upstream of a region of genomic DNA identical to the wild-type COP1 DNA sequence. Transcription and splicing of the mutant COP1 gene was predicted to produce the COP1′ transcript, whereas transcription from an internal promoter in the 2.5-kb region of DNA located between the duplicated regions of COP1 would produce the wild-type COP1 transcript. The presence of small quantities of wild-type COP1 transcripts may reduce the severity of the phenotype produced by the mutated COP1′ protein. The genomic DNA sequences of the COP1 gene from wild-type and lip1 peas and the cDNA sequences of COP1 and COP1′ transcripts have been submitted to the EMBL database under the EMBL accession numbers AJ276591, AJ276592, AJ289773, and AJ289774, respectively. PMID:11041887

Impaired fertilizing ability of superoxide dismutase 1-deficient mouse sperm during in vitro fertilization.

PubMed

Tsunoda, Satoshi; Kawano, Natsuko; Miyado, Kenji; Kimura, Naoko; Fujii, Junichi

2012-11-01

The oxidative modification of gametes by a reactive oxygen species is a major deleterious factor that decreases the successful rate of in vitro fertilization. Superoxide dismutase 1 (SOD1) plays a pivotal role in antioxidation by scavenging the superoxide anion, and its deficiency causes infertility in female mice, but the significance of the enzyme in male mice remains unclear. In the present study, we characterized Sod1(-/-) (Sod1-KO) male reproductive organs and compiled the first report of the impaired fertilizing ability of Sod1-KO sperm in in vitro fertilization. Insemination of wild-type oocytes with Sod1-KO sperm exhibited lower rates of fertility compared with insemination by wild-type sperm. The low fertilizing ability found for Sod1-KO sperm was partially rescued by reductant 2-mercaptoethanol, which suggested the oxidative modification of sperm components. The numbers of motile and progressive sperm decreased during the in vitro fertilization process, and a decline in ATP content and elevation in lipid peroxidation occurred in the Sod1-KO sperm in an incubation time-dependent manner. Tyrosine phosphorylation, which is a hallmark for sperm capacitation, was also impaired in the Sod1-KO sperm. These results collectively suggest that machinery involved in sperm capacitation and motility are vulnerable to oxidative damage during the in vitro fertilization process, which could increase the rate of inefficient fertilization.

Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

NASA Technical Reports Server (NTRS)

Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

2002-01-01

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

Up-Regulation of Phosphoinositide Metabolism in Tobacco Cells Constitutively Expressing the Human Type I Inositol Polyphosphate 5-Phosphatase1

PubMed Central

Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.

2002-01-01

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP3) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP3. The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP3 compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP3 in both wild-type and transgenic cells. However, even with stimulation, InsP3 levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP3 signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP2), the lipid precursor of InsP3, was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP2 metabolism showed that the activity of the PtdInsP2-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of 32P into PtdInsP2 in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP2 synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP2 synthesis as a regulatory step in this system. PMID:12177493

Discordance between bovine leukemia virus tax immortalization in vitro and oncogenicity in vivo.

PubMed

Twizere, J C; Kerkhofs, P; Burny, A; Portetelle, D; Kettmann, R; Willems, L

2000-11-01

Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area, Japan.

PubMed

Shyaka, Anselme; Kusumoto, Akiko; Chaisowwong, Warangkhana; Okouchi, Yoshiki; Fukumoto, Shinya; Yoshimura, Aya; Kawamoto, Keiko

2015-08-01

The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.

Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area, Japan

PubMed Central

SHYAKA, Anselme; KUSUMOTO, Akiko; CHAISOWWONG, Warangkhana; OKOUCHI, Yoshiki; FUKUMOTO, Shinya; YOSHIMURA, Aya; KAWAMOTO, Keiko

2015-01-01

The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells. PMID:25843040

The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism

PubMed Central

Labrador, Juan Pablo; Azcoitia, Valeria; Tuckermann, Jan; Lin, Calvin; Olaso, Elvira; Mañes, Santos; Brückner, Katja; Goergen, Jean-Louis; Lemke, Greg; Yancopoulos, George; Angel, Peter; Martínez-A, Carlos; Klein, Rüdiger

2001-01-01

The discoidin domain receptor 2 (DDR2) is a member of a subfamily of receptor tyrosine kinases whose ligands are fibrillar collagens, and is widely expressed in postnatal tissues. We have generated DDR2-deficient mice to establish the in vivo functions of this receptor, which have remained obscure. These mice exhibit dwarfism and shortening of long bones. This phenotype appears to be caused by reduced chondrocyte proliferation, rather than aberrant differentiation or function. In a skin wound healing model, DDR2–/– mice exhibit a reduced proliferative response compared with wild-type littermates. In vitro, fibroblasts derived from DDR2–/– mutants proliferate more slowly than wild-type fibroblasts, a defect that is rescued by introduction of wild-type but not kinase-dead DDR2 receptor. Together our results suggest that DDR2 acts as an extracellular matrix sensor to modulate cell proliferation. PMID:11375938

Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts

NASA Technical Reports Server (NTRS)

Eckes, B.; Dogic, D.; Colucci-Guyon, E.; Wang, N.; Maniotis, A.; Ingber, D.; Merckling, A.; Langa, F.; Aumailley, M.; Delouvee, A.;

Wild-type H- and N-Ras promote mutant K-Ras driven tumorigenesis by modulating the DNA damage response

PubMed Central

Grabocka, Elda; Pylayeva-Gupta, Yuliya; Jones, Mathew JK; Lubkov, Veronica; Yemanaberhan, Eyoel; Taylor, Laura; Jeng, Hao Hsuan; Bar-Sagi, Dafna

2014-01-01

SUMMARY Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anti-cancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways, and consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo. PMID:24525237

Mice with GFAP-targeted loss of neurofibromin demonstrate increased axonal MET expression with aging.

PubMed

Su, Weiping; Xing, Rubing; Guha, Abhijit; Gutmann, David H; Sherman, Larry S

2007-05-01

Neurofibromatosis 1 (NF1) is a common genetic disease that predisposes patients to peripheral nerve tumors and central nervous system (CNS) abnormalities including low-grade astrocytomas and cognitive disabilities. Using mice with glial fibrillary acidic protein (GFAP)-targeted Nf1 loss (Nf1(GFAP)CKO mice), we found that Nf1(-/-) astrocytes proliferate faster and are more invasive than wild-type astrocytes. In light of our previous finding that aberrant expression of the MET receptor tyrosine kinase contributes to the invasiveness of human NF1-associated malignant peripheral nerve sheath tumors, we sought to determine whether MET expression is aberrant in the brains of Nf1 mutant mice. We found that Nf1(-/-) astrocytes express slightly more MET than wild-type cells in vitro, but do not express elevated MET in situ. However, fiber tracts containing myelinated axons in the hippocampus, midbrain, cerebral cortex, and cerebellum express higher than normal levels of MET in older (> or =6 months) Nf1(GFAP)CKO mice. Both Nf1(GFAP)CKO and wild-type astrocytes induced MET expression in neurites of wild-type hippocampal neurons in vitro, suggesting that astrocyte-derived signals may induce MET in Nf1 mutant mice. Because the Nf1 gene product functions as a RAS GTPase, we examined MET expression in the brains of mice with GFAP-targeted constitutively active forms of RAS. MET was elevated in axonal fiber tracts in mice with active K-RAS but not H-RAS. Collectively, these data suggest that loss of Nf1 in either astrocytes or GFAP(+) neural progenitor cells results in increased axonal MET expression, which may contribute to the CNS abnormalities in children and adults with NF1. (c) 2007 Wiley-Liss, Inc.

In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

PubMed

Yasugi, Mayo; Sugahara, Yuki; Hoshi, Hidenobu; Kondo, Kaori; Talukdar, Prabhat K; Sarker, Mahfuzur R; Yamamoto, Shigeki; Kamata, Yoichi; Miyake, Masami

2015-08-01

Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Regulation of Small Mitochondrial DNA Replicative Advantage by Ribonucleotide Reductase in Saccharomyces cerevisiae

PubMed Central

Bradshaw, Elliot; Yoshida, Minoru; Ling, Feng

2017-01-01

Small mitochondrial genomes can behave as selfish elements by displacing wild-type genomes regardless of their detriment to the host organism. In the budding yeast Saccharomyces cerevisiae, small hypersuppressive mtDNA transiently coexist with wild-type in a state of heteroplasmy, wherein the replicative advantage of the small mtDNA outcompetes wild-type and produces offspring without respiratory capacity in >95% of colonies. The cytosolic enzyme ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and its inhibition has been correlated with increased petite colony formation, reflecting loss of respiratory function. Here, we used heteroplasmic diploids containing wild-type (rho+) and suppressive (rho−) or hypersuppressive (HS rho−) mitochondrial genomes to explore the effects of RNR activity on mtDNA heteroplasmy in offspring. We found that the proportion of rho+ offspring was significantly increased by RNR overexpression or deletion of its inhibitor, SML1, while reducing RNR activity via SML1 overexpression produced the opposite effects. In addition, using Ex Taq and KOD Dash polymerases, we observed a replicative advantage for small over large template DNA in vitro, but only at low dNTP concentrations. These results suggest that dNTP insufficiency contributes to the replicative advantage of small mtDNA over wild-type and cytosolic dNTP synthesis by RNR is an important regulator of heteroplasmy involving small mtDNA molecules in yeast. PMID:28717049

Requirement for erythroblast-macrophage protein (Emp) in definitive erythropoiesis.

PubMed

Soni, Shivani; Bala, Shashi; Hanspal, Manjit

2008-01-01

Emp, erythroblast-macrophage protein was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation. More recent studies have shown that targeted disruption of Emp leads to abnormal erythropoiesis in the fetal liver, and fetal demise. To further address the activity of Emp in the hematopoietic lineage in adult bone marrow, we conducted fetal liver HSC reconstitution assay. Emp null fetal liver cells were transplanted into lethally irradiated wild-type sibling mice, and assessed the erythropoietic activity. We found that Emp null cells rescued lethally irradiated mice with efficiency comparable to that of wild-type cells. However, the recipients of Emp null cells showed abnormal erythropoiesis as indicated by the presence of persistent anemia, extensive extramedullary erythropoiesis, and increased apoptosis of erythroid precursors. Extramedullary erythropoiesis suggests perturbed interactions between the Emp-deficient hematopoietic cells and the wild-type niche. Furthermore, in spleen colony-forming unit assays, proliferation rates of the Emp null cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Emp null livers. Morphologic examination showed that Emp null CFU-E-derived erythroblasts were immature compared to those derived from wild-type CFU-Es, suggesting that loss of Emp function in erythroid cells results in impaired proliferation and terminal differentiation. These results demonstrate that Emp plays a cell intrinsic role in the erythroid lineage.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

PubMed Central

2012-01-01

Background The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib. PMID:23234355

A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

PubMed Central

Yamamoto, N; Naraparaju, V R

1996-01-01

Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice. PMID:8881764

Mek inhibition results in marked antitumor activity against metastatic melanoma patient-derived melanospheres and in melanosphere-generated xenografts

PubMed Central

2013-01-01

One of the key oncogenic pathways involved in melanoma aggressiveness, development and progression is the RAS/BRAF/MEK pathway, whose alterations are found in most patients. These molecular anomalies are promising targets for more effective anti-cancer therapies. Some Mek inhibitors showed promising antitumor activity, although schedules and doses associated with low systemic toxicity need to be defined. In addition, it is now accepted that cancers can arise from and be maintained by the cancer stem cells (CSC) or tumor-initiating cells (TIC), commonly expanded in vitro as tumorspheres from several solid tumors, including melanoma (melanospheres). Here, we investigated the potential targeting of MEK pathway by exploiting highly reliable in vitro and in vivo pre-clinical models of melanomas based on melanospheres, as melanoma initiating cells (MIC) surrogates. MEK inhibition, through PD0325901, provided a successful strategy to affect survival of mutated-BRAF melanospheres and growth of wild type-BRAF melanospheres. A marked citotoxicity was observed in differentated melanoma cells regardless BRAF mutational status. PD0325901 treatment, dramatically inhibited growth of melanosphere-generated xenografts and determined impaired tumor vascularization of both mutated- and wild type-BRAF tumors, in the absence of mice toxicity. These results suggest that MEK inhibition might represent a valid treatment option for patients with both mutated- or wild type-BRAF melanomas, affecting tumor growth through multiple targets. PMID:24238212

Evaluation of ompA and pgtE genes in determining pathogenicity in Salmonella enterica serovar Enteritidis.

PubMed

Zhou, Y; Zhou, J; Wang, D; Gao, Q; Mu, X; Gao, S; Liu, X

2016-12-01

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is a major causative agent of gastroenteritis in humans. This important food-borne pathogen also colonises the intestinal tracts of poultry and can spread systemically, especially in chickens. To identify the S. Enteritidis virulence genes involved in infection and colonisation of chickens, chromosomal deletion mutants of the ompA and pgtE genes, which encode essential components of omptins, were constructed. There were no significant differences between the wild-type and ompA and pgtE mutants in a series of in vitro assays, including an intracellular survival assay, survival in specific-pathogen-free (SPF) chicken serum, and in vitro competition assays. In contrast, in vivo competition assays revealed that ompA and pgtE mutants underwent attenuated growth in liver, cardiac blood, spleen, lung, and kidney compared to a wild-type strain (CVCC3378). When tested in SPF chickens, ompA or pgtE gene inactivation substantially reduced organ colonisation and delayed systemic infection compared with the wild-type strain. Colonisation was restored in S. Enteritidis mutants by reintroduction of the whole ompA or pgtE gene with the native promoters. The results of this study demonstrate that ompA and pgtE play an important role in the pathogenesis of S. Enteritidis and its ability to infect chickens. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of 22 Novel CYP2D6 Variants Found in the Chinese Population on the Bufuralol and Dextromethorphan Metabolisms In Vitro.

PubMed

Cai, Jie; Dai, Da-Peng; Geng, Pei-Wu; Wang, Shuang-Hu; Wang, Hao; Zhan, Yun-Yun; Huang, Xiang-Xin; Hu, Guo-Xin; Cai, Jian-Ping

2016-03-01

Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that metabolizes a large number of therapeutic drugs. To date, more than 100 CYP2D6 allelic variants have been reported. Among these variants, we recently identified 22 novel variants in the Chinese population. The aim of this study was to functionally characterize the enzymatic activity of these variants in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6.1 and other variants (CYP2D6.2, CYP2D6.10 and 22 novel CYP2D6 variants) at high levels. Then, the insect microsomes containing expressed CYP2D6 proteins were incubated with bufuralol or dextromethorphan at 37°C for 20 or 25 min., respectively. After termination, the metabolites were extracted and used for the detection with high-performance liquid chromatography. Among the 24 CYP2D6 variants tested, two variants (CYP2D6.92 and CYP2D6.96) were found to be catalytically inactive. The remaining 22 variants exhibited significantly decreased intrinsic clearance values for bufuralol 1'-hydroxylation and 20 variants showed significantly lower intrinsic clearance values for dextromethorphan O-demethylation than those of the wild-type CYP2D6.1. Our in vitro results suggest that most of the variants exhibit significantly reduced catalytic activities compared with the wild-type, and these data provide valuable information for personalized medicine in Chinese and other Asian populations. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Increased susceptibility to fatigue of slow- and fast-twitch muscles from mice lacking the MG29 gene.

PubMed

Nagaraj, R Y; Nosek, C M; Brotto, M A; Nishi, M; Takeshima, H; Nosek, T M; Ma, J

2000-11-09

Mitsugumin 29 (MG29), a major protein component of the triad junction in skeletal muscle, has been identified to play roles in the formation of precise junctional membrane structures important for efficient signal conversion in excitation-contraction (E-C) coupling. We carried out several experiments to not only study the role of MG29 in normal muscle contraction but also to determine its role in muscle fatigue. We compared the in vitro contractile properties of three muscles types, extensor digitorum longus (EDL) (fast-twitch muscle), soleus (SOL) (slow-twitch muscle), and diaphragm (DPH) (mixed-fiber muscle), isolated from mice lacking the MG29 gene and wild-type mice prior to and after fatigue. Our results indicate that the mutant EDL and SOL muscles, but not DPH, are more susceptible to fatigue than the wild-type muscles. The mutant muscles not only fatigued to a greater extent but also recovered significantly less than the wild-type muscles. Following fatigue, the mutant EDL and SOL muscles produced lower twitch forces than the wild-type muscles; in addition, fatiguing produced a downward shift in the force-frequency relationship in the mutant mice compared with the wild-type controls. Our results indicate that fatiguing affects the E-C components of the mutant EDL and SOL muscles, and the effect of fatigue in these mutant muscles could be primarily due to an alteration in the intracellular Ca homeostasis.

Matrix metalloproteinase (MMP)-9 in cancer-associated fibroblasts (CAFs) is suppressed by omega-3 polyunsaturated fatty acids in vitro and in vivo.

PubMed

Taguchi, Ayumi; Kawana, Kei; Tomio, Kensuke; Yamashita, Aki; Isobe, Yosuke; Nagasaka, Kazunori; Koga, Kaori; Inoue, Tomoko; Nishida, Haruka; Kojima, Satoko; Adachi, Katsuyuki; Matsumoto, Yoko; Arimoto, Takahide; Wada-Hiraike, Osamu; Oda, Katsutoshi; Kang, Jing X; Arai, Hiroyuki; Arita, Makoto; Osuga, Yutaka; Fujii, Tomoyuki

2014-01-01

Cancer associated fibroblasts (CAFs) are responsible for tumor growth, angiogenesis, invasion, and metastasis. Matrix metalloproteinase (MMP)-9 secreted from cancer stroma populated by CAFs is a prerequisite for cancer angiogenesis and metastasis. Omega-3 polyunsaturated fatty acids (omega-3 PUFA) have been reported to have anti-tumor effects on diverse types of malignancies. Fat-1 mice, which can convert omega-6 to omega-3 PUFA independent of diet, are useful to investigate the functions of endogenous omega-3 PUFA. To examine the effect of omega-3 PUFA on tumorigenesis, TC-1 cells, a murine epithelial cell line immortalized by human papillomavirus (HPV) oncogenes, were injected subcutaneously into fat-1 or wild type mice. Tumor growth and angiogenesis of the TC-1 tumor were significantly suppressed in fat-1 compared to wild type mice. cDNA microarray of the tumors derived from fat-1 and wild type mice revealed that MMP-9 is downregulated in fat-1 mice. Immunohistochemical study demonstrated immunoreactivity for MMP-9 in the tumor stromal fibroblasts was diffusely positive in wild type whereas focal in fat-1 mice. MMP-9 was expressed in primary cultured fibroblasts isolated from fat-1 and wild type mice but was not expressed in TC-1 cells. Co-culture of fibroblasts with TC-1 cells enhanced the expression and the proteinase activity of MMP-9, although the protease activity of MMP-9 in fat-1-derived fibroblasts was lower than that in wild type fibroblasts. Our data suggests that omega-3 PUFAs suppress MMP-9 induction and tumor angiogenesis. These findings may provide insight into mechanisms by which omega-3 PUFAs exert anti-tumor effects by modulating tumor microenvironment.

Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models.

PubMed

Wang, Hui Qin; Halilovic, Ensar; Li, Xiaoyan; Liang, Jinsheng; Cao, Yichen; Rakiec, Daniel P; Ruddy, David A; Jeay, Sebastien; Wuerthner, Jens U; Timple, Noelito; Kasibhatla, Shailaja; Li, Nanxin; Williams, Juliet A; Sellers, William R; Huang, Alan; Li, Fang

2017-04-20

The efficacy of ALK inhibitors in patients with ALK -mutant neuroblastoma is limited, highlighting the need to improve their effectiveness in these patients. To this end, we sought to develop a combination strategy to enhance the antitumor activity of ALK inhibitor monotherapy in human neuroblastoma cell lines and xenograft models expressing activated ALK. Herein, we report that combined inhibition of ALK and MDM2 induced a complementary set of anti-proliferative and pro-apoptotic proteins. Consequently, this combination treatment synergistically inhibited proliferation of TP53 wild-type neuroblastoma cells harboring ALK amplification or mutations in vitro, and resulted in complete and durable responses in neuroblastoma xenografts derived from these cells. We further demonstrate that concurrent inhibition of MDM2 and ALK was able to overcome ceritinib resistance conferred by MYCN upregulation in vitro and in vivo. Together, combined inhibition of ALK and MDM2 may provide an effective treatment for TP53 wild-type neuroblastoma with ALK aberrations.

Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene.

PubMed

Poussier, Stéphane; Thoquet, Philippe; Trigalet-Demery, Danièle; Barthet, Séverine; Meyer, Damien; Arlat, Matthieu; Trigalet, André

2003-08-01

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild-type pathogenic to a non-pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild-type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.

A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase.

PubMed

Liebherr, Raphaela B; Renner, Max; Gorris, Hans H

2014-04-23

The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.

In vitro evolution of a ribozyme that contains 5-bromouridine

NASA Technical Reports Server (NTRS)

Dai, X.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

2000-01-01

The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice.

PubMed

Bandara, Aloka B; DeShazer, David; Inzana, Thomas J; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

2008-09-01

Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344DeltasacB. A portion of the ctpA gene (encoding CtpA) of strain 23344DeltasacB was deleted by gene replacement to generate strain 23344DeltasacBDeltactpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344DeltasacB, the ctpA mutant 23344DeltasacBDeltactpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

A Novel CCR5 Mutation Common in Sooty Mangabeys Reveals SIVsmm Infection of CCR5-Null Natural Hosts and Efficient Alternative Coreceptor Use In Vivo

PubMed Central

Riddick, Nadeene E.; Hermann, Emilia A.; Loftin, Lamorris M.; Elliott, Sarah T.; Wey, Winston C.; Cervasi, Barbara; Taaffe, Jessica; Engram, Jessica C.; Li, Bing; Else, James G.; Li, Yingying; Hahn, Beatrice H.; Derdeyn, Cynthia A.; Sodora, Donald L.; Apetrei, Cristian; Paiardini, Mirko; Silvestri, Guido; Collman, Ronald G.

2010-01-01

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species. PMID:20865163

A novel CCR5 mutation common in sooty mangabeys reveals SIVsmm infection of CCR5-null natural hosts and efficient alternative coreceptor use in vivo.

PubMed

Riddick, Nadeene E; Hermann, Emilia A; Loftin, Lamorris M; Elliott, Sarah T; Wey, Winston C; Cervasi, Barbara; Taaffe, Jessica; Engram, Jessica C; Li, Bing; Else, James G; Li, Yingying; Hahn, Beatrice H; Derdeyn, Cynthia A; Sodora, Donald L; Apetrei, Cristian; Paiardini, Mirko; Silvestri, Guido; Collman, Ronald G

2010-08-26

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species.

E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis.

PubMed

Park, Kyeong-Su; Kim, Ju Hee; Shin, Hee Won; Chung, Kyung-Sook; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

2015-10-26

Missense mutation of VHL gene is frequently detected in type 2 VHL diseases and linked to a wide range of pVHL functions and stability. Certain mutant pVHLs retain ability to regulate HIFs but lose their function by instability. In this case, regulating of degradation of mutant pVHLs, can be postulated as therapeutic method. The stability and cellular function of missense mutant pVHLs were determine in HEK293T transient expressing cell and 786-O stable cell line. Ubiquitination assay of mutant VHL proteins was performed in vitro system. Anticancer effect of adenovirus mediated shUCP expressing was evaluated using ex vivo mouse xenograft assay. Three VHL missense mutants (V155A, L158Q, and Q164R) are directly ubiquitinated by E2-EPF UCP (UCP) in vitro. Mutant pVHLs are more unstable than wild type in cell. Missense mutant pVHLs interact with UCP directly in both in vitro and cellular systems. Lacking all of lysine residues of pVHL result in resistance to ubiquitination thereby increase its stability. Missense mutant pVHLs maintained the function of E3 ligase to ubiquitinate HIF-1α in vitro. In cells expressing mutant pVHLs, Glut-1 and VEGF were relatively upregulated compared to their levels in cells expressing wild-type. Depletion of UCP restored missense mutant pVHLs levels and inhibited cell growth. Adenovirus-mediated shUCP RNA delivery inhibited tumor growth in ex vivo mouse xenograft model. These data suggest that targeting of UCP can be one of therapeutic method in type 2 VHL disease caused by unstable but functional missense mutant pVHL.

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor*

PubMed Central

Matsushita, Yuki; Sakamoto, Kei; Tamamura, Yoshihiro; Shibata, Yasuaki; Minamizato, Tokutaro; Kihara, Tasuku; Ito, Masako; Katsube, Ken-ichi; Hiraoka, Shuichi; Koseki, Haruhiko; Harada, Kiyoshi; Yamaguchi, Akira

2013-01-01

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy. PMID:23653360

Profiling of anthocyanins in transgenic purple-fleshed sweet potatoes by HPLC-MS/MS.

PubMed

Ge, Jingqiu; Hu, Yijie; Wang, Hongxia; Huang, Yuanshe; Zhang, Peng; Liao, Zhihua; Chen, Min

2017-11-01

Anthocyanins in purple-fleshed sweet potato (PSP) are beneficial to human health. The leaf color (Lc) gene is a transcription factor involved in regulating anthocyanin biosynthesis. The anthocyanin profiles of wild-type PSP of Ayamurasaki and its three Lc-transgenic lines were investigated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). In vitro antioxidant activities of wild-type and Lc-transgenic lines, including reducing power activity, DPPH radical scavenging activity, hydroxyl radical scavenging activity, linoleic acid autoxidation inhibition activity, ABTS free radical scavenging activity and oxygen radical absorbance capacity activity, were measured. The results showed that the total anthocyanin contents increased 1.5-1.9 times in three transgenic lines compared with that in wild-type PSP. Seventeen anthocyanins were found in wild-type PSP, while 19 in Lc-transgenic lines including cyanidin-based, peonidin-based and pelargonidin-based anthocyanins. Three pelargonidin-based anthocyanins were detected in three Lc-transgenic lines. Among them, the relative contents of cyanidin-based and pelargonidin-based anthocyanins increased 1.9-2.0 and 3.4-4.5 times respectively, while peonidin-based anthocyanins decreased 1.8-1.9 times in Lc-transgenic lines, compared with wild-type PSP. PSP from wild-type Ayamurasaki and three Lc-transgenic lines exhibited potent antioxidant activities, whereas there was no distinct difference among them. The transgene Lc significantly increased the content of total anthocyanins and remarkably changed the anthocyanin profiles in Ayamurasaki. Such novel and high content of anthocyanins obtained in the Lc-transgenic lines with potent antioxidant activities may provide unique functional products with potential helpful for human health. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Matrilysin (Matrix Metalloproteinase-7) Regulates Anti-Inflammatory and Antifibrotic Pulmonary Dendritic Cells That Express CD103 (αEβ7-Integrin)

PubMed Central

Manicone, Anne M.; Huizar, Isham; McGuire, John K.

2009-01-01

The E-cadherin receptor CD103 (αEβ7-integrin) is expressed on specific populations of pulmonary dendritic cells (DC) and T cells. However, CD103 function in the lung is not well understood. Matrilysin (MMP-7) expression is increased in lung injury and cleaves E-cadherin from injured lung epithelium. Thus, to assess matrilysin effects on CD103-E-cadherin interactions in lung injury, wild-type, CD103−/−, and Mmp7−/− mice, in which E-cadherin isn’t cleaved in the lung, were treated with bleomycin or bleomycin with nFMLP to reverse the defect in acute neutrophil influx seen in Mmp7−/− mice. Pulmonary CD103+ DC were significantly increased in injured wild-type compared with Mmp7−/− mice, and CD103+ leukocytes showed significantly enhanced interaction with E-cadherin on injured wild-type epithelium than with Mmp7−/− epithelium in vitro and in vivo. Bleomycin-treated CD103−/− mice had persistent neutrophilic inflammation, increased fibrosis, and increased mortality compared with wild-type mice, a phenotype that was partially recapitulated in bleomycin/nFMLP-treated Mmp7−/− mice. Soluble E-cadherin increased IL-12 and IL-10 and reduced IL-6 mRNA expression in wild-type bone marrow-derived DC but not in CD103−/− bone marrow-derived DC. Similar mRNA patterns were seen in lungs of bleomycin-injured wild-type, but not CD103−/− or Mmp7−/−, mice. In conclusion, matrilysin regulates pulmonary localization of DC that express CD103, and E-cadherin cleavage may activate CD103+ DC to limit inflammation and inhibit fibrosis. PMID:19893044

A Chrysodeixis chalcites Single-Nucleocapsid Nucleopolyhedrovirus Population from the Canary Islands Is Genotypically Structured To Maximize Survival

PubMed Central

Bernal, Alexandra; Simón, Oihane; Williams, Trevor; Muñoz, Delia

2013-01-01

A Chrysodeixis chalcites single-nucleocapsid nucleopolyhedrovirus wild-type isolate from the Canary Islands, Spain, named ChchSNPV-TF1 (ChchTF1-wt), appears to have great potential as the basis for a biological insecticide for control of the pest. An improved understanding of the genotypic structure of this wild-type strain population should facilitate the selection of genotypes for inclusion in a bioinsecticidal product. Eight genetically distinct genotypes were cloned in vitro: ChchTF1-A to ChchTF1-H. Quantitative real-time PCR (qPCR) analysis confirmed that ChchTF1-A accounted for 36% of the genotypes in the wild-type population. In bioassays, ChchTF1-wt occlusion bodies (OBs) were significantly more pathogenic than any of the component single-genotype OBs, indicating that genotype interactions were likely responsible for the pathogenicity phenotype of wild-type OBs. However, the wild-type population was slower killing and produced higher OB yields than any of the single genotypes alone. These results strongly suggested that the ChchTF1-wt population is structured to maximize its transmission efficiency. Experimental OB mixtures and cooccluded genotype mixtures containing the most abundant and the rarest genotypes, at frequencies similar to those at which they were isolated, revealed a mutualistic interaction that restored the pathogenicity of OBs. In OB and cooccluded mixtures containing only the most abundant genotypes, ChchTF1-ABC, OB pathogenicity was even greater than that of wild-type OBs. The ChchTF1-ABC cooccluded mixture killed larvae 33 h faster than the wild-type population and remained genotypically and biologically stable throughout five successive passages in vivo. In conclusion, the ChchTF1-ABC mixture shows great potential as the active ingredient of a bioinsecticide to control C. chalcites in the Canary Islands. PMID:24096419

Streptococcus mutans in a Wild, Sucrose-Eating Rat Population

PubMed Central

Coykendall, Alan L.; Specht, Patricia A.; Samol, Harry H.

1974-01-01

Streptococcus mutans, an organism implicated in dental caries and not previously found outside of man and certain laboratory animals, was isolated from the mouths of wild rats which ate sugar cane. The strains isolated fermented mannitol and sorbitol, and failed to grow in 6.5% NaCl or at 45 C. They formed in vitro plaques on nichrome wires when grown in sucrose broth. They also stored intracellular polysaccharide which could be catabolized by washed, resting cells. Deoxyribonucleic acid-deoxyribonucleic acid reassociations revealed two genetic types. One type shared extensive deoxyribonucleic acid base sequences with S. mutans strains HS6 and OMZ61, two members of a genetic type found in man and laboratory hamsters. The other type seemed unrelated to any S. mutans genetic type previously encountered. It is concluded that the ecological triad of tooth-sucrose-S. mutans is not a phenomenon unique to man and experimental animals. Images PMID:4601769

Effects of Chitosan-Essential Oil Coatings on Safety and Quality of Fresh Blueberries

USDA-ARS?s Scientific Manuscript database

Chitosan coating plus different essential oils was developed and applied to fresh blueberries, in order to find environmentally friendly and healthy treatments to preserve fresh fruit quality and safety during postharvest storage. Studies were first performed in vitro where wild-type Escherichia col...

COMPARISON OF IN VITRO-CULTURED AND WILD-TYPE PERKINSUS MARINUS III. FECAL ELIMINATION AND ITS ROLE IN TRANSMISSION

EPA Science Inventory

Perkinsus marinus, a pathogen of the eastern oyster Crassostrea virginica, is transmitted directly among oysters. Previous studies found viable P. marinus parasites in the feces and
pseudofeces of oysters within hours of injection with parasites, suggesting that the parasite ...

COMPARISON OF IN VITRO-CULTURED AND WILD-TYPE PERKINSUS MARINUS. II: DOSING METHODS AND HOST RESPONSE

EPA Science Inventory

Endoparasites must breach host barriers to establish infection and then must survive host internal defenses to cause disease. Such barriers may frustrate attempts to experimentally transmit parasites by ?natural' methods. In addition, the host's condition may affect a study's out...

Antifungal Susceptibility Testing of Aspergillus spp. by Using a Composite Correlation Index (CCI)-Based Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method Appears To Not Offer Benefit over Traditional Broth Microdilution Testing

PubMed Central

Gitman, Melissa R.; McTaggart, Lisa; Spinato, Joanna; Poopalarajah, Rahgavi; Lister, Erin; Husain, Shahid

2017-01-01

ABSTRACT Aspergillus spp. cause serious invasive lung infections, and Aspergillus fumigatus is the most commonly encountered clinically significant species. Voriconazole is considered to be the drug of choice for treating A. fumigatus infections; however, rising resistance rates have been reported. We evaluated a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based method for the differentiation between wild-type and non-wild-type isolates of 20 Aspergillus spp. (including 2 isolates of Aspergillus ustus and 1 of Aspergillus calidoustus that were used as controls due their intrinsic low azole susceptibility with respect to the in vitro response to voriconazole). At 30 and 48 h of incubation, there was complete agreement between Cyp51A sequence analysis, broth microdilution, and MALDI-TOF MS classification of isolates as wild type or non-wild type. In this proof-of-concept study, we demonstrated that MALDI-TOF MS can be used to accurately detect A. fumigatus strains with reduced voriconazole susceptibility. However, rather than proving to be a rapid and simple method for antifungal susceptibility testing, this particular MS-based method showed no benefit over conventional testing methods. PMID:28404678

Antifungal Susceptibility Testing of Aspergillus spp. by Using a Composite Correlation Index (CCI)-Based Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Method Appears To Not Offer Benefit over Traditional Broth Microdilution Testing.

PubMed

Gitman, Melissa R; McTaggart, Lisa; Spinato, Joanna; Poopalarajah, Rahgavi; Lister, Erin; Husain, Shahid; Kus, Julianne V

2017-07-01

Aspergillus spp. cause serious invasive lung infections, and Aspergillus fumigatus is the most commonly encountered clinically significant species. Voriconazole is considered to be the drug of choice for treating A. fumigatus infections; however, rising resistance rates have been reported. We evaluated a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method for the differentiation between wild-type and non-wild-type isolates of 20 Aspergillus spp. (including 2 isolates of Aspergillus ustus and 1 of Aspergillus calidoustus that were used as controls due their intrinsic low azole susceptibility with respect to the in vitro response to voriconazole). At 30 and 48 h of incubation, there was complete agreement between Cyp51A sequence analysis, broth microdilution, and MALDI-TOF MS classification of isolates as wild type or non-wild type. In this proof-of-concept study, we demonstrated that MALDI-TOF MS can be used to accurately detect A. fumigatus strains with reduced voriconazole susceptibility. However, rather than proving to be a rapid and simple method for antifungal susceptibility testing, this particular MS-based method showed no benefit over conventional testing methods. © Crown copyright 2017.

Antagonism of scavenger receptor CD36 by 5A peptide prevents chronic kidney disease progression in mice independent of blood pressure regulation

PubMed Central

Souza, Ana Carolina P.; Bocharov, Alexander V.; Baranova, Irina; Vishnyakova, Tatyana; Huang, Yuning G.; Wilkins, Kenneth J.; Hu, Xuzhen; Street, Jonathan M.; Alvarez-Prats, Alejandro; Mullick, Adam E.; Patterson, Amy P.; Remaley, Alan; Eggerman, Thomas L.; Yuen, Peter S.T.; Star, Robert A.

2016-01-01

Scavenger receptor CD36 participates in lipid metabolism and inflammatory pathways important for cardiovascular disease and chronic kidney disease (CKD). Few pharmacological agents are available to slow the progression of CKD. However, apolipoprotein AI-mimetic peptide 5A antagonizes CD36 in vitro. To test the efficacy of 5A, and to test the role of CD36 during CKD, we compared wild type to CD36 knockout mice and wild type mice treated with 5A, in a progressive CKD model that resembles human disease. Knockout and 5A-treated wild type mice were protected from CKD progression without changes in blood pressure and had reductions in cardiovascular risk surrogate markers that are associated with CKD. Treatment with 5A did not further protect CD36 knockout mice from CKD progression, implicating CD36 as its main site of action. In a separate model of kidney fibrosis, 5A-treated wild type mice had less macrophage infiltration and interstitial fibrosis. Peptide 5A exerted anti-inflammatory effects in the kidney and decreases renal expression of inflammasome genes. Thus, CD36 is a new therapeutic target for CKD and its associated cardiovascular risk factors. Peptide 5A may be a promising new agent to slow CKD progression. PMID:26994575

Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation.

PubMed

Kirby, S; Walton, W; Smithies, O

2000-06-15

In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM. (Blood. 2000;95:3710-3715)

Zn(II)-curc targets p53 in thyroid cancer cells.

PubMed

Garufi, Alessia; D'Orazi, Valerio; Crispini, Alessandra; D'Orazi, Gabriella

2015-10-01

TP53 mutation is a common event in many cancers, including thyroid carcinoma. Defective p53 activity promotes cancer resistance to therapies and a more malignant phenotype, acquiring oncogenic functions. Rescuing the function of mutant p53 (mutp53) protein is an attractive anticancer therapeutic strategy. Zn(II)-curc is a novel small molecule that has been shown to target mutp53 protein in several cancer cells, but its effect in thyroid cancer cells remains unclear. Here, we investigated whether Zn(II)-curc could affect p53 in thyroid cancer cells with both p53 mutation (R273H) and wild-type p53. Zn(II)-curc induced mutp53H273 downregulation and reactivation of wild-type functions, such as binding to canonical target promoters and target gene transactivation. This latter effect was similar to that induced by PRIMA-1. In addition, Zn(II)-curc triggered p53 target gene expression in wild-type p53-carrying cells. In combination treatments, Zn(II)-curc enhanced the antitumor activity of chemotherapeutic drugs, in both mutant and wild-type-carrying cancer cells. Taken together, our data indicate that Zn(II)-curc promotes the reactivation of p53 in thyroid cancer cells, providing in vitro evidence for a potential therapeutic approach in thyroid cancers.

Hfq variant with altered RNA binding functions

PubMed Central

Ziolkowska, Katarzyna; Derreumaux, Philippe; Folichon, Marc; Pellegrini, Olivier; Régnier, Philippe; Boni, Irina V.; Hajnsdorf, Eliane

2006-01-01

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS–lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq–RNA interaction but has probably no effect on the folding and the oligomerization of the protein. PMID:16449205

FrnE, a Cadmium-Inducible Protein in Deinococcus radiodurans, Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo

PubMed Central

Khairnar, Nivedita P.; Joe, Min-Ho; Misra, H. S.; Lim, Sang-Yong

2013-01-01

Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ∼15- and ∼3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ∼2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42°C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation. PMID:23603741

Regulation of Cl(-) secretion by AMPK in vivo.

PubMed

Kongsuphol, Patthara; Hieke, Bernhard; Ousingsawat, Jiraporn; Almaca, Joana; Viollet, Benoit; Schreiber, Rainer; Kunzelmann, Karl

2009-03-01

Previous in vitro studies suggested that Cl(-) currents produced by the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) are inhibited by the alpha1 isoform of the adenosine monophosphate (AMP)-stimulated kinase (AMPK). AMPK is a serine/threonine kinase that is activated during metabolic stress. It has been proposed as a potential mediator for transport-metabolism coupling in epithelial tissues. All previous studies have been performed in vitro and thus little is known about the regulation of Cl(-) secretion by AMPK in vivo. Using AMPKalpha1(-/-) mice and wild-type littermates, we demonstrate that phenformin, an activator of AMPK, strongly inhibits cAMP-activated Cl(-) secretion in mouse airways and colon, when examined in ex vivo in Ussing chamber recordings. However, phenformin was equally effective in AMPKalpha1(-/-) and wild-type animals, suggesting additional AMPK-independent action of phenformin. Phenformin inhibited CFTR Cl(-) conductance in basolaterally permeabilized colonic epithelium from AMPKalpha1(+/+) but not AMPKalpha1(-/-) mice. The inhibitor of AMPK compound C enhanced CFTR-mediated Cl(-) secretion in epithelial tissues of AMPKalpha1(-/-) mice, but not in wild-type littermates. There was no effect on Ca(2+)-mediated Cl(-) secretion, activated by adenosine triphosphate or carbachol. Moreover CFTR-dependent Cl(-) secretion was enhanced in the colon of AMPKalpha1(-/-) mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl(-) secretion mediated by CFTR is controlled by AMPK in vivo.

MELK expression correlates with tumor mitotic activity but is not required for cancer growth

PubMed Central

Smith, Joan C; Palladino, Ann C

2018-01-01

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. Previously, we established that triple-negative breast cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK proliferate at wild-type levels in vitro (Lin et al., 2017). Here, we generate several additional knockout clones of MELK and demonstrate that across cancer types, cells lacking MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of cytotoxic chemotherapies, and in vivo. By combining our MELK-knockout clones with a recently described, highly specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cohorts of cancer patients identifies MELK expression as a correlate of tumor mitotic activity, explaining its association with poor clinical prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies. PMID:29417930

Light entrainment of the murine intraocular pressure circadian rhythm utilizes non-local mechanisms.

PubMed

Tsuchiya, Shunsuke; Buhr, Ethan D; Higashide, Tomomi; Sugiyama, Kazuhisa; Van Gelder, Russell N

2017-01-01

Intraocular pressure (IOP) is known to have a strong circadian rhythm, yet how light/dark cycles entrain this rhythm is unknown. The purpose of this study was to assess whether, like the retina, the mammalian ciliary body and IOP clocks have an intrinsic ability to entrain to light/dark cycles. Iris-ciliary body complexes were obtained from period2:luciferase (PER2::LUC) mice and cultured to measure bioluminescence rhythmicity. Pairs of the iris-ciliary body complex were exposed to antiphasic 9:15 h light/dark cycle in vitro. After 4 days of exposure to light/dark cycles, bioluminescence was recorded to establish their circadian phases. In addition, pairs of the iris-ciliary body complex co-cultured with the retinas or corneas of wild-type mice were also investigated. The IOP circadian changes of free-running Opn4-/-;rd1/rd1 mice whose behavior was antiphasic to wild-type were measured by a rebound tonometry, and compared with wild-type mice. Opn3, Opn4, and Opn5 mRNA expression in the iris-ciliary body were analyzed using RT-PCR. The iris/ciliary body complex expressed Opn3, Opn4, and Opn5 mRNA; however, unlike in retina and cornea, neither the iris-CB complex nor the co-cultured complex was directly entrained by light-dark cycle in vitro. The diurnal IOP change of Opn4-/-;rd1/rd1 mice showed an antiphasic pattern to wild-type mice and their rhythms followed the whole-animal behavioral rhythm. Despite expressing mRNA for several non-visual opsins, circadian rhythms of the iris-ciliary body complex of mice do not entrain directly to light-dark cycles ex vivo. Unlike retina, the iris/ciliary body clocks of blind mice remain synchronized to the organismal behavioral rhythm rather than local light-dark cycles. These results suggest that IOP rhythm entrainment is mediated by a systemic rather than local signal in mice.

Light entrainment of the murine intraocular pressure circadian rhythm utilizes non-local mechanisms

PubMed Central

Tsuchiya, Shunsuke; Buhr, Ethan D.; Higashide, Tomomi; Sugiyama, Kazuhisa

2017-01-01

Purpose Intraocular pressure (IOP) is known to have a strong circadian rhythm, yet how light/dark cycles entrain this rhythm is unknown. The purpose of this study was to assess whether, like the retina, the mammalian ciliary body and IOP clocks have an intrinsic ability to entrain to light/dark cycles. Methods Iris-ciliary body complexes were obtained from period2:luciferase (PER2::LUC) mice and cultured to measure bioluminescence rhythmicity. Pairs of the iris-ciliary body complex were exposed to antiphasic 9:15 h light/dark cycle in vitro. After 4 days of exposure to light/dark cycles, bioluminescence was recorded to establish their circadian phases. In addition, pairs of the iris-ciliary body complex co-cultured with the retinas or corneas of wild-type mice were also investigated. The IOP circadian changes of free-running Opn4-/-;rd1/rd1 mice whose behavior was antiphasic to wild-type were measured by a rebound tonometry, and compared with wild-type mice. Opn3, Opn4, and Opn5 mRNA expression in the iris-ciliary body were analyzed using RT-PCR. Results The iris/ciliary body complex expressed Opn3, Opn4, and Opn5 mRNA; however, unlike in retina and cornea, neither the iris-CB complex nor the co-cultured complex was directly entrained by light-dark cycle in vitro. The diurnal IOP change of Opn4-/-;rd1/rd1 mice showed an antiphasic pattern to wild-type mice and their rhythms followed the whole-animal behavioral rhythm. Conclusions Despite expressing mRNA for several non-visual opsins, circadian rhythms of the iris-ciliary body complex of mice do not entrain directly to light-dark cycles ex vivo. Unlike retina, the iris/ciliary body clocks of blind mice remain synchronized to the organismal behavioral rhythm rather than local light-dark cycles. These results suggest that IOP rhythm entrainment is mediated by a systemic rather than local signal in mice. PMID:28934261

Lack of rivaroxaban influence on a prothrombinase-based assay for the detection of activated C protein resistance: an Italian ex vivo and in vitro study in normal subjects and factor V Leiden carriers.

PubMed

Gessoni, G; Valverde, S; Valle, L; Gessoni, F; Caruso, P; Valle, R

2017-08-01

Activated protein C resistance (APCr) leads to hypercoagulability and is due, often but not exclusively, to Factor V Leiden (FVL). The aim of this study was to assess the ex vivo and in vitro interference of the direct factor Xa inhibitor rivaroxaban (RIV) on a prothrombinase-based assay for APCr detection. An ex vivo study was performed on fresh plasma samples obtained from 44 subjects with FV wild-type and seven with FVL heterozygous, all treated with RIV. An in vitro study was performed on 15 plasma samples (six from normal subjects, six from heterozygous, and three from homozygous FVL carriers, all frozen specimens) spiked with RIV. RIV concentration was evaluated using a chromogenic assay, and APCr was evaluated by a prothrombinase-based assay. No significant interference of RIV on APCr results obtained by a prothrombinase-based assay was observed for drug concentrations up to 400 ng/mL in FV wild-type and FVL carriers (homozygous and heterozygous). These results were confirmed both ex vivo and in vitro. RIV did not significantly interfere with the prothrombinase-based assay used for the assessment of APCr, and this was observed to occur independently of FV status. However, only concentrations up to 400 ng/mL were tested and, therefore, what occurs in the presence of higher doses remains to be investigated. © 2017 John Wiley & Sons Ltd.

Characterization of the RcsC sensor kinase from Erwinia amylovora and other enterobacteria

USDA-ARS?s Scientific Manuscript database

RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine kinase domain and a receiver domain. We have previously demonstrated that, while the Erwinia amylovora rcsC mutant produces more amylovoran than the wild type strain in vitro, the mutant remains avirulent on both immature pea...

Edwardsiella ictaluri Encodes an Acid Activated Urease that is Required for Intracellular Replication in Channel Catfish Ictalurus punctatus Macrophages

USDA-ARS?s Scientific Manuscript database

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative ureasepathogenicity island containing 9 open reading frames, including urea and ammonium transporters. In vitro studies with the wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significa...

Developmental toxicity and serum levels of perfluorononanoic acid in the wild-type and PPAR-alpha knockout mouse after gestational exposure

EPA Science Inventory

Perfluorononanoic acid (PFNA) is a perfluoroalkyl acid detected in.the environment and in tissues of humans and wildlife. PFNA activates peroxisome proliferator-activated receptor-alpha (PPARa) in vitro and negatively impacts development and survival of CD1 mice. Our objective wa...

MicroRNA-146a Deficiency Protects against Listeria monocytogenes Infection by Modulating the Gut Microbiota.

PubMed

Du, Chong-Tao; Gao, Wei; Ma, Ke; Yu, Shui-Xing; Li, Na; Yan, Shi-Qing; Zhou, Feng-Hua; Liu, Zhen-Zhen; Chen, Wei; Lei, Lian-Cheng; Yang, Yong-Jun; Han, Wen-Yu

2018-03-26

The gut microbiota and microRNAs play important roles in the defense against infection. However, the role of miR-146a in L. monocytogenes infection and gut microbiota remains unclear. We tried to determine whether miR-146a controlled L. monocytogenes infection by regulating the gut microbiota. Wild-type and miR-146a-deficient mice or macrophages were used to characterize the impact of miR-146a on animal survival, cell death, bacterial clearance, and gut microbiota following L. monocytogenes challenge. We found that L. monocytogenes infection induced miR-146a expression both in vitro and in vivo. When compared to wild-type mice, miR-146a-deficient mice were more resistant to L. monocytogenes infection. MiR-146a deficiency in macrophages resulted in reduced invasion and intracellular survival of L. monocytogenes . High-throughput sequencing of 16S rRNA revealed that the gut microbiota composition differed between miR-146a-deficient and wild-type mice. Relative to wild-type mice, miR-146a-deficient mice had decreased levels of the Proteobacteria phylum, Prevotellaceae family, and Parasutterella genus, and significantly increased short-chain fatty acid producing bacteria, including the genera Alistipes , Blautia , Coprococcus_1, and Ruminococcus_1 . Wild-type mice co-housed with miR-146a-deficient mice had increased resistance to L. monocytogenes , indicating that miR-146a deficiency guides the gut microbiota to alleviate infection. Together, these results suggest that miR-146a deficiency protects against L. monocytogenes infection by regulating the gut microbiota.

Adenosine A2B Receptor Deficiency Promotes Host Defenses against Gram-Negative Bacterial Pneumonia

PubMed Central

Barletta, Kathryn E.; Cagnina, R. Elaine; Burdick, Marie D.; Linden, Joel

2012-01-01

Rationale: Activation of the adenosine A2B receptor (A2BR) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. Objectives: To test the hypothesis that absence of adenosine A2B receptor signaling promotes host defense against bacterial pneumonia. Methods: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A2BR. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. Measurements and Main Results: A2BR–/– mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow–derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A2BR–/– and wild-type mice, but A2BR–/– neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A2BR–/– mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. Conclusions: These data suggest that the absence of A2BR signaling enhances antimicrobial activity in gram-negative bacterial pneumonia. PMID:22997203

Enhancing Human Spermine Synthase Activity by Engineered Mutations

PubMed Central

Zhang, Zhe; Zheng, Yueli; Petukh, Margo; Pegg, Anthony; Ikeguchi, Yoshihiko; Alexov, Emil

2013-01-01

Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing. PMID:23468611

Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation.

PubMed

Tian, Na; Liu, Shuoqian; Li, Juan; Xu, Wenwen; Yuan, Lin; Huang, Jianan; Liu, Zhonghua

2014-08-01

Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation. © 2013 Scandinavian Plant Physiology Society.

Phi-value analysis of apo-azurin folding: comparison between experiment and theory.

PubMed

Zong, Chenghang; Wilson, Corey J; Shen, Tongye; Wolynes, Peter G; Wittung-Stafshede, Pernilla

2006-05-23

Pseudomonas aeruginosa azurin is a 128-residue beta-sandwich metalloprotein; in vitro kinetic experiments have shown that it folds in a two-state reaction. Here, we used a variational free energy functional to calculate the characteristics of the transition state ensemble (TSE) for folding of the apo-form of P. aeruginosa azurin and investigate how it responds to thermal and mutational changes. The variational method directly yields predicted chevron plots for wild-type and mutant apo-forms of azurin. In parallel, we performed in vitro kinetic-folding experiments on the same set of azurin variants using chemical perturbation. Like the wild-type protein, all apo-variants fold in apparent two-state reactions both in calculations and in stopped-flow mixing experiments. Comparisons of phi (phi) values determined from the experimental and theoretical chevron parameters reveal an excellent agreement for most positions, indicating a polarized, highly structured TSE for folding of P. aeruginosa apo-azurin. We also demonstrate that careful analysis of side-chain interactions is necessary for appropriate theoretical description of core mutants.

Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models

PubMed Central

Wang, Hui Qin; Halilovic, Ensar; Li, Xiaoyan; Liang, Jinsheng; Cao, Yichen; Rakiec, Daniel P; Ruddy, David A; Jeay, Sebastien; Wuerthner, Jens U; Timple, Noelito; Kasibhatla, Shailaja; Li, Nanxin; Williams, Juliet A; Sellers, William R; Huang, Alan; Li, Fang

2017-01-01

The efficacy of ALK inhibitors in patients with ALK-mutant neuroblastoma is limited, highlighting the need to improve their effectiveness in these patients. To this end, we sought to develop a combination strategy to enhance the antitumor activity of ALK inhibitor monotherapy in human neuroblastoma cell lines and xenograft models expressing activated ALK. Herein, we report that combined inhibition of ALK and MDM2 induced a complementary set of anti-proliferative and pro-apoptotic proteins. Consequently, this combination treatment synergistically inhibited proliferation of TP53 wild-type neuroblastoma cells harboring ALK amplification or mutations in vitro, and resulted in complete and durable responses in neuroblastoma xenografts derived from these cells. We further demonstrate that concurrent inhibition of MDM2 and ALK was able to overcome ceritinib resistance conferred by MYCN upregulation in vitro and in vivo. Together, combined inhibition of ALK and MDM2 may provide an effective treatment for TP53 wild-type neuroblastoma with ALK aberrations. DOI: http://dx.doi.org/10.7554/eLife.17137.001 PMID:28425916

Reassessment of murine APOBEC1 as a retrovirus restriction factor in vivo.

PubMed

Barrett, Bradley S; Guo, Kejun; Harper, Michael S; Li, Sam X; Heilman, Karl J; Davidson, Nicholas O; Santiago, Mario L

2014-11-01

APOBEC1 is a cytidine deaminase involved in cholesterol metabolism that has been linked to retrovirus restriction, analogous to the evolutionarily-related APOBEC3 proteins. In particular, murine APOBEC1 was shown to inhibit Friend retrovirus (FV) in vitro, generating high levels of C-to-T and G-to-A mutations. These observations raised the possibility that FV infection might be altered in APOBEC1-null mice. To examine this question directly, we infected wild-type and APOBEC1-null mice with FV complex and evaluated acute infection levels. Surprisingly, APOBEC1-null mice exhibited similar cellular infection levels and plasma viremia relative to wild-type mice. Moreover, next-generation sequencing analyses revealed that in contrast to APOBEC3, APOBEC1 did not enhance retroviral C-to-T and G-to-A mutational frequencies in genomic DNA. Thus, APOBEC1 neither inhibited nor significantly drove the molecular evolution of FV in vivo. Our findings reinforce that not all retrovirus restriction factors characterized as potent in vitro may be functionally relevant in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA Repair Modulates The Vulnerability of The Developing Brain to Alkylating Agents

PubMed Central

Kisby, G.E.; Olivas, A.; Park, T.; Churchwell, M.; Doerge, D.; Samson, L. D.; Gerson, S.L.; Turker, M.S.

2009-01-01

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag-/-) or O6-methylguanine methyltransferase (Mgmt-/-), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt-/- neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag-/- neurons were for the most part significantly less sensitive than wild type or Mgmt-/- neurons to MAM and HN2. Aag-/- neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt-/- mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM treated Aag-/- or MGMT overexpressing (MgmtTg+) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in MgmtTg+ mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant. PMID:19162564

The role of pili in Bacillus cereus intraocular infection.

PubMed

Callegan, Michelle C; Parkunan, Salai Madhumathi; Randall, C Blake; Coburn, Phillip S; Miller, Frederick C; LaGrow, Austin L; Astley, Roger A; Land, Craig; Oh, So-Young; Schneewind, Olaf

2017-06-01

Bacterial endophthalmitis is a potentially blinding intraocular infection. The bacterium Bacillus cereus causes a devastating form of this disease which progresses rapidly, resulting in significant inflammation and loss of vision within a few days. The outer surface of B. cereus incites the intraocular inflammatory response, likely through interactions with innate immune receptors such as TLRs. This study analyzed the role of B. cereus pili, adhesion appendages located on the bacterial surface, in experimental endophthalmitis. To test the hypothesis that the presence of pili contributed to intraocular inflammation and virulence, we analyzed the progress of experimental endophthalmitis in mouse eyes infected with wild type B. cereus (ATCC 14579) or its isogenic pilus-deficient mutant (ΔbcpA-srtD-bcpB or ΔPil). One hundred CFU were injected into the mid-vitreous of one eye of each mouse. Infections were analyzed by quantifying intraocular bacilli and retinal function loss, and by histology from 0 to 12 h postinfection. In vitro growth and hemolytic phenotypes of the infecting strains were also compared. There was no difference in hemolytic activity (1:8 titer), motility, or in vitro growth (p > 0.05, every 2 h, 0-18 h) between wild type B. cereus and the ΔPil mutant. However, infected eyes contained greater numbers of wild type B. cereus than ΔPil during the infection course (p ≤ 0.05, 3-12 h). Eyes infected with wild type B. cereus experienced greater losses in retinal function than eyes infected with the ΔPil mutant, but the differences were not always significant. Eyes infected with ΔPil or wild type B. cereus achieved similar degrees of severe inflammation. The results indicated that the intraocular growth of pilus-deficient B. cereus may have been better controlled, leading to a trend of greater retinal function in eyes infected with the pilus-deficient strain. Although this difference was not enough to significantly alter the severity of the inflammatory response, these results suggest a potential role for pili in protecting B. cereus from clearance during the early stages of endophthalmitis, which is a newly described virulence mechanism for this organism and this infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

PubMed

Noguchi, Fumihito; Nakajima, Takeshi; Inui, Shigeki; Reddy, Janardan K; Itami, Satoshi

2014-01-01

MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair follicle stem cells to epidermal regeneration after wounding in 6-month-old Med1(epi-/-) mice. This study sheds light on the novel function of MED1 in keratinocytes and suggests a possible new therapeutic approach for skin wound healing and aging.

Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia.

PubMed

Lerman, Oren Z; Galiano, Robert D; Armour, Mary; Levine, Jamie P; Gurtner, Geoffrey C

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.

Cellular Dysfunction in the Diabetic Fibroblast

PubMed Central

Lerman, Oren Z.; Galiano, Robert D.; Armour, Mary; Levine, Jamie P.; Gurtner, Geoffrey C.

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O2), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 ± 1.3 pg/ml versus 34.8 ± 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients. PMID:12507913

Assessing the Role of STAT3 in DC Differentiation and Autologous DC Immunotherapy in Mouse Models of GBM

PubMed Central

Assi, Hikmat; Espinosa, Jaclyn; Suprise, Sarah; Sofroniew, Michael; Doherty, Robert; Zamler, Daniel; Lowenstein, Pedro R.; Castro, Maria G.

2014-01-01

Cellular microenvironments, particularly those found in tumors, elicit a tolerogenic DC phenotype which can attenuate immune responses. Central to this process is the STAT3-mediated signaling cascade. As a transcription factor and oncogene, STAT3 promotes the expression of genes which allow tumor cells to proliferate, migrate and evade apoptosis. More importantly, activation of STAT3 in tumor infiltrating immune cells has been shown to be responsible, in part, for their immune-suppressed phenotype. The ability of STAT3 to orchestrate a diverse set of immunosuppressive instructions has made it an attractive target for cancer vaccines. Using a conditional hematopoietic knockout mouse model of STAT3, we evaluated the impact of STAT3 gene ablation on the differentiation of dendritic cells from bone marrow precursors. We also assessed the impact of STAT3 deletion on phagocytosis, maturation, cytokine secretion and antigen presentation by GM-CSF derived DCs in vitro. In addition to in vitro studies, we compared the therapeutic efficacy of DC vaccination using STAT3 deficient DCs to wild type counterparts in an intracranial mouse model of GBM. Our results indicated the following pleiotropic functions of STAT3: hematopoietic cells which lacked STAT3 were unresponsive to Flt3L and failed to differentiate as DCs. In contrast, STAT3 was not required for GM-CSF induced DC differentiation as both wild type and STAT3 null bone marrow cells gave rise to similar number of DCs. STAT3 also appeared to regulate the response of GM-CSF derived DCs to CpG. STAT3 null DCs expressed high levels of MHC-II, secreted more IL-12p70, IL-10, and TNFα were better antigen presenters in vitro. Although STAT3 deficient DCs displayed an enhanced activated phenotype in culture, they elicited comparable therapeutic efficacy in vivo compared to their wild type counterparts when utilized in vaccination paradigms in mice bearing intracranial glioma tumors. PMID:24806510

Assessing the role of STAT3 in DC differentiation and autologous DC immunotherapy in mouse models of GBM.

PubMed

Assi, Hikmat; Espinosa, Jaclyn; Suprise, Sarah; Sofroniew, Michael; Doherty, Robert; Zamler, Daniel; Lowenstein, Pedro R; Castro, Maria G

2014-01-01

Cellular microenvironments, particularly those found in tumors, elicit a tolerogenic DC phenotype which can attenuate immune responses. Central to this process is the STAT3-mediated signaling cascade. As a transcription factor and oncogene, STAT3 promotes the expression of genes which allow tumor cells to proliferate, migrate and evade apoptosis. More importantly, activation of STAT3 in tumor infiltrating immune cells has been shown to be responsible, in part, for their immune-suppressed phenotype. The ability of STAT3 to orchestrate a diverse set of immunosuppressive instructions has made it an attractive target for cancer vaccines. Using a conditional hematopoietic knockout mouse model of STAT3, we evaluated the impact of STAT3 gene ablation on the differentiation of dendritic cells from bone marrow precursors. We also assessed the impact of STAT3 deletion on phagocytosis, maturation, cytokine secretion and antigen presentation by GM-CSF derived DCs in vitro. In addition to in vitro studies, we compared the therapeutic efficacy of DC vaccination using STAT3 deficient DCs to wild type counterparts in an intracranial mouse model of GBM. Our results indicated the following pleiotropic functions of STAT3: hematopoietic cells which lacked STAT3 were unresponsive to Flt3L and failed to differentiate as DCs. In contrast, STAT3 was not required for GM-CSF induced DC differentiation as both wild type and STAT3 null bone marrow cells gave rise to similar number of DCs. STAT3 also appeared to regulate the response of GM-CSF derived DCs to CpG. STAT3 null DCs expressed high levels of MHC-II, secreted more IL-12p70, IL-10, and TNFα were better antigen presenters in vitro. Although STAT3 deficient DCs displayed an enhanced activated phenotype in culture, they elicited comparable therapeutic efficacy in vivo compared to their wild type counterparts when utilized in vaccination paradigms in mice bearing intracranial glioma tumors.

Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: Implications for therapy

PubMed Central

Tovar, Christian; Rosinski, James; Filipovic, Zoran; Higgins, Brian; Kolinsky, Kenneth; Hilton, Holly; Zhao, Xiaolan; Vu, Binh T.; Qing, Weiguo; Packman, Kathryn; Myklebost, Ola; Heimbrook, David C.; Vassilev, Lyubomir T.

2006-01-01

The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53–MDM2 interaction. PMID:16443686

Reduced Infectivity in Cattle for an Outer Membrane Protein Mutant of Anaplasma marginale

PubMed Central

Brayton, Kelly A.; Magunda, Forgivemore; Munderloh, Ulrike G.; Kelley, Karen L.; Barbet, Anthony F.

2015-01-01

Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10::himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species. PMID:25595772

Hepatitis B "e" antigen-mediated inhibition of HBV replication fitness and transcription efficiency in vitro.

PubMed

Samal, Jasmine; Kandpal, Manish; Vivekanandan, Perumal

2015-10-01

A mutation at nucleotide 1896 (G1896A) is the most common cause for the loss of HBeAg. In contrast to clinical data, cell culture studies report a high-replicating phenotype for the G1896A mutant. Differences between the wild-type and the G1896A mutant in early steps of HBV replication including the synthesis of pre-genomic RNA and transcripts have not been investigated. The G1896A mutant is associated with higher replication fitness, transcription efficiency and higher levels of secreted HBsAg than the wild-type. Interestingly, trans-complementation of the G1896A mutant with HBeAg lowers the replication fitness and transcriptionefficiency to levels comparable to that of the wild-type. Our results highlight the role of HBeAg in modulating the early steps in HBV replication. In sum, our findings highlight the role of HBeAg in regulating hepatitis B virus replication fitness and transcription efficiency and new insights on the early steps of replication in the G1896A mutant. Copyright © 2015 Elsevier Inc. All rights reserved.

Hydrogen Cyanide Produced by Pseudomonas chlororaphis O6 Exhibits Nematicidal Activity against Meloidogyne hapla

PubMed Central

Kang, Beom Ryong; Anderson, Anne J.; Kim, Young Cheol

2018-01-01

Root-knot nematodes (Meloidogyne spp.) are parasites that attack many field crops and orchard trees, and affect both the quantity and quality of the products. A root-colonizing bacterium, Pseudomonas chlororaphis O6, possesses beneficial traits including strong nematicidal activity. To determine the molecular mechanisms involved in the nematicidal activity of P. chlororaphis O6, we constructed two mutants; one lacking hydrogen cyanide production, and a second lacking an insecticidal toxin, FitD. Root drenching with wild-type P. chlororaphis O6 cells caused juvenile mortality in vitro and in planta. Efficacy was not altered in the fitD mutant compared to the wild-type but was reduced in both bioassays for the mutant lacking hydrogen cyanide production. The reduced number of galls on tomato plants caused by the wild-type strain was comparable to that of a standard chemical nematicide. These findings suggest that hydrogen cyanide-producing root colonizers, such as P. chlororaphis O6, could be formulated as “green” nematicides that are compatible with many crops and offer agricultural sustainability. PMID:29422786

Synthesis, Docking, In Vitro and In Vivo Antimalarial Activity of Hybrid 4-aminoquinoline-1,3,5-triazine Derivatives Against Wild and Mutant Malaria Parasites.

PubMed

Bhat, Hans Raj; Singh, Udaya Pratap; Gahtori, Prashant; Ghosh, Surajit Kumar; Gogoi, Kabita; Prakash, Anil; Singh, Ramendra K

2015-09-01

A new series of hybrid 4-aminoquinoline-1,3,5-triazine derivatives was synthesized by a four-step reaction. Target compounds were screened for in vitro antimalarial activity against chloroquine-sensitive (3D-7) and chloroquine-resistant (RKL-2) strains of Plasmodium falciparum. Compounds exhibited, by and large, good antimalarial activity against the resistant strain, while two of them, that is 8g and 8a, displayed higher activity against both the strains of P. falciparum. Additionally, docking study was performed on both wild (1J3I.pdb) and quadruple mutant (N51I, C59R, S108 N, I164L, 3QG2.pdb) type pf-DHFR-TS to highlight the structural features of hybrid molecules. © 2014 John Wiley & Sons A/S.

Src Homology 2–containing 5-Inositol Phosphatase (SHIP) Suppresses an Early Stage of Lymphoid Cell Development through Elevated Interleukin-6 Production by Myeloid Cells in Bone Marrow

PubMed Central

Nakamura, Koji; Kouro, Taku; Kincade, Paul W.; Malykhin, Alexander; Maeda, Kazuhiko; Coggeshall, K. Mark

2004-01-01

The Src homology (SH)2–containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP−/− animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP−/− mice. Lineage-negative marrow (Lin−) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP−/− mice. Furthermore, culture supernatants of SHIP−/− Lin− cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP−/− Lin− cells contained more IL-6 transcripts than wild-type Lin− cells, and neutralizing anti–IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP−/− Lin− cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin− bone marrow cells reproduced the phenotype of SHIP−/− bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow. PMID:14718513

Possible involvement of inefficient cleavage of preprovasopressin by signal peptidase as a cause for familial central diabetes insipidus.

PubMed Central

Ito, M; Oiso, Y; Murase, T; Kondo, K; Saito, H; Chinzei, T; Racchi, M; Lively, M O

1993-01-01

A transition of G to A at nucleotide position 279 in exon 1 of the vasopressin gene has been identified in patients with familial central diabetes insipidus. The mutation predicts an amino acid substitution of Thr (ACG) for Ala (GCG) at the COOH terminus of the signal peptide in preprovasopression (preproVP). Translation in vitro of wild-type and mutant mRNAs produced 19-kD preproVPs. When translated in the presence of canine pancreatic rough microsomes, wild-type preproVP was converted to a 21-kD protein, whereas the mutant mRNA produced proteins of 21 kD and 23 kD. NH2-terminal amino acid sequence analysis revealed that the 21-kD proteins from the wild-type and the mutants were proVPs generated by the proteolytic cleavage of the 19-residue signal peptide and the addition of carbohydrate. Accordingly, mutant preproVP was cleaved at the correct site after Thr-19, but the efficiency of cleavage by signal peptidase was < 25% that observed for the wild-type preproVP, resulting in the formation of a predominant glycosylated but uncleaved 23-kD product. These data suggest that inefficient processing of preproVP produced by the mutant allele is possibly involved in the pathogenesis of diabetes insipidus in the affected individuals. Images PMID:8514868

Controlled synthesis of the DSF cell–cell signal is required for biofilm formation and virulence in Xanthomonas campestris

PubMed Central

Torres, Pablo S; Malamud, Florencia; Rigano, Luciano A; Russo, Daniela M; Marano, María Rosa; Castagnaro, Atilio P; Zorreguieta, Angeles; Bouarab, Kamal; Dow, John Maxwell; Vojnov, Adrián A

2007-01-01

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell–cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence. PMID:17635553

The Candida albicans Pho4 Transcription Factor Mediates Susceptibility to Stress and Influences Fitness in a Mouse Commensalism Model

PubMed Central

Urrialde, Verónica; Prieto, Daniel; Pla, Jesús; Alonso-Monge, Rebeca

2016-01-01

The Pho4 transcription factor is required for growth under low environmental phosphate concentrations in Saccharomyces cerevisiae. A characterization of Candida albicans pho4 mutants revealed that these cells are more susceptible to both osmotic and oxidative stress and that this effect is diminished in the presence of 5% CO2 or anaerobiosis, reflecting the relevance of oxygen metabolism in the Pho4-mediated response. A pho4 mutant was as virulent as wild type strain when assayed in the Galleria mellonella infection model and was even more resistant to murine macrophages in ex vivo killing assays. The lack of Pho4 neither impairs the ability to colonize the murine gut nor alters the localization in the gastrointestinal tract. However, we found that Pho4 influenced the colonization of C. albicans in the mouse gut in competition assays; pho4 mutants were unable to attain high colonization levels when inoculated simultaneously with an isogenic wild type strain. Moreover, pho4 mutants displayed a reduced adherence to the intestinal mucosa in a competitive ex vivo assays with wild type cells. In vitro competitive assays also revealed defects in fitness for this mutant compared to the wild type strain. Thus, Pho4, a transcription factor involved in phosphate metabolism, is required for adaptation to stress and fitness in C. albicans. PMID:27458452

Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

NASA Technical Reports Server (NTRS)

Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

2002-01-01

It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM(+/-)) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM(+/-) cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage.

Effect of IAA on in vitro growth and colonization of Nostoc in plant roots

PubMed Central

Hussain, Anwar; Shah, Syed T.; Rahman, Hazir; Irshad, Muhammad; Iqbal, Amjad

2015-01-01

Nostoc is widely known for its ability to fix atmospheric nitrogen and the establishment of symbiotic relationship with a wide range of plants from various taxonomic groups. Several strains of Nostoc produce phytohormones that promote growth of its plant partners. Nostoc OS-1 was therefore selected for study because of the presence of putative ipdC gene that encodes a key enzyme to produce Indole-3-acetic acid (IAA). The results indicated that both cellular and released IAA was found high with increasing incubation time and reached to a peak value (i.e., 21 pmol mg-1ch-a) on the third week as determined by UPLC-ESI-MS/MS. Also the Nostoc OS-1 strain efficiently colonized the roots and promoted the growth of rice as well as wheat under axenic conditions and induced ipdC gene that suggested the possible involvement of IAA in these phenotypes. To confirm the impact of IAA on root colonization efficiency and plant promoting phenotypes of Nostoc OS-1, an ipdC knockout mutant was generated by homologous recombinant method. The amount of releasing IAA, in vitro growth, root colonization, and plant promoting efficiency of the ipdC knockout mutant was observed significantly lower than wild type strain under axenic conditions. Importantly, these phenotypes were restored to wild-type levels when the ipdC knockout mutant was complemented with wild type ipdC gene. These results together suggested that ipdC and/or synthesized IAA of Nostoc OS-1 is required for its efficient root colonization and plant promoting activity. PMID:25699072

Edwardsiella ictaluri Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish (Ictalurus punctatus) Macrophages▿

PubMed Central

Booth, Natha J.; Beekman, Judith B.; Thune, Ronald L.

2009-01-01

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome. PMID:19749068

A novel ICK mutation causes ciliary disruption and lethal endocrine-cerebro-osteodysplasia syndrome.

PubMed

Oud, Machteld M; Bonnard, Carine; Mans, Dorus A; Altunoglu, Umut; Tohari, Sumanty; Ng, Alvin Yu Jin; Eskin, Ascia; Lee, Hane; Rupar, C Anthony; de Wagenaar, Nathalie P; Wu, Ka Man; Lahiry, Piya; Pazour, Gregory J; Nelson, Stanley F; Hegele, Robert A; Roepman, Ronald; Kayserili, Hülya; Venkatesh, Byrappa; Siu, Victoria M; Reversade, Bruno; Arts, Heleen H

2016-01-01

Endocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology. Through homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G > T; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells. Through identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy.

THE ROLE OF ELECTRICAL SIGNALS IN MURINE CORNEAL WOUND RE-EPITHELIALISATION

PubMed Central

Kucerova, R.; Walczysko, P.; Reid, B.; Ou, J.; Leiper, L. J.; Rajnicek, A. M.; McCaig, C. D.; Zhao, M.; Collinson, J. M.

2011-01-01

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for electric fields in wound re-epithelialisation, using the Pax6+/− mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type and Pax6+/− corneal epithelial cells showed increased migration speeds in response to applied electric fields in vitro. However, only Pax6+/+ cells demonstrated directional galvanotaxis towards the cathode, with activation of pSrc signalling, polarised to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6+/− corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as wild-type. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for wild-type or mutant corneas. Furthermore, during healing in vivo, no polarisation of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous electric fields do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signalling initiation. PMID:20945376

Engineering thermal stability of L-asparaginase by in vitro directed evolution.

PubMed

Kotzia, Georgia A; Labrou, Nikolaos E

2009-03-01

L-asparaginase (EC 3.5.1.1, L-ASNase) catalyses the hydrolysis of l-Asn, producing L-Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukaemia. In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability. A library of enzyme variants was created by a staggered extension process using the genes that code for the L-ASNases from Erwinia chrysanthemi and Erwinia carotovora. The amino acid sequences of the parental L-ASNases show 77% identity, but their half-inactivation temperature (T(m)) differs by 10 degrees C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The T(m) of this variant was 55.8 degrees C, whereas the wild-type enzyme has a T(m) of 46.4 degrees C. At 50 degrees C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respectively. Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability. Screening of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu mutation confers optimal thermostability.

Wild-type phosphatase and tensin homolog deleted on chromosome 10 improved the sensitivity of cells to rapamycin through regulating phosphorylation of Akt in esophageal squamous cell carcinoma.

PubMed

Lu, Z; Wang, J; Zheng, Y; Yang, S; Liu, M; Chen, X; Wang, C; Hou, G

2017-02-01

Esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed cancers in China, but the etiology and mode of carcinogenesis of this disease remain poorly understood. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), as a negative regulator of Akt/mTOR pathway, frequently mutates or is inactive in many cancers. Although mTOR has been thought a promising cancer therapeutic target, the sensitivity of tumor cells to rapamycin was still to be revaluated. In this study, we measured the effects of rapamycin on cell proliferation and phosphorylation of Akt in ESCC cells with varying degrees of differentiation. And then, the relationship between PTEN status and the sensitivity of cells to rapamycin was investigated in EC9706 cells with or without wild-type PTEN in vitro and in vivo. The results demonstrated ESCC cells with poor differentiation were insensitive to rapamycin of high concentration and rapamycin obviously promoted the phosphorylation of Akt in these cells, but it had no obvious effects on p-Akt in cells with well differentiation. Also, we showed that wild-type PTEN improved the sensitivity of poor differentiation cells to rapamycin through inhibiting phosphorylation of Akt in vitro and in vivo. This study explored the possible molecular mechanism of some ESCC cells insensitive to rapamycin and provided a measure for treating ESCC patients with PTEN inactivation using mTOR inhibitors. © 2015 International Society for Diseases of the Esophagus.

Annexin 1 Modulates Monocyte-Endothelial Cell Interaction In Vitro and Cell Migration In Vivo in the Human SCID Mouse Transplantation Model1

PubMed Central

Perretti, Mauro; Ingegnoli, Francesca; Wheller, Samantha K.; Blades, Mark C.; Solito, Egle; Pitzalis, Costantino

2015-01-01

The effect of the glucocorticoid inducible protein annexin 1 (ANXA1) on the process of monocytic cell migration was studied using transfected U937 cells expressing variable protein levels. An antisense (AS) (36.4AS; ~50% less ANXA1) and a sense (S) clone (15S; overexpressing the bioactive 24-kDa fragment) together with the empty plasmid CMV clone were obtained and compared with wild-type U937 cells in various models of cell migration in vitro and in vivo. 15S-transfected U937 cells displayed a reduced (50%) degree of trans-endothelial migration in response to stromal cell-derived factor-1α (CXC chemokine ligand 12 (CXCL12)). In addition, the inhibitory role of endogenous ANXA1 on U937 cell migration in vitro was confirmed by the potentiating effect of a neutralizing anti-ANXA1 serum. Importantly, overexpression of ANXA1 in clone 15S inhibited the extent of cell migration into rheumatoid synovial grafts transplanted into SCID mice. ANXA1 inhibitory effects were not due to modifications in adhesion molecule or CXCL12 receptor (CXCR4) expression as shown by the similar amounts of surface molecules found in transfected and wild-type U937 cells. Likewise, an equal chemotactic response to CXCL12 in vitro excluded an intrinsic defect in cell motility in clones 15S and 36.4AS. These data strongly support the notion that ANXA1 critically interferes with a leukocyte endothelial step essential for U937 cell, and possibly monocyte, transmigration both in vitro and in vivo. PMID:12165536

Investigations of fungicide exposure and pollen type on bee health: Insights from in vitro-reared solitary bees

USDA-ARS?s Scientific Manuscript database

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while th...

Identification and Function of Ets Target Genes Involved in Lung Cancer Progression

DTIC Science & Technology

2013-10-01

Upadhyay et al., 2006). Tissues with Lkb1 mutations have higher Pea3 expression than wild type tissues (Upadhyay et al., 2006). In vitro, Pea3...with rotation. AG beads (Santa Cruz Biotechnology, Inc.), BSA (100 μg/ml), and salmon sperm (500 μg/ml) were added to samples. Samples were rotated (4

Evaluation of the impact of quorum sensing transcriptional regulator SdiA on long-term persistence and fecal shedding of Escherichia coli O157:H7 in weaned calves.

PubMed

Sharma, V K; Bearson, S M D

2013-04-01

Escherichia coli O157:H7 (O157) colonization of bovine intestine is mediated through the locus of enterocyte effacement (LEE)-encoded type III secretion system and secreted virulence proteins that promote colonization of the recto-anal junction (RAJ) of the large intestine of cattle. The quorum sensing transcriptional regulator SdiA, a homolog of LuxR, has been shown in vitro to repress LEE strongly when overexpressed from a multi-copy recombinant plasmid or when its activity is enhanced by the binding of N-acyl-L-homoserine lactones (AHLs), the quorum sensing signals that are detected by SdiA. Since LEE has been shown to be essential for colonization and persistence of O157 in bovine intestine, we examined whether a mutation in sdiA, which normally represses LEE in vitro, would also exert negative effect on colonization and long-term persistence of O157 in weaned calves. Ten-week old weaned calves (n = 4/group) were inoculated orally with 10(10) cfu of either the wild-type or sdiA mutant strain. Initial fecal shedding of the sdiA mutant and the wild-type strain were similar in magnitude and declined during the first 2 weeks post-inoculation. The sdiA mutant was detected in feces of only one of the four calves at low levels (≥10(2) cfu/g feces) from days 19 - 27 post-inoculation, whereas, the fecal shedding of the wild-type strain persisted at approximately 4-logs in all four calves from days 19 - 27. We also confirmed that SdiA represses ler, which encodes a positive transcriptional regulator of LEE, in response to AHLs, and reduces adherence of O157 to HEp-2 cells. In conclusion, this study demonstrates that although in vitro the sdiA gene represses LEE and LEE-mediated adherence to cultured cells, the presence of sdiA is necessary for colonization of bovine large intestine that in turn promotes persistent fecal shedding of O157 by these animals. Published by Elsevier Ltd.

Expression Analysis of a Highly Adherent and Cytotoxic Small Colony Variant of Pseudomonas aeruginosa Isolated from a Lung of a Patient with Cystic Fibrosis†

PubMed Central

von Götz, Franz; Häussler, Susanne; Jordan, Doris; Saravanamuthu, Senthil Selvan; Wehmhöner, Dirk; Strüßmann, André; Lauber, Joerg; Attree, Ina; Buer, Jan; Tümmler, Burkhard; Steinmetz, Ivo

2004-01-01

The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants. PMID:15175297

Autophagy and Oxidative Stress in Gliomas with IDH1 Mutations

PubMed Central

Gilbert, Misty R.; Liu, Yinxing; Neltner, Janna; Pu, Hong; Morris, Andrew; Sunkara, Manjula; Pittman, Thomas; Kyprianou, Natasha; Horbinski, Craig

2013-01-01

IDH1 mutations in gliomas associate with longer survival. Prooxidant and antiproliferative effects of IDH1 mutations and its D-2-hydroxyglutarate (2-HG) product have been described in vitro, but inconsistently observed. It is also unclear whether overexpression of mutant IDH1 in wild-type cells accurately phenocopies the effects of endogenous IDH1-mutations on tumor apoptosis and autophagy. Herein we investigated the effects of 2-HG and mutant IDH1 overexpression on proliferation, apoptosis, oxidative stress, and autophagy in IDH1 wild-type glioma cells, and compared those results with patient-derived tumors. 2-HG reduced viability and proliferation of U87MG and LN18 cells, triggered apoptosis in LN18 cells, and autophagy in U87MG cells. In vitro studies and flank xenografts of U87MG cells overexpressing R132H IDH1 exhibited increased oxidative stress, including increases of both manganese superoxide dismutase (MnSOD) and p62. Patient-derived IDH1-mutant tumors showed no significant differences in apoptosis or autophagy, but showed p62 accumulation and actually trended toward reduced MnSOD expression. These data indicate that mutant IDH1 and 2-HG can induce oxidative stress, autophagy, and apoptosis, but these effects vary greatly according to cell type. PMID:24150401

The Sep1 Mutant of Saccharomyces Cerevisiae Arrests in Pachytene and Is Deficient in Meiotic Recombination

PubMed Central

Tishkoff, D. X.; Rockmill, B.; Roeder, G. S.; Kolodner, R. D.

1995-01-01

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae promotes homologous pairing of DNA in vitro and sep1 mutants display pleiotropic phenotypes in both vegetative and meiotic cells. In this study, we examined in detail the ability of the sep1 mutant to progress through meiosis I prophase and to undergo meiotic recombination. In meiotic return-to-growth experiments, commitment to meiotic recombination began at the same time in wild type and mutant; however, recombinants accumulated at decreased rates in the mutant. Gene conversion eventually reached nearly wild-type levels, whereas crossing over reached 15-50% of wild type. In an assay of intrachromosomal pop-out recombination, the sep1, dmc1 and rad51 single mutations had only small effects; however, pop-out recombination was virtually eliminated in the sep1 dmc1 and sep1 rad51 double mutants, providing evidence for multiple recombination pathways. Analysis of meiotic recombination intermediates indicates that the sep1 mutant is deficient in meiotic double-strand break repair. In a physical assay, the formation of mature reciprocal recombinants in the sep1 mutant was delayed relative to wild type and ultimately reached only 50% of the wild-type level. Electron microscopic analysis of meiotic nuclear spreads indicates that the sep1δ mutant arrests in pachytene, with apparently normal synaptonemal complex. This arrest is RAD9-independent. We hypothesize that the Sep1 protein participates directly in meiotic recombination and that other strand exchange enzymes, acting in parallel recombination pathways, are able to substitute partially for the absence of the Sep1 protein. PMID:7713413

Role of Maltose Enzymes in Glycogen Synthesis by Escherichia coli▿

PubMed Central

Park, Jong-Tae; Shim, Jae-Hoon; Tran, Phuong Lan; Hong, In-Hee; Yong, Hwan-Ung; Oktavina, Ershita Fitria; Nguyen, Hai Dang; Kim, Jung-Wan; Lee, Tae Soo; Park, Sung-Hoon; Boos, Winfried; Park, Kwan-Hwa

2011-01-01

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase. PMID:21421758

Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo

PubMed Central

1994-01-01

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed. PMID:8294513

Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola

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

Cho, Yangrae; Ohm, Robin A.; Grigoriev, Igor V.

Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. To identify molecular determinants of pathogenicity, we created non-pathogenic mutants of a transcription factor-encoding gene, AbPf2. The frequency and timing of germination and appressorium formation on host plants were similar between the non-pathogenic abpf2 mutants and wild-type A. brassicicola. The mutants were also similar in vitro to wild-type A. brassicicola in terms of vegetative growth, conidium production, and responses to a phytoalexin, reactive oxygen species and osmolites. The hyphae of the mutants grew slowly but did not cause disease symptoms on the surface of host plants. Transcripts of the AbPf2more » gene increased exponentially soon after wild-type conidia contacted their host plants . A small amount of AbPf2 protein, as monitored using GFP fusions, was present in young, mature conidia. The protein level decreased during saprophytic growth, but increased and was located primarily in fungal nuclei during pathogenesis. Levels of the proteins and transcripts sharply decreased following colonization of host tissues beyond the initial infection site. When expression of the transcription factor was induced in the wild-type during early pathogenesis, 106 fungal genes were also induced in the wild-type but not in the abpf2 mutants. Notably, 33 of the 106 genes encoded secreted proteins, including eight putative effector proteins. Plants inoculated with abpf2 mutants expressed higher levels of genes associated with photosynthesis, the pentose phosphate pathway and primary metabolism, but lower levels of defense-related genes. Our results suggest that AbPf2 is an important regulator of pathogenesis, but does not affect other cellular processes in A. brassicicola.« less

Differential Inhibition of Ex-Vivo Tumor Kinase Activity by Vemurafenib in BRAF(V600E) and BRAF Wild-Type Metastatic Malignant Melanoma

PubMed Central

Tahiri, Andliena; Røe, Kathrine; Ree, Anne H.; de Wijn, Rik; Risberg, Karianne; Busch, Christian; Lønning, Per E.; Kristensen, Vessela; Geisler, Jürgen

2013-01-01

Background Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed. Methodology In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status. Results Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro. Conclusions Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers. PMID:24023633

Activation of VIP signaling enhances immunosuppressive effect of MDSCs on CMV-induced adaptive immunity.

PubMed

Forghani, Parvin; Petersen, Christopher T; Waller, Edmund K

2017-10-10

Vasoactive intestinal peptide (VIP) is recognized as a potent anti-inflammatory factor which affects both the innate and adaptive arms of the immune system. These effects include, but are not limited to, inhibition of T cell proliferation and disruption of immune homeostasis. Myeloid-derived suppressor cells (MDSC) are an immune regulatory cell type that has been described in settings of cancer and infectious disease._Here we demonstrate a reduced circulating monocytic MDSCs in the VIP -/- vs. wild type MCMV. VIP-/- MDSCs secretes less NO upon stimulation with LPS and interferon that relatively lose the ability to suppress T cells activation in vitro compared to wild type MDSCs._Considering the importance of VIP in immunomodulation, the possible effect of VIP in the suppressive function of MDSC populations following CMV infection remains unknown. We describe the possible role of VIP in the regulation of anti-CMV activity of T cells through the activation of MDSCs.

Mutation of the Kunitz-type proteinase inhibitor domain in the amyloid β-protein precursor abolishes its anti-thrombotic properties in vivo.

PubMed

Xu, Feng; Davis, Judianne; Hoos, Michael; Van Nostrand, William E

2017-07-01

Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid β-protein precursor (AβPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AβPP are abundant in brain. Regions of AβPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AβPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo. To determine the extent of KPI function of AβPP in regulating cerebral thrombosis we generated a recombinant reactive center KPI R13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AβPP knock out and AβPP/KPI R13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage. Recombinant mutant KPI R13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AβPP/KPI R13I mutant mice were similarly deficient as AβPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage. We demonstrate that the anti-thrombotic function of AβPP primarily resides in the KPI activity of the protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

A hybrid two-component system protein from Azospirillum brasilense Sp7 was involved in chemotaxis.

PubMed

Cui, Yanhua; Tu, Ran; Wu, Lixian; Hong, Yuanyuan; Chen, Sanfeng

2011-09-20

We here report the sequence and functional analysis of org35 of Azospirillum brasilense Sp7, which was originally identified to be able to interact with NifA in yeast-two-hybrid system. The org35 encodes a hybrid two-component system protein, including N-terminal PAS domains, a histidine kinase (HPK) domain and a response regulator (RR) domain in C-terminal. To determine the function of the Org35, a deletion-insertion mutant in PAS domain [named Sp7353] and a complemental strain Sp7353C were constructed. The mutant had reduced chemotaxis ability compared to that of wild-type, and the complemental strain was similar to the wild-type strain. These data suggested that the A. brasilense org35 played a key role in chemotaxis. Variants containing different domains of the org35 were expressed, and the functions of these domains were studied in vitro. Phosphorylation assays in vitro demonstrated that the HPK domain of Org35 possessed the autokinase activity and that the phosphorylated HPK was able to transfer phosphate groups to the RR domain. The result indicated Org35 was a phosphorylation-communicating protein. Copyright © 2010 Elsevier GmbH. All rights reserved.

Differential transcription patterns in wild-type and glycoprotein G-deleted infectious laryngotracheitis viruses.

PubMed

Mahmoudian, Alireza; Markham, Philip F; Noormohammadi, Amir H; Devlin, Joanne M; Browning, Glenn F

2013-01-01

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in poultry throughout the world. Recently the role of glycoprotein G (gG) in ILTV pathogenesis has been investigated and it has been shown to have chemokine-binding activity. An ILTV vaccine candidate deficient in gG has been developed and the deletion has been shown to alter the host's immune response to the virus. To understand the effect of the gG gene on transcription of other viral genes, the global expression profile of 72 ILTV genes in gG-deleted and wild-type ILTVs were investigated both in vivo and in vitro using quantitative reverse transcription-polymerase chain reaction. Several genes were differentially expressed in the different viruses in LMH cell cultures or in the tracheas of infected birds, and the expression of a number of genes, including ICP27, gC, gJ, Ul7 and UL40, differed significantly both in vivo and in vitro, suggesting that they had direct or indirect roles in virulence. This study has provided insights into the interactions between gG and other ILTV genes that may have a role in virulence.

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia

PubMed Central

Foster, K. Wade; Liu, Zhaoli; Nail, Clinton D.; Li, Xingnan; Fitzgerald, Thomas J.; Bailey, Sarah K.; Frost, Andra R.; Louro, Iuri D.; Townes, Tim M.; Paterson, Andrew J.; Kudlow, Jeffrey E.; Lobo-Ruppert, Susan M.; Ruppert, J. Michael

2006-01-01

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions. PMID:15674344

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia.

PubMed

Foster, K Wade; Liu, Zhaoli; Nail, Clinton D; Li, Xingnan; Fitzgerald, Thomas J; Bailey, Sarah K; Frost, Andra R; Louro, Iuri D; Townes, Tim M; Paterson, Andrew J; Kudlow, Jeffrey E; Lobo-Ruppert, Susan M; Ruppert, J Michael

2005-02-24

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes, KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions.

Type 2 diabetes impairs the ability of skeletal muscle pericytes to augment postischemic neovascularization in db/db mice.

PubMed

Hayes, Katherine L; Messina, Louis M; Schwartz, Lawrence M; Yan, Jinglian; Burnside, Amy S; Witkowski, Sarah

2018-05-01

Peripheral artery disease is an atherosclerotic occlusive disease that causes limb ischemia and has few effective noninterventional treatments. Stem cell therapy is promising, but concomitant diabetes may limit its effectiveness. We evaluated the therapeutic potential of skeletal muscle pericytes to augment postischemic neovascularization in wild-type and type 2 diabetic (T2DM) mice. Wild-type C57BL/6J and leptin receptor spontaneous mutation db/db T2DM mice underwent unilateral femoral artery excision to induce limb ischemia. Twenty-four hours after ischemia induction, CD45 - CD34 - CD146 + skeletal muscle pericytes or vehicle controls were transplanted into ischemic hindlimb muscles. At postoperative day 28, pericyte transplantation augmented blood flow recovery in wild-type mice (79.3 ± 5% vs. 61.9 ± 5%; P = 0.04), but not in T2DM mice (48.6% vs. 46.3 ± 5%; P = 0.51). Pericyte transplantation augmented collateral artery enlargement in wild-type (26.7 ± 2 μm vs. 22.3 ± 1 μm, P = 0.03), but not T2DM mice (20.4 ± 1.4 μm vs. 18.5 ± 1.2 μm, P = 0.14). Pericyte incorporation into collateral arteries was higher in wild-type than in T2DM mice ( P = 0.002). Unexpectedly, pericytes differentiated into Schwann cells in vivo. In vitro, Insulin increased Nox2 expression and decreased tubular formation capacity in human pericytes. These insulin-induced effects were reversed by N-acetylcysteine antioxidant treatment. In conclusion, T2DM impairs the ability of pericytes to augment neovascularization via decreased collateral artery enlargement and impaired engraftment into collateral arteries, potentially via hyperinsulinemia-induced oxidant stress. While pericytes show promise as a unique form of stem cell therapy to increase postischemic neovascularization, characterizing the molecular mechanisms by which T2DM impairs their function is essential to achieve their therapeutic potential.

Human T-Cell Lymphotropic Virus Type 1 Open Reading Frame II-Encoded p30II Is Required for In Vivo Replication: Evidence of In Vivo Reversion

PubMed Central

Silverman, Lee R.; Phipps, Andrew J.; Montgomery, Andrew; Ratner, Lee; Lairmore, Michael D.

2004-01-01

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma and exhibits high genetic stability in vivo. HTLV-1 contains four open reading frames (ORFs) in its pX region. ORF II encodes two proteins, p30II and p13II, both of which are incompletely characterized. p30II localizes to the nucleus or nucleolus and has distant homology to the transcription factors Oct-1, Pit-1, and POU-M1. In vitro studies have demonstrated that at low concentrations, p30II differentially regulates cellular and viral promoters through an interaction with CREB binding protein/p300. To determine the in vivo significance of p30II, we inoculated rabbits with cell lines expressing either a wild-type clone of HTLV-1 (ACH.1) or a clone containing a mutation in ORF II, which eliminated wild-type p30II expression (ACH.30.1). ACH.1-inoculated rabbits maintained higher HTLV-1-specific antibody titers than ACH.30.1-inoculated rabbits, and all ACH.1-inoculated rabbits were seropositive for HTLV-1, whereas only two of six ACH.30.1-inoculated rabbits were seropositive. Provirus could be consistently PCR amplified from peripheral blood mononuclear cell (PBMC) DNA in all ACH.1-inoculated rabbits but in only three of six ACH.30.1-inoculated rabbits. Quantitative competitive PCR indicated higher PBMC proviral loads in ACH.1-inoculated rabbits. Interestingly, sequencing of ORF II from PBMC of provirus-positive ACH.30.1-inoculated rabbits revealed a reversion to wild-type sequence with evidence of early coexistence of mutant and wild-type sequence. Our data provide evidence that HTLV-1 must maintain its key accessory genes to survive in vivo and that in vivo pressures select for maintenance of wild-type ORF II gene products during the early course of infection. PMID:15047799

Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli.

PubMed

Borst, D W; Blumenthal, R M; Matthews, R G

1996-12-01

Most studies of global regulatory proteins are performed in vitro or involve phenotypic comparisons between wild-type and mutant strains. We report the use of strains in which the gene for the leucine-responsive regulatory protein (lrp) is transcribed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters for the purpose of continuously varying the in vivo concentration of Lrp. To obtain a broad range of Lrp concentrations, strains were employed that contained the lrp fusion either in the chromosome (I. C. Blomfield, P. J. Calie, K. J. Eberhardt, M. S. McClain, and B. I. Eisenstein, J. Bacteriol. 175:27-36, 1993) or on a multicopy plasmid. Western blot (immunoblot) analysis with polyclonal antiserum to Lrp confirmed that Lrp levels could be varied more than 70-fold by growing the strains in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium containing different amounts of IPTG. Expression of an Lrp-regulated gltB::lacZ operon fusion was measured over this range of Lrp concentrations. beta-Galactosidase activity rose with increasing Lrp levels up to the level of Lrp found in wild-type strains, at which point expression is maximal. The presence of leucine in the medium increased the level of Lrp necessary to achieve half-maximal expression of the gltB::lacZ fusion, as predicted by earlier in vitro studies (B. R. Ernsting, J. W. Denninger, R. M. Blumenthal, and R. G. Matthews, J. Bacteriol. 175:7160-7169, 1993). Interestingly, levels of Lrp greater than those in wild-type cells interfered with activation of gltB::lacZ expression. The growth rate of cultures correlated with the intracellular Lrp concentration: levels of Lrp either lower or higher than wild-type levels resulted in significantly slower growth rates. Thus, the level of Lrp in the cell appears to be optimal for rapid growth in minimal medium, and the gltBDF control region is designed to give maximal expression at this Lrp level.

Role of Klebsiella pneumoniae Type 1 and Type 3 Fimbriae in Colonizing Silicone Tubes Implanted into the Bladders of Mice as a Model of Catheter-Associated Urinary Tract Infections

PubMed Central

Murphy, Caitlin N.; Mortensen, Martin S.; Krogfelt, Karen A.

2013-01-01

Catheter-associated urinary tract infections are biofilm-mediated infections that cause a significant economic and health burden in nosocomial environments. Using a newly developed murine model of this type of infection, we investigated the role of fimbriae in implant-associated urinary tract infections by the Gram-negative bacterium Klebsiella pneumoniae, which is a proficient biofilm former and a commonly isolated nosocomial pathogen. Studies have shown that type 1 and type 3 fimbriae are involved in attachment and biofilm formation in vitro, and these fimbrial types are suspected to be important virulence factors during infection. To test this hypothesis, the virulence of fimbrial mutants was assessed in independent challenges in which mouse bladders were inoculated with the wild type or a fimbrial mutant and in coinfection studies in which the wild type and fimbrial mutants were inoculated together to assess the results of a direct competition in the urinary tract. Using these experiments, we were able to show that both fimbrial types serve to enhance colonization and persistence. Additionally, a double mutant had an additive colonization defect under some conditions, indicating that both fimbrial types have unique roles in the attachment and persistence in the bladder and on the implant itself. All of these mutants were outcompeted by the wild type in coinfection experiments. Using these methods, we are able to show that type 1 and type 3 fimbriae are important colonization factors in the murine urinary tract when an implanted silicone tube is present. PMID:23753626

SUSTAINABLE DEVELOPMENT OF AMOMUM VILLOSUM: A SYSTEMATIC INVESTIGATION ON THREE DIFFERENT PRODUCTION MODES.

PubMed

Lai, Yun-Feng; Chen, Ling-Xiao; Chen, Yu-Ning; Zhao, Jing; Leong, Fong; Li, Xi-Wen; Yang, Qing; Li, Peng; Hu, Hao

2016-01-01

Amomum Villosum (A. Villosum ), called Chunsharen in Chinese, is widely used in treating gastrointestinal disease. Its clinical benefits have been confirmed by both in vitro and in vivo studies. Facing the shortage of wild A. Villosum , artificial cultivating and natural fostering have been practiced in recent years. Therefore, it would be wondered whether the three different types of A. Villosum are comparable or not, particularly the herbal qualities, technological challenges, ecological impacts and economic benefits. In this study, we combined quality research by using GC-MS, and field investigation to provide a systematic assessment about the three types of A. Villosum from these four aspects. It found that the wild type had low output and was in an endangered situation. The artificial cultivation had larger agriculturing area with higher productivity, but faced the ecological challenges. Lastly, the natural fostering type generated the highest economic benefit and relatively low ecological impact. In addition, the natural fostering type had relatively better quality than the other types. Therefore, it suggests that natural fostering can be applied for long-term sustainable development of A. Villosum .

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

Liu, Fabao; Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071; You, Xiaona

Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migrationmore » in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.« less

Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants

PubMed Central

Vagasi, Alexandra S.; Rahman, Mohammad M.; Chaudhari, Snehal N.; Kipreos, Edward T.

2017-01-01

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016. PMID:28868332

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

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

Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen speciesmore » compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.« less

Mutation design of a thermophilic Rubisco based on three-dimensional structure enhances its activity at ambient temperature.

PubMed

Fujihashi, Masahiro; Nishitani, Yuichi; Kiriyama, Tomohiro; Aono, Riku; Sato, Takaaki; Takai, Tomoyuki; Tagashira, Kenta; Fukuda, Wakao; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

2016-10-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Identifying the Deleterious Effect of Rare LHX4 Allelic Variants, a Challenging Issue

PubMed Central

Rochette, Claire; Jullien, Nicolas; Saveanu, Alexandru; Caldagues, Emmanuelle; Bergada, Ignacio; Braslavsky, Debora; Pfeifer, Marija; Reynaud, Rachel; Herman, Jean-Paul; Barlier, Anne; Brue, Thierry; Enjalbert, Alain; Castinetti, Frederic

2015-01-01

LHX4 is a LIM homeodomain transcription factor involved in the early steps of pituitary ontogenesis. To date, 8 heterozygous LHX4 mutations have been reported as responsible of combined pituitary hormone deficiency (CPHD) in Humans. We identified 4 new LHX4 heterozygous allelic variants in patients with congenital hypopituitarism: W204X, delK242, N271S and Q346R. Our objective was to determine the role of LHX4 variants in patients’ phenotypes. Heterologous HEK293T cells were transfected with plasmids encoding for wild-type or mutant LHX4. Protein expression was analysed by Western Blot, and DNA binding by electro-mobility shift assay experiments. Target promoters of LHX4 were cotransfected with wild type or mutant LHX4 to test the transactivating abilities of each variant. Our results show that the W204X mutation was associated with early GH and TSH deficiencies and later onset ACTH deficiency. It led to a truncated protein unable to bind to alpha-Gsu promoter binding consensus sequence. W204X was not able to activate target promoters in vitro. Cotransfection experiments did not favour a dominant negative effect. In contrast, all other mutants were able to bind the promoters and led to an activation similar as that observed with wild type LHX4, suggesting that they were likely polymorphisms. To conclude, our study underlines the need for functional in vitro studies to ascertain the role of rare allelic variants of LHX4 in disease phenotypes. It supports the causative role of the W204X mutation in CPHD and adds up childhood onset ACTH deficiency to the clinical spectrum of the various phenotypes related to LHX4 mutations. PMID:25955177

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

PubMed Central

Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

2013-01-01

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

Antagonism of CD11b with neutrophil inhibitory factor (NIF) inhibits vascular lesions in diabetic retinopathy.

PubMed

Veenstra, Alexander A; Tang, Jie; Kern, Timothy S

2013-01-01

Leukocytes and proteins that govern leukocyte adhesion to endothelial cells play a causal role in retinal abnormalities characteristic of the early stages of diabetic retinopathy, including diabetes-induced degeneration of retinal capillaries. Leukocyte integrin αmβ2 (CD11b/CD18, MAC1), a protein mediating adhesion, has been shown to mediate damage to endothelial cells by activated leukocytes in vitro. We hypothesized that Neutrophil Inhibitory Factor (NIF), a selective antagonist of integrin αmβ2, would inhibit the diabetes-induced degeneration of retinal capillaries by inhibiting the excessive interaction between leukocytes and retinal endothelial cells in diabetes. Wild type animals and transgenic animals expressing NIF were made diabetic with streptozotocin and assessed for diabetes-induced retinal vascular abnormalities and leukocyte activation. To assess if the leukocyte blocking therapy compromised the immune system, animals were challenged with bacteria. Retinal superoxide production, leukostasis and leukocyte superoxide production were increased in wild type mice diabetic for 10 weeks, as was the ability of leukocytes isolated from diabetic animals to kill retinal endothelial cells in vitro. Retinal capillary degeneration was significantly increased in wild type mice diabetic 40 weeks. In contrast, mice expressing NIF did not develop any of these abnormalities, with the exception that non-diabetic and diabetic mice expressing NIF generated greater amounts of superoxide than did similar mice not expressing NIF. Importantly, NIF did not significantly impair the ability of mice to clear an opportunistic bacterial challenge, suggesting that NIF did not compromise immune surveillance. We conclude that antagonism of CD11b (integrin αmβ2) by NIF is sufficient to inhibit early stages of diabetic retinopathy, while not compromising the basic immune response.

Antagonism of CD11b with Neutrophil Inhibitory Factor (NIF) Inhibits Vascular Lesions in Diabetic Retinopathy

PubMed Central

Veenstra, Alexander A.; Tang, Jie; Kern, Timothy S.

2013-01-01

Leukocytes and proteins that govern leukocyte adhesion to endothelial cells play a causal role in retinal abnormalities characteristic of the early stages of diabetic retinopathy, including diabetes-induced degeneration of retinal capillaries. Leukocyte integrin αmβ2 (CD11b/CD18, MAC1), a protein mediating adhesion, has been shown to mediate damage to endothelial cells by activated leukocytes in vitro. We hypothesized that Neutrophil Inhibitory Factor (NIF), a selective antagonist of integrin αmβ2, would inhibit the diabetes-induced degeneration of retinal capillaries by inhibiting the excessive interaction between leukocytes and retinal endothelial cells in diabetes. Wild type animals and transgenic animals expressing NIF were made diabetic with streptozotocin and assessed for diabetes-induced retinal vascular abnormalities and leukocyte activation. To assess if the leukocyte blocking therapy compromised the immune system, animals were challenged with bacteria. Retinal superoxide production, leukostasis and leukocyte superoxide production were increased in wild type mice diabetic for 10 weeks, as was the ability of leukocytes isolated from diabetic animals to kill retinal endothelial cells in vitro. Retinal capillary degeneration was significantly increased in wild type mice diabetic 40 weeks. In contrast, mice expressing NIF did not develop any of these abnormalities, with the exception that non-diabetic and diabetic mice expressing NIF generated greater amounts of superoxide than did similar mice not expressing NIF. Importantly, NIF did not significantly impair the ability of mice to clear an opportunistic bacterial challenge, suggesting that NIF did not compromise immune surveillance. We conclude that antagonism of CD11b (integrin αmβ2) by NIF is sufficient to inhibit early stages of diabetic retinopathy, while not compromising the basic immune response. PMID:24205223

Isothiocyanates Reduce Mercury Accumulation via an Nrf2-Dependent Mechanism during Exposure of Mice to Methylmercury

PubMed Central

Toyama, Takashi; Shinkai, Yasuhiro; Yasutake, Akira; Uchida, Koji; Yamamoto, Masayuki

2011-01-01

Background: Methylmercury (MeHg) exhibits neurotoxicity through accumulation in the brain. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) plays an important role in reducing the cellular accumulation of MeHg. Objectives: We investigated the protective effect of isothiocyanates, which are known to activate Nrf2, on the accumulation of mercury after exposure to MeHg in vitro and in vivo. Methods: We used primary mouse hepatocytes in in vitro experiments and mice as an in vivo model. We used Western blotting, luciferase assays, atomic absorption spectrometry assays, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays, and we identified toxicity in mice based on hind-limb flaccidity and mortality. Results: The isothiocyanates 6-methylsulfinylhexyl isothiocyanate (6-HITC) and sulforaphane (SFN) activated Nrf2 and up-regulated downstream proteins associated with MeHg excretion, such as glutamate-cysteine ligase, glutathione S-transferase, and multidrug resistance–associated protein, in primary mouse hepatocytes. Under these conditions, intracellular glutathione levels increased in wild-type but not Nrf2-deficient primary mouse hepatocytes. Pretreatment with 6-HITC and SFN before MeHg exposure suppressed cellular accumulation of mercury and cytotoxicity in wild-type but not Nrf2-deficient primary mouse hepatocytes. In comparison, in vivo administration of MeHg to Nrf2-deficient mice resulted in increased sensitivity to mercury concomitant with an increase in mercury accumulation in the brain and liver. Injection of SFN before administration of MeHg resulted in a decrease in mercury accumulation in the brain and liver of wild-type, but not Nrf2-deficient, mice. Conclusions: Through activation of Nrf2, 6-HITC and SFN can suppress mercury accumulation and intoxication caused by MeHg intake. PMID:21382770

Effects of CYP2D6 Status on Harmaline Metabolism, Pharmacokinetics and Pharmacodynamics, and a Pharmacogenetics-Based Pharmacokinetic Model

PubMed Central

Wu, Chao; Jiang, Xi-Ling; Shen, Hong-Wu; Yu, Ai-Ming

2009-01-01

Harmaline is a β-carboline alkaloid showing neuroprotective and neurotoxic properties. Our recent studies have revealed an important role for cytochrome P450 2D6 (CYP2D6) in harmaline O-demethylation. This study, therefore, aimed to delineate the effects of CYP2D6 phenotype/genotype on harmaline metabolism, pharmacokinetics (PK) and pharmacodynamics (PD), and to develop a pharmacogenetics mechanism-based compartmental PK model. In vitro kinetic studies on metabolite formation in human CYP2D6 extensive metabolizer (EM) and poor metabolizer (PM) hepatocytes indicated that harmaline O-demethylase activity (Vmax/Km) was about 9-fold higher in EM hepatocytes. Substrate depletion showed mono-exponential decay trait, and estimated in vitro harmaline clearance (CLint, μL/min/106 cells) was significantly lower in PM hepatocytes (28.5) than EM hepatocytes (71.1). In vivo studies in CYP2D6-humanized and wild-type mouse models showed that wild-type mice were subjected to higher and longer exposure to harmaline (5 and 15 mg/kg; i.v. and i.p.), and more severe hypothermic responses. The PK/PD data were nicely described by our pharmacogenetics-based PK model involving the clearance of drug by CYP2D6 (CLCYP2D6) and other mechanisms (CLother), and an indirect response PD model, respectively. Wild-type mice were also more sensitive to harmaline in marble-burying tests, as manifested by significantly lower ED50 and steeper Hill slope. These findings suggest that distinct CYP2D6 status may cause considerable variations in harmaline metabolism, PK and PD. In addition, the pharmacogenetics-based PK model may be extended to define PK difference caused by other polymorphic drug-metabolizing enzyme in different populations. PMID:19445902

Dual targeting of wild-type and mutant p53 by small molecule RITA results in the inhibition of N-Myc and key survival oncogenes and kills neuroblastoma cells in vivo and in vitro.

PubMed

Burmakin, Mikhail; Shi, Yao; Hedström, Elisabeth; Kogner, Per; Selivanova, Galina

2013-09-15

Restoration of the p53 function in tumors is a promising therapeutic strategy due to the high potential of p53 as tumor suppressor and the fact that established tumors depend on p53 inactivation for their survival. Here, we addressed the question whether small molecule RITA can reactivate p53 in neuroblastoma and suppress the growth of neuroblastoma cells in vitro and in vivo. The ability of RITA to inhibit growth and to induce apoptosis was shown in seven neuroblastoma cell lines. Mechanistic studies were carried out to determine the p53 dependence and the molecular mechanism of RITA-induced apoptosis in neuroblastoma, using cell viability assays, RNAi silencing, co-immunoprecipitation, qPCR, and Western blotting analysis. In vivo experiments were conducted to study the effect of RITA on human neuroblastoma xenografts in mice. RITA induced p53-dependent apoptosis in a set of seven neuroblastoma cell lines, carrying wild-type or mutant p53; it activated p53 and triggered the expression of proapoptotic p53 target genes. Importantly, p53 activated by RITA inhibited several key oncogenes that are high-priority targets for pharmacologic anticancer strategies in neuroblastoma, including N-Myc, Aurora kinase, Mcl-1, Bcl-2, Wip-1, MDM2, and MDMX. Moreover, RITA had a strong antitumor effect in vivo. Reactivation of wild-type and mutant p53 resulting in the induction of proapoptotic factors along with ablation of key oncogenes by compounds such as RITA may be a highly effective strategy to treat neuroblastoma. ©2013 AACR.

Atm heterozygous mice are more sensitive to radiation-induced cataracts than are their wild-type counterparts

PubMed Central

Worgul, Basil V.; Smilenov, Lubomir; Brenner, David J.; Junk, Anna; Zhou, Wei; Hall, Eric J.

2002-01-01

It is important to know whether the human population includes genetically predisposed radiosensitive subsets. In vitro studies have shown that cells from individuals homozygous for ataxia telangiectasia (A-T) are much more radiosensitive than cells from unaffected individuals. Although cells heterozygous for the ATM gene (ATM+/−) may be slightly more radiosensitive in vitro, it remained to be determined whether the greater susceptibility of ATM+/− cells translates into an increased sensitivity for late effects in vivo, though there is a suggestion that radiotherapy patients that are heterozygous for the ATM gene may be more at risk of developing late normal tissue damage. We chose cataractogenesis in the lens as a means to assay for the effects of ATM deficiency in a late-responding tissue. One eye of wild-type, Atm heterozygous and homozygous knockout mice was exposed to 0.5-, 1.0-, 2.0-, or 4.0-Gy x rays. The animals were followed weekly for cataract development by conventional slit-lamp biomicroscopy. Cataract development in the animals of all three groups was strongly dependent on dose. The lenses of homozygous mice were the first to opacify at any given dose. Most important in the present context is that cataracts appeared earlier in the heterozygous versus wild-type animals. The data suggest that ATM heterozygotes in the human population may also be radiosensitive. This may influence the choice of individuals destined to be exposed to higher than normal doses of radiation, such as astronauts, and may also suggest that radiotherapy patients who are ATM heterozygotes could be predisposed to increased late normal tissue damage. PMID:12119422

Immunohistochemical Characterization of Connexin43 Expression in a Mouse Model of Diabetic Retinopathy and in Human Donor Retinas

PubMed Central

Mugisho, Odunayo O.; Green, Colin R.; Zhang, Jie; Binz, Nicolette; Acosta, Monica L.; Rakoczy, Elizabeth

2017-01-01

Diabetic retinopathy (DR) develops due to hyperglycemia and inflammation-induced vascular disruptions in the retina with connexin43 expression patterns in the disease still debated. Here, the effects of hyperglycemia and inflammation on connexin43 expression in vitro in a mouse model of DR and in human donor tissues were evaluated. Primary human retinal microvascular endothelial cells (hRMECs) were exposed to high glucose (HG; 25 mM) or pro-inflammatory cytokines IL-1β and TNF-α (10 ng/mL each) or both before assessing connexin43 expression. Additionally, connexin43, glial fibrillary acidic protein (GFAP), and plasmalemma vesicular associated protein (PLVAP) were labeled in wild-type (C57BL/6), Akita (diabetic), and Akimba (DR) mouse retinas. Finally, connexin43 and GFAP expression in donor retinas with confirmed DR was compared to age-matched controls. Co-application of HG and cytokines increased connexin43 expression in hRMECs in line with results seen in mice, with no significant difference in connexin43 or GFAP expression in Akita but higher expression in Akimba compared to wild-type mice. On PLVAP-positive vessels, connexin43 was higher in Akimba but unchanged in Akita compared to wild-type mice. Connexin43 expression appeared higher in donor retinas with confirmed DR compared to age-matched controls, similar to the distribution seen in Akimba mice and correlating with the in vitro results. Although connexin43 expression seems reduced in diabetes, hyperglycemia and inflammation present in the pathology of DR seem to increase connexin43 expression, suggesting a causal role of connexin43 channels in the disease progression. PMID:29186067

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney

PubMed Central

Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

2017-01-01

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis. PMID:27758862

Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2

PubMed Central

2010-01-01

Background Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions. PMID:20122244

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

PubMed

Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A; Moraes, Carlos T; Sanderson, Thomas H; Stemmler, Timothy L; Grossman, Lawrence I; Kagan, Valerian E; Brunzelle, Joseph S; Salomon, Arthur R; Edwards, Brian F P; Hüttemann, Maik

2017-01-06

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨ m hyperpolarization, a known cause of ROS and trigger of apoptosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Period 2 gene deletion abolishes β-endorphin neuronal response to ethanol

PubMed Central

Agapito, Maria; Mian, Nadia; Boyadjieva, Nadka I.; Sarkar, Dipak K.

2010-01-01

Background Ethanol exposure during early life has been shown to permanently alter the circadian expression of clock regulatory genes and the β-endorphin precursor proopiomelanocortin (POMC) gene in the hypothalamus. Ethanol also alters the stress- and immune-regulatory functions of β-endorphin neurons in laboratory rodents. Our aim was to determine whether the circadian clock regulatory Per2 gene modulates the action of ethanol on β-endorphin neurons in mice. Methods Per2 mutant (mPer2Brdml) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol-regulated β-endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The β-endorphin neuronal activity following acute and chronic ethanol treatments, was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme-linked immunosorbent assay (ELISA). Results Per2 mutant mice showed a higher basal level of β-endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison to control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory β-endorphin secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH β-endorphin levels to acute and chronic ethanol challenges in vivo. Conclusions These results suggest for the first time that the Per2 gene may be critically involved in regulating β-endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating β-endorphin neuronal responses to ethanol. PMID:20586752

Growth Arrest-Specific Protein 6 is Hepatoprotective Against Ischemia/Reperfusion Injury

PubMed Central

Llacuna, Laura; Bárcena, Cristina; Bellido-Martín, Lola; Fernández, Laura; Stefanovic, Milica; Marí, Montserrat; García-Ruiz, Carmen; Fernández-Checa, José C.; de Frutos, Pablo García; Morales, Albert

2010-01-01

Growth arrest-specific gene 6 (GAS6) promotes growth and cell survival during tissue repair and development in different organs, including the liver. However, the specific role of GAS6 in liver ischemia/reperfusion (I/R) injury has not been previously addressed. Here, we report an early increase in serum GAS6 levels following I/R exposure. Moreover, unlike wild type mice, Gas6-/- mice were highly sensitive to partial hepatic I/R, with 90% of mice dying within 12 hours of reperfusion due to massive hepatocellular injury. I/R induced early hepatic AKT phosphorylation in wild type but not in Gas6-/- mice, without significant changes in JNK phosphorylation or nuclear NF-κB translocation, whereas hepatic IL-1β and TNF mRNA levels were higher in Gas6-/- mice compared to wild type mice. In line with the in vivo data, in vitro studies indicated that GAS6 induced AKT phosphorylation in primary mouse hepatocytes protecting them from hypoxia-induced cell death, while GAS6 diminished lipopolysaccharide (LPS)-induced cytokine expression (IL-1β and TNF) in murine macrophages. Finally, in vivo recombinant GAS6 treatment not only rescued GAS6 knockout mice from I/R-induced severe liver damage, but also attenuated hepatic damage in wild type mice following I/R. In conclusion, our data uncover GAS6 as a new player in liver I/R injury, emerging as a potential therapeutic target to reduce post-ischemic hepatic damage. PMID:20730776

Mechanisms Relevant to the Enhanced Virulence of a Dihydroxynaphthalene-Melanin Metabolically Engineered Entomopathogen

PubMed Central

Tseng, Min-Nan; Chung, Chia-Ling; Tzean, Shean-Shong

2014-01-01

The entomopathogenic fungus Metarhizium anisopliae MA05-169 is a transformant strain that has been metabolically engineered to express dihydroxynaphthalene-melanin biosynthesis genes. In contrast to the wild type strain, the transformant displays a greater resistance to environmental stress and a higher virulence toward target insect host. However, the underlying mechanisms for these characteristics remain unclear; hence experiments were initiated to explore the possible mechanism(s) through physiological and molecular approaches. Although both transformant and wild type strains could infect and share the same insect host range, the former germinated faster and produced more appressoria than the latter, both in vivo and in vitro. The transformant showed a significantly shorter median lethal time (LT50) when infecting the diamondback moth (Plutella xylostella) and the striped flea beetle (Phyllotreta striolata), than the wild type. Additionally, the transformant was more tolerant to reactive oxygen species (ROS), produced 40-fold more orthosporin and notably overexpressed the transcripts of the pathogenicity-relevant hydrolytic enzymes (chitinase, protease, and phospholipase) genes in vivo. In contrast, appressorium turgor pressure and destruxin A content were slightly decreased compared to the wild type. The transformant's high anti-stress tolerance, its high virulence against five important insect pests (cowpea aphid Aphis craccivora, diamondback moth Pl. xylostella, striped flea beetle Ph. striolata, and silverleaf whitefly Bemisia argentifolii) and its capacity to colonize the root system are key properties for its potential bio-control field application. PMID:24662974

Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

PubMed Central

Sniatecki, Jan J.; Goloborodko, Evgeny; Steege, Andreas; Zavaritskaya, Olga; Vetter, Jan M.; Grus, Franz H.; Patzak, Andreas; Wess, Jürgen; Pfeiffer, Norbert

2011-01-01

Purpose. To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. Methods. Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor–deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. Results. Only mRNA for the M3 receptor was detected in retinal arterioles. Thus, M3 receptor–deficient mice (M3R−/−) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R−/− mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R−/−, mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non–subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor Nω-nitro-l-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Conclusions. These findings provide evidence that endothelial M3 receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase. PMID:21873683

Neutropenia restores virulence to an attenuated Cu,Zn superoxide dismutase-deficient Haemophilus ducreyi strain in the swine model of chancroid.

PubMed

San Mateo, L R; Toffer, K L; Orndorff, P E; Kawula, T H

1999-10-01

Haemophilus ducreyi causes chancroid, a sexually transmitted cutaneous genital ulcer disease associated with increased heterosexual transmission of human immunodeficiency virus. H. ducreyi expresses a periplasmic copper-zinc superoxide dismutase (Cu, Zn SOD) that protects the bacterium from killing by exogenous superoxide in vitro. We hypothesized that the Cu,Zn SOD would protect H. ducreyi from immune cell killing, enhance survival, and affect ulcer development in vivo. In order to test this hypothesis and study the role of the Cu,Zn SOD in H. ducreyi pathogenesis, we compared a Cu,Zn SOD-deficient H. ducreyi strain to its isogenic wild-type parent with respect to survival and ulcer development in immunocompetent and immunosuppressed pigs. The Cu,Zn SOD-deficient strain was recovered from significantly fewer inoculated sites and in significantly lower numbers than the wild-type parent strain or a merodiploid (sodC+ sodC) strain after infection of immunocompetent pigs. In contrast, survival of the wild-type and Cu,Zn SOD-deficient strains was not significantly different in pigs that were rendered neutropenic by treatment with cyclophosphamide. Ulcer severity in pigs was not significantly different between sites inoculated with wild type and sites inoculated with Cu,Zn SOD-deficient H. ducreyi. Our data suggest that the periplasmic Cu,Zn SOD is an important virulence determinant in H. ducreyi, protecting the bacterium from host immune cell killing and contributing to survival and persistence in the host.

Neutropenia Restores Virulence to an Attenuated Cu,Zn Superoxide Dismutase-Deficient Haemophilus ducreyi Strain in the Swine Model of Chancroid

PubMed Central

San Mateo, Lani R.; Toffer, Kristen L.; Orndorff, Paul E.; Kawula, Thomas H.

1999-01-01

Haemophilus ducreyi causes chancroid, a sexually transmitted cutaneous genital ulcer disease associated with increased heterosexual transmission of human immunodeficiency virus. H. ducreyi expresses a periplasmic copper-zinc superoxide dismutase (Cu,Zn SOD) that protects the bacterium from killing by exogenous superoxide in vitro. We hypothesized that the Cu,Zn SOD would protect H. ducreyi from immune cell killing, enhance survival, and affect ulcer development in vivo. In order to test this hypothesis and study the role of the Cu,Zn SOD in H. ducreyi pathogenesis, we compared a Cu,Zn SOD-deficient H. ducreyi strain to its isogenic wild-type parent with respect to survival and ulcer development in immunocompetent and immunosuppressed pigs. The Cu,Zn SOD-deficient strain was recovered from significantly fewer inoculated sites and in significantly lower numbers than the wild-type parent strain or a merodiploid (sodC+ sodC) strain after infection of immunocompetent pigs. In contrast, survival of the wild-type and Cu,Zn SOD-deficient strains was not significantly different in pigs that were rendered neutropenic by treatment with cyclophosphamide. Ulcer severity in pigs was not significantly different between sites inoculated with wild type and sites inoculated with Cu,Zn SOD-deficient H. ducreyi. Our data suggest that the periplasmic Cu,Zn SOD is an important virulence determinant in H. ducreyi, protecting the bacterium from host immune cell killing and contributing to survival and persistence in the host. PMID:10496915

The Klebsiella pneumoniae O Antigen Contributes to Bacteremia and Lethality during Murine Pneumonia

PubMed Central

Shankar-Sinha, Sunita; Valencia, Gabriel A.; Janes, Brian K.; Rosenberg, Jessica K.; Whitfield, Chris; Bender, Robert A.; Standiford, Ted J.; Younger, John G.

2004-01-01

Bacterial surface carbohydrates are important pathogenic factors in gram-negative pneumonia infections. Among these factors, O antigen has been reported to protect pathogens against complement-mediated killing. To examine further the role of O antigen, we insertionally inactivated the gene encoding a galactosyltransferase necessary for serotype O1 O-antigen synthesis (wbbO) from Klebsiella pneumoniae 43816. Analysis of the mutant lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the absence of O antigen. In vitro, there were no detectable differences between wild-type K. pneumoniae and the O-antigen-deficient mutant in regard to avid binding by murine complement C3 or resistance to serum- or whole-blood-mediated killing. Nevertheless, the 72-h 50% lethal dose of the wild-type strain was 30-fold greater than that of the mutant (2 × 103 versus 6 × 104 CFU) after intratracheal injection in ICR strain mice. Despite being less lethal, the mutant organism exhibited comparable intrapulmonary proliferation at 24 h compared to the level of the wild type. Whole-lung chemokine expression (CCL3 and CXCL2) and bronchoalveolar inflammatory cell content were also similar between the two infections. However, whereas the wild-type organism produced bacteremia within 24 h of infection in every instance, bacteremia was not seen in mutant-infected mice. These results suggest that during murine pneumonia caused by K. pneumoniae, O antigen contributes to lethality by increasing the propensity for bacteremia and not by significantly changing the early course of intrapulmonary infection. PMID:14977947

Critical role of IFN-gamma in CFA-mediated protection of NOD mice from diabetes development.

PubMed

Mori, Yoshiko; Kodaka, Tetsuro; Kato, Takako; Kanagawa, Edith M; Kanagawa, Osami

2009-11-01

IFN-gamma signaling-deficient non-obese diabetic (NOD) mice develop diabetes with similar kinetics to those of wild-type NOD mice. However, the immunization of IFN-gamma signaling-deficient NOD mice with CFA failed to induce long-term protection, whereas wild-type NOD mice receiving CFA remained diabetes-free. CFA also failed to protect IFN-gamma receptor-deficient (IFN-gammaR(-/-)) NOD mice from the autoimmune rejection of transplanted islets, as it does in diabetic NOD mice, and from disease transfer by spleen cells from diabetic NOD mice. These data clearly show that the pro-inflammatory cytokine IFN-gamma is necessary for the CFA-mediated protection of NOD mice from diabetes. There is no difference in the T(h)1/T(h)17 balance between IFN-gammaR(-/-) NOD and wild-type NOD mice. There is also no difference in the total numbers and percentages of regulatory T (Treg) cells in the lymph node CD4(+) T-cell populations between IFN-gammaR(-/-) NOD and wild-type NOD mice. However, pathogenic T cells lacking IFN-gammaR are resistant to the suppressive effect of Treg cells, both in vivo and in vitro. Therefore, it is likely that CFA-mediated protection against diabetes development depends on a change in the balance between Treg cells and pathogenic T cells, and IFN-gamma signaling seems to control the susceptibility of pathogenic T cells to the inhibitory activity of Treg cells.

Structural basis of nSH2 regulation and lipid binding in PI3Kα.

PubMed

Miller, Michelle S; Schmidt-Kittler, Oleg; Bolduc, David M; Brower, Evan T; Chaves-Moreira, Daniele; Allaire, Marc; Kinzler, Kenneth W; Jennings, Ian G; Thompson, Philip E; Cole, Philip A; Amzel, L Mario; Vogelstein, Bert; Gabelli, Sandra B

2014-07-30

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.

Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers

PubMed Central

Cox, David G.; Simard, Jacques; Sinnett, Daniel; Hamdi, Yosr; Soucy, Penny; Ouimet, Manon; Barjhoux, Laure; Verny-Pierre, Carole; McGuffog, Lesley; Healey, Sue; Szabo, Csilla; Greene, Mark H.; Mai, Phuong L.; Andrulis, Irene L.; Thomassen, Mads; Gerdes, Anne-Marie; Caligo, Maria A.; Friedman, Eitan; Laitman, Yael; Kaufman, Bella; Paluch, Shani S.; Borg, Åke; Karlsson, Per; Stenmark Askmalm, Marie; Barbany Bustinza, Gisela; Nathanson, Katherine L.; Domchek, Susan M.; Rebbeck, Timothy R.; Benítez, Javier; Hamann, Ute; Rookus, Matti A.; van den Ouweland, Ans M.W.; Ausems, Margreet G.E.M.; Aalfs, Cora M.; van Asperen, Christi J.; Devilee, Peter; Gille, Hans J.J.P.; Peock, Susan; Frost, Debra; Evans, D. Gareth; Eeles, Ros; Izatt, Louise; Adlard, Julian; Paterson, Joan; Eason, Jacqueline; Godwin, Andrew K.; Remon, Marie-Alice; Moncoutier, Virginie; Gauthier-Villars, Marion; Lasset, Christine; Giraud, Sophie; Hardouin, Agnès; Berthet, Pascaline; Sobol, Hagay; Eisinger, François; Bressac de Paillerets, Brigitte; Caron, Olivier; Delnatte, Capucine; Goldgar, David; Miron, Alex; Ozcelik, Hilmi; Buys, Saundra; Southey, Melissa C.; Terry, Mary Beth; Singer, Christian F.; Dressler, Anne-Catharina; Tea, Muy-Kheng; Hansen, Thomas V.O.; Johannsson, Oskar; Piedmonte, Marion; Rodriguez, Gustavo C.; Basil, Jack B.; Blank, Stephanie; Toland, Amanda E.; Montagna, Marco; Isaacs, Claudine; Blanco, Ignacio; Gayther, Simon A.; Moysich, Kirsten B.; Schmutzler, Rita K.; Wappenschmidt, Barbara; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Niederacher, Dieter; Sutter, Christian; Gadzicki, Dorothea; Fiebig, Britta; Caldes, Trinidad; Laframboise, Rachel; Nevanlinna, Heli; Chen, Xiaoqing; Beesley, Jonathan; Spurdle, Amanda B.; Neuhausen, Susan L.; Ding, Yuan C.; Couch, Fergus J.; Wang, Xianshu; Peterlongo, Paolo; Manoukian, Siranoush; Bernard, Loris; Radice, Paolo; Easton, Douglas F.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Stoppa-Lyonnet, Dominique; Mazoyer, Sylvie; Sinilnikova, Olga M.

2011-01-01

Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA1 gene would modify the risk of breast cancer in carriers of BRCA1 mutations. A total of 9874 BRCA1 mutation carriers were available in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) for haplotype analyses of BRCA1. Women carrying the rare allele of single nucleotide polymorphism rs16942 on the wild-type copy of BRCA1 were at decreased risk of breast cancer (hazard ratio 0.86, 95% confidence interval 0.77–0.95, P = 0.003). Promoter in vitro assays of the major BRCA1 haplotypes showed that common polymorphisms in the regulatory region alter its activity and that this effect may be attributed to the differential binding affinity of nuclear proteins. In conclusion, variants on the wild-type copy of BRCA1 modify risk of breast cancer among carriers of BRCA1 mutations, possibly by altering the efficiency of BRCA1 transcription. PMID:21890493

Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for cinnamyl alcohol dehydrogenase.

PubMed

Fornalé, Silvia; Capellades, Montserrat; Encina, Antonio; Wang, Kan; Irar, Sami; Lapierre, Catherine; Ruel, Katia; Joseleau, Jean-Paul; Berenguer, Jordi; Puigdomènech, Pere; Rigau, Joan; Caparrós-Ruiz, David

2012-07-01

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

Interactions between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and nontransformed tomato roots of either wild-type or AM-defective phenotypes in monoxenic cultures.

PubMed

Bago, Alberto; Cano, Custodia; Toussaint, Jean-Patrick; Smith, Sally; Dickson, Sandy

2006-09-01

Monoxenic symbioses between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and two nontransformed tomato root organ cultures (ROCs) were established. Wild-type tomato ROC from cultivar "RioGrande 76R" was employed as a control for mycorrhizal colonization and compared with its mutant line (rmc), which exhibits a highly reduced mycorrhizal colonization (rmc) phenotype. Structural features of the two root lines were similar when grown either in soil or under in vitro conditions, indicating that neither monoxenic culturing nor the rmc mutation affected root development or behavior. Colonization by G. intraradices in monoxenic culture of the wild-type line was low (<10%) but supported extensive development of extraradical mycelium, branched absorbing structures, and spores. The reduced colonization of rmc under monoxenic conditions (0.6%) was similar to that observed previously in soil. Extraradical development of runner hyphae was low and proportional to internal colonization. Few spores were produced. These results might suggest that carbon transfer may be modified in the rmc mutant. Our results support the usefulness of monoxenically obtained mycorrhizas for investigation of AM colonization and intraradical symbiotic functioning.

P-glycoprotein, but not Multidrug Resistance Protein 4, Plays a Role in the Systemic Clearance of Irinotecan and SN-38 in Mice

PubMed Central

Tagen, Michael; Zhuang, Yanli; Zhang, Fan; Harstead, K. Elaine; Shen, Jun; Schaiquevich, Paula; Fraga, Charles H.; Panetta, John C.; Waters, Christopher M.; Stewart, Clinton F.

2015-01-01

The ATP-binding cassette transporters P-glycoprotein (ABCB1, MDR1) and multidrug resistance protein 4 (MRP4) efflux irinotecan and its active metabolite SN-38 in vitro, and thus may contribute to system clearance of these compounds. Mdr1a/b−/−, Mrp4−/−, and wild-type mice were administered 20 or 40 mg/kg irinotecan, and plasma samples were collected for 6 hours. Irinotecan and SN-38 lactone and carboxylate were quantitated and data were analyzed with nonlinear mixed-effects modeling. Mdr1a/b genotype was a significant covariate for the clearance of both irinotecan lactone and SN-38 lactone. Exposures to irinotecan lactone and SN-38 lactone after a 40 mg/kg dose were 1.6-fold higher in Mdr1a/b−/− mice compared to wild-type mice. Plasma concentrations of irinotecan lactone, irinotecan carboxylate, and SN-38 lactone in Mrp4−/− mice were similar to the wild-type controls. These results suggest that P-gp plays a role in irinotecan and SN-38 elimination, but Mrp4 does not affect irinotecan or SN-38 plasma pharmacokinetics. PMID:20583968

Products of dark CO sub 2 fixation in pea root nodules support bacteroid metabolism. [Pisum sativum L

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

Rosendahl, L.; Pedersen, W.B.; Vance, C.P.

1990-05-01

Products of the nodule cytosol in vivo dark ({sup 14}C)CO{sub 2} fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv Bodil) nodules. The distribution of the metabolites of the dark CO{sub 2} fixation products was compared in effective (fix{sup +}) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix{sup {minus}}) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The {sup 14}C incorporation from ({sup 14}C)CO{sub 2} was about threefold greater in themore » wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the {sup 14}C label in the cytosol was found in organic acids in both symbioses. The results indicate a central role for nodule cytosol dark CO{sub 2} fixation in the supply of the bacteroids with dicarboxylic acids.« less

Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

PubMed Central

Law, G. H. Erica; Gandelman, Olga A.; Tisi, Laurence C.; Lowe, Christopher R.; Murray, James A. H.

2006-01-01

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications. PMID:16551268

Site-specific characterization of envelope protein N-glycosylation on Sanofi Pasteur's tetravalent CYD dengue vaccine.

PubMed

Dubayle, Jean; Vialle, Sandrine; Schneider, Diane; Pontvianne, Jérémy; Mantel, Nathalie; Adam, Olivier; Guy, Bruno; Talaga, Philippe

2015-03-10

Recently, several virus studies have shown that protein glycosylation play a fundamental role in the virus-host cell interaction. Glycosylation characterization of the envelope proteins in both insect and mammalian cell-derived dengue virus (DENV) has established that two potential glycosylation residues, the asparagine 67 and 153 can potentially be glycosylated. Moreover, it appears that the glycosylation of these two residues can influence dramatically the virus production and the infection spreading in either mosquito or mammalian cells. The Sanofi Pasteur tetravalent dengue vaccine (CYD) consists of four chimeric viruses produced in mammalian vero cells. As DENV, the CYDs are able to infect human monocyte-derived dendritic cells in vitro via C-type lectins cell-surface molecules. Despite the importance of this interaction, the specific glycosylation pattern of the DENV has not been clearly documented so far. In this paper, we investigated the structure of the N-linked glycans in the four CYD serotypes. Using MALDI-TOF analysis, the N-linked glycans of CYDs were found to be a mix of high-mannose, hybrid and complex glycans. Site-specific N-glycosylation analysis of CYDs using nanoLC-ESI-MS/MS demonstrates that both asparagine residues 67 and 153 are glycosylated. Predominant glycoforms at asparagine 67 are high mannose-type structures while mainly complex- and hybrid-type structures are detected at asparagine 153. In vitro studies have shown that the immunological consequences of infection by the CYD dengue viruses 1-4 versus the wild type parents are comparable in human monocyte-derived dendritic cells. Our E-protein glycan characterizations of CYD are consistent with those observations from the wild type parents and thus support in vitro studies. In addition, these data provide new insights for the role of glycans in the dengue virus-host cell interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phenotypic and genotypic characterization of influenza virus mutants selected with the sialidase fusion protein DAS181

PubMed Central

Triana-Baltzer, Gallen B.; Sanders, Rebecca L.; Hedlund, Maria; Jensen, Kellie A.; Aschenbrenner, Laura M.; Larson, Jeffrey L.; Fang, Fang

2011-01-01

Background Influenza viruses (IFVs) frequently achieve resistance to antiviral drugs, necessitating the development of compounds with novel mechanisms of action. DAS181 (Fludase®), a sialidase fusion protein, may have a reduced potential for generating drug resistance due to its novel host-targeting mechanism of action. Methods IFV strains B/Maryland/1/59 and A/Victoria/3/75 (H3N2) were subjected to >30 passages under increasing selective pressure with DAS181. The DAS181-selected IFV isolates were characterized in vitro and in mice. Results Despite extensive passaging, DAS181-selected viruses exhibited a very low level of resistance to DAS181, which ranged between 3- and 18-fold increase in EC50. DAS181-selected viruses displayed an attenuated phenotype in vitro, as exhibited by slower growth, smaller plaque size and increased particle to pfu ratios relative to wild-type virus. Further, the DAS181 resistance phenotype was unstable and was substantially reversed over time upon DAS181 withdrawal. In mice, the DAS181-selected viruses exhibited no greater virulence than their wild-type counterparts. Genotypic and phenotypic analysis of DAS181-selected viruses revealed mutations in the haemagglutinin (HA) and neuraminidase (NA) molecules and also changes in HA and NA function. Conclusions Results indicate that resistance to DAS181 is minimal and unstable. The DAS181-selected IFV isolates exhibit reduced fitness in vitro, likely due to altered HA and NA functions. PMID:21097900

MRG15, a component of HAT and HDAC complexes, is essential for proliferation and differentiation of neural precursor cells.

PubMed

Chen, Meizhen; Takano-Maruyama, Masumi; Pereira-Smith, Olivia M; Gaufo, Gary O; Tominaga, Kaoru

2009-05-15

Neurogenesis during development depends on the coordinated regulation of self-renewal and differentiation of neural precursor cells (NPCs). Chromatin regulation is a key step in self-renewal activity and fate decision of NPCs. However, the molecular mechanism or mechanisms of this regulation is not fully understood. Here, we demonstrate for the first time that MRG15, a chromatin regulator, is important for proliferation and neural fate decision of NPCs. Neuroepithelia from Mrg15-deficient embryonic brain are much thinner than those from control, and apoptotic cells increase in this region. We isolated NPCs from Mrg15-deficient and wild-type embryonic whole brains and produced neurospheres to measure the self-renewal and differentiation abilities of these cells in vitro. Neurospheres culture from Mrg15-deficient embryo grew less efficiently than those from wild type. Measurement of proliferation by means of BrdU (bromodeoxyuridine) incorporation revealed that Mrg15-deficient NPCs have reduced proliferation ability and apoptotic cells do not increase during in vitro culture. The reduced proliferation of Mrg15-deficient NPCs most likely accounts for the thinner neuroepithelia in Mrg15-deficient embryonic brain. Moreover, we also demonstrate Mrg15-deficient NPCs are defective in differentiation into neurons in vitro. Our results demonstrate that MRG15 has more than one function in neurogenesis and defines a novel role for this chromatin regulator that integrates proliferation and cell-fate determination in neurogenesis during development. Copyright 2008 Wiley-Liss, Inc.

CD36 Is a Matrix Metalloproteinase-9 Substrate That Stimulates Neutrophil Apoptosis and Removal During Cardiac Remodeling.

PubMed

DeLeon-Pennell, Kristine Y; Tian, Yuan; Zhang, Bai; Cates, Courtney A; Iyer, Rugmani Padmanabhan; Cannon, Presley; Shah, Punit; Aiyetan, Paul; Halade, Ganesh V; Ma, Yonggang; Flynn, Elizabeth; Zhang, Zhen; Jin, Yu-Fang; Zhang, Hui; Lindsey, Merry L

2016-02-01

After myocardial infarction, the left ventricle undergoes a wound healing response that includes the robust infiltration of neutrophils and macrophages to facilitate removal of dead myocytes as well as turnover of the extracellular matrix. Matrix metalloproteinase (MMP)-9 is a key enzyme that regulates post-myocardial infarction left ventricular remodeling. Infarct regions from wild-type and MMP-9 null mice (n=8 per group) analyzed by glycoproteomics showed that of 541 N-glycosylated proteins quantified, 45 proteins were at least 2-fold upregulated or downregulated with MMP-9 deletion (all P<0.05). Cartilage intermediate layer protein and platelet glycoprotein 4 (CD36) were identified as having the highest fold increase in MMP-9 null mice. By immunoblotting, CD36 but not cartilage intermediate layer protein decreased steadily during the time course post-myocardial infarction, which identified CD36 as a candidate MMP-9 substrate. MMP-9 was confirmed in vitro and in vivo to proteolytically degrade CD36. In vitro stimulation of day 7 post-myocardial infarction macrophages with MMP-9 or a CD36-blocking peptide reduced phagocytic capacity. Dual immunofluorescence revealed concomitant accumulation of apoptotic neutrophils in the MMP-9 null group compared with wild-type group. In vitro stimulation of isolated neutrophils with MMP-9 decreased neutrophil apoptosis, indicated by reduced caspase-9 expression. Our data reveal a new cell-signaling role for MMP-9 through CD36 degradation to regulate macrophage phagocytosis and neutrophil apoptosis. © 2015 American Heart Association, Inc.

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

PubMed

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

2001-01-01

Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y < 215D = 215S = 215T. As detected by real-time nucleic acid sequence-based amplification with two molecular beacons, the addition of AZT or stavudine (d4T) to the viral cultures favored the 215Y mutant in a dose-dependent manner. Our results illustrate that infection with nucleoside analogue-resistant HIV leads in newly infected individuals to mutants that are sensitive to nucleoside analogues, but only a single mutation removed from drug-resistant HIV. Such mutants were shown to be transmissible, stable, and prone to rapid selection for resistance to AZT or d4T as soon as antiretroviral therapy was administered. Monitoring of patients for the presence of new HIV-1 wild types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.

An interaction between L-prostaglandin D synthase and arrestin increases PGD2 production.

PubMed

Mathurin, Karine; Gallant, Maxime A; Germain, Pascale; Allard-Chamard, Hugues; Brisson, Jessy; Iorio-Morin, Christian; de Brum Fernandes, Artur; Caron, Marc G; Laporte, Stéphane A; Parent, Jean-Luc

2011-01-28

L-type prostaglandin synthase (L-PGDS) produces PGD(2), a lipid mediator involved in neuromodulation and inflammation. Here, we show that L-PGDS and arrestin-3 (Arr3) interact directly and can be co-immunoprecipitated endogenously from MG-63 osteoblasts. Perinuclear L-PGDS/Arr3 co-localization is observed in PGD(2)-producing MG-63 cells and is induced by the addition of the L-PGDS substrate or co-expression of COX-2 in HEK293 cells. Inhibition of L-PGDS activity in MG-63 cells triggers redistribution of Arr3 and L-PGDS to the cytoplasm. Perinuclear localization of L-PGDS is detected in wild-type mouse embryonic fibroblasts (MEFs) but is more diffused in MEFs-arr-2(-/-)-arr-3(-/-). Arrestin-3 promotes PGD(2) production by L-PGDS in vitro. IL-1β-induced PGD(2) production is significantly lower in MEFs-arr-2(-/-)-arr-3(-/-) than in wild-type MEFs but can be rescued by expressing Arr2 or Arr3. A peptide corresponding to amino acids 86-100 of arrestin-3 derived from its L-PGDS binding domain stimulates L-PGDS-mediated PGD(2) production in vitro and in MG-63 cells. We report the first characterization of an interactor/modulator of a PGD(2) synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases.

Effect of a structurally modified human granulocyte colony stimulating factor, G-CSFa, on leukopenia in mice and monkeys

PubMed Central

2011-01-01

Background Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, and differentiation of neutrophilic granulocyte precursors, Recombinant G-CSF has been used for the treatment of congenital and therapy-induced neutropenia and stem cell mobilization. Due to its intrinsic instability, recombinant G-CSF needs to be excessively and/or frequently administered to patients in order to maintain a plasma concentration high enough to achieve therapeutic effects. Therefore, there is a need for the development of G-CSF derivatives that are more stable and active in vivo. Methods Using site-direct mutagenesis and recombinant DNA technology, a structurally modified derivative of human G-CSF termed G-CSFa was obtained. G-CSFa contains alanine 17 (instead of cysteine 17 as in wild-type G-CSF) as well as four additional amino acids including methionine, arginine, glycine, and serine at the amino-terminus. Purified recombinant G-CSFa was tested for its in vitro activity using cell-based assays and in vivo activity using both murine and primate animal models. Results In vitro studies demonstrated that G-CSFa, expressed in and purified from E. coli, induced a much higher proliferation rate than that of wild-type G-CSF at the same concentrations. In vivo studies showed that G-CSFa significantly increased the number of peripheral blood leukocytes in cesium-137 irradiated mice or monkeys with neutropenia after administration of clyclophosphamide. In addition, G-CSFa increased neutrophil counts to a higher level in monkeys with a concomitant slower declining rate than that of G-CSF, indicating a longer half-life of G-CSFa. Bone marrow smear analysis also confirmed that G-CSFa was more potent than G-CSF in the induction of granulopoiesis in bone marrows of myelo-suppressed monkeys. Conclusion G-CSFa, a structurally modified form of G-CSF, is more potent in stimulating proliferation and differentiation of myeloid cells of the granulocytic lineage than the wild-type counterpart both in vitro and in vivo. G-CSFa can be explored for the development of a new generation of recombinant therapeutic drug for leukopenia. PMID:21668998

Effect of a structurally modified human granulocyte colony stimulating factor, G-CSFa, on leukopenia in mice and monkeys.

PubMed

Jiang, Yongping; Jiang, Wenhong; Qiu, Yuchang; Dai, Wei

2011-06-13

Granulocyte colony stimulating factor (G-CSF) regulates survival, proliferation, and differentiation of neutrophilic granulocyte precursors, Recombinant G-CSF has been used for the treatment of congenital and therapy-induced neutropenia and stem cell mobilization. Due to its intrinsic instability, recombinant G-CSF needs to be excessively and/or frequently administered to patients in order to maintain a plasma concentration high enough to achieve therapeutic effects. Therefore, there is a need for the development of G-CSF derivatives that are more stable and active in vivo. Using site-direct mutagenesis and recombinant DNA technology, a structurally modified derivative of human G-CSF termed G-CSFa was obtained. G-CSFa contains alanine 17 (instead of cysteine 17 as in wild-type G-CSF) as well as four additional amino acids including methionine, arginine, glycine, and serine at the amino-terminus. Purified recombinant G-CSFa was tested for its in vitro activity using cell-based assays and in vivo activity using both murine and primate animal models. In vitro studies demonstrated that G-CSFa, expressed in and purified from E. coli, induced a much higher proliferation rate than that of wild-type G-CSF at the same concentrations. In vivo studies showed that G-CSFa significantly increased the number of peripheral blood leukocytes in cesium-137 irradiated mice or monkeys with neutropenia after administration of cyclophosphamide. In addition, G-CSFa increased neutrophil counts to a higher level in monkeys with a concomitant slower declining rate than that of G-CSF, indicating a longer half-life of G-CSFa. Bone marrow smear analysis also confirmed that G-CSFa was more potent than G-CSF in the induction of granulopoiesis in bone marrows of myelo-suppressed monkeys. G-CSFa, a structurally modified form of G-CSF, is more potent in stimulating proliferation and differentiation of myeloid cells of the granulocytic lineage than the wild-type counterpart both in vitro and in vivo. G-CSFa can be explored for the development of a new generation of recombinant therapeutic drug for leukopenia.

HIV-1-based defective lentiviral vectors efficiently transduce human monocytes-derived macrophages and suppress replication of wild-type HIV-1

PubMed Central

Zeng, Lingbing; Planelles, Vicente; Sui, Ziye; Gartner, Suzanne; Maggirwar, Sanjay B.; Dewhurst, Stephen; Ye, Linbai; Nerurkar, Vivek R.; Yanagihara, Richard; Lu, Yuanan

2010-01-01

Background Human monocytes play an important role in mediating human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS), and monocytes-derived macrophages (MDM) represent a major viral reservoir within the brain and other target organs. Current gene transduction of MDM is hindered by a limited efficiency. In this study we established a lentiviral vector-based technique for improved gene transfer into human MDM cultures in vitro and demonstrated significant protection of transduced MDM from super-infection with wild-type HIV-1. Methods HIV-1-based lentiviral vector stocks were prepared in 293T cells by the established calcium phosphate transfection method. Human monocytes were isolated from donors' blood by Ficoll-Paque separation and cultured in vitro. To establish an effective technique for vector-mediated gene transfer, primary cultures of human MDM were transduced at varying multiplicities of infection (MOI) and at a range of time points following initial isolation of cells (time-in-culture). Transduced cells were then examined for transgene (green fluorescent protein (GFP)) expression by fluorescent microscopy and reverse transcription polymerase chain reaction (RT-PCR). These cultures were then exposed to wild-type HIV-1, and viral replication was quantitated by p24 assay; production of neurotoxic effector molecules by the transduced MDM was also examined, using indicator neurons. Results We have demonstrated that primary human MDM could be efficiently transduced (>50%) with concentrated HIV-1-based defective lentiviral vectors (DLV). Furthermore, DLV-mediated gene transduction was stable, and the transduced cells exhibited no apparent difference from normal MDM in terms of their morphology, viability and neurotoxin secretion. Challenge of DLV-transduced MDM cultures with HIV-1Ba-L revealed a 4- to 5-fold reduction in viral replication, as measured by p24 antigen production. This effect was associated with the mobilization of the GFP-expressing DLV construct by the wild-type virus. Conclusions These data demonstrate the inhibition of HIV-1 replication in primary MDM, by a DLV vector that lacks any anti-HIV-1 transgene. These findings lay the initial groundwork for future studies on the ability of DLV-modified monocytes to introduce anti-HIV-1 genes into the CNS. Lentiviral vector-mediated gene delivery to the CNS by monocytes/macrophages is a promising, emerging strategy for treating neuro-AIDS. PMID:16142830

Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.

PubMed

Behnan, Jinan; Isakson, Pauline; Joel, Mrinal; Cilio, Corrado; Langmoen, Iver A; Vik-Mo, Einar O; Badn, Wiaam

2014-05-01

The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients. © 2013 AlphaMed Press.

Role of P-glycoprotein and breast cancer resistance protein-1 in the brain penetration and brain pharmacodynamic activity of the novel phosphatidylinositol 3-kinase inhibitor GDC-0941.

PubMed

Salphati, Laurent; Lee, Leslie B; Pang, Jodie; Plise, Emile G; Zhang, Xiaolin

2010-09-01

2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of the phosphatidylinositol 3-kinase (PI3K) pathway currently evaluated in the clinic as an anticancer agent. The objectives of this study were to determine in vitro whether GDC-0941 was a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) and to investigate the impact of these transporters on the pharmacokinetics, brain penetration, and activity of GDC-0941 in FVBn mice (wild-type) and Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) knockout mice. Studies with Madin-Darby canine kidney cells transfected with P-gp or Bcrp1 established that this compound was a substrate of both transporters. After administrations to mice, GDC-0941 brain-to-plasma ratio ranged from 0.02 to 0.06 in the wild-type and Bcrp1(-/-) mice and was modestly higher in the Mdr1a/b(-/-) mice, ranging from 0.08 to 0.11. In contrast, GDC-0941 brain-to-plasma ratio in Mdr1a/b(-/-)/Bcrp1(-/-) triple knockout mice was 30-fold higher than in the wild-type mice. The plasma clearance of GDC-0941 was similar in wild-type and all knockout mice, ranging from 15 to 25 ml/(min . kg) in the wild-type mice and from 18 to 35 ml/(min . kg) in the knockout mice. Exposure after oral administration was comparable in the four strains of mice. The PI3K pathway was markedly inhibited in the brain of Mdr1a/b(-/-)/Bcrp1(-/-) mice for up to 6 h postdose, as evidenced by a 60% suppression of the phosphorylated Akt signal, whereas no inhibition was detected in the brain of wild-type mice. The concerted effects of P-gp and Bcrp1 in restricting GDC-0941 access and pathway modulation in mouse brain may have implications for the treatment of patients with brain tumors.

High frequency electromagnetic fields (GSM signals) affect gene expression levels in tumor suppressor p53-deficient embryonic stem cells.

PubMed

Czyz, Jaroslaw; Guan, Kaomei; Zeng, Qinghua; Nikolova, Teodora; Meister, Armin; Schönborn, Frank; Schuderer, Jürgen; Kuster, Niels; Wobus, Anna M

2004-05-01

Effects of electromagnetic fields (EMF) simulating exposure to the Global System for Mobile Communications (GSM) signals were studied using pluripotent embryonic stem (ES) cells in vitro. Wild-type ES cells and ES cells deficient for the tumor suppressor p53 were exposed to pulse modulated EMF at 1.71 GHz, lower end of the uplink band of GSM 1800, under standardized and controlled conditions, and transcripts of regulatory genes were analyzed during in vitro differentiation. Two dominant GSM modulation schemes (GSM-217 and GSM-Talk), which generate temporal changes between GSM-Basic (active during talking phases) and GSM-DTX (active during listening phases thus simulating a typical conversation), were applied to the cells at and below the basic safety limits for local exposures as defined for the general public by the International Commission on Nonionizing Radiation Protection (ICNIRP). GSM-217 EMF induced a significant upregulation of mRNA levels of the heat shock protein, hsp70 of p53-deficient ES cells differentiating in vitro, paralleled by a low and transient increase of c-jun, c-myc, and p21 levels in p53-deficient, but not in wild-type cells. No responses were observed in either cell type after EMF exposure to GSM-Talk applied at similar slot-averaged specific absorption rates (SAR), but at lower time-averaged SAR values. Cardiac differentiation and cell cycle characteristics were not affected in embryonic stem and embryonic carcinoma cells after exposure to GSM-217 EMF signals. Our data indicate that the genetic background determines cellular responses to GSM modulated EMF. Bioelectromagnetics 25:296-307, 2004. Copyright 2004 Wiley-Liss, Inc.

Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study.

PubMed

Beck, Raphaël; Pedrosa, Rozangela Curi; Dejeans, Nicolas; Glorieux, Christophe; Levêque, Philippe; Gallez, Bernard; Taper, Henryk; Eeckhoudt, Stéphane; Knoops, Laurent; Calderon, Pedro Buc; Verrax, Julien

2011-10-01

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening

PubMed Central

Cheong, Wing-Lam; Tsang, Ming-San; So, Pui-Kin; Chung, Wai-Hong; Leung, Yun-Chung; Chan, Pak-Ho

2014-01-01

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening. PMID:25074398

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

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

Ohkawa, Mayumi; Ohno, Yoshiya; Masuko, Kazue

Highlights: {yields} We established LAT1 amino-acid transporter-disrupted DT40 cells. {yields} LAT1-disrupted cells showed slow growth and lost the oncogenicity. {yields} siRNA and mAb inhibited human tumor growth in vitro and in vivo. {yields} LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1{supmore » -/-}) cell clones, derived from a heterozygous LAT1{sup +/-} clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1{sup -/-} DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1{sup -/-} cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1{sup -/-} DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1{sup -/-} DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1{sup +/-} DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.« less

Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

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

Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.

1988-01-01

By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alterationmore » of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).« less

The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase.

PubMed

Edgren, Tomas; Nordlund, Stefan

2004-04-01

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.

Susceptibility of Glucokinase-MODY Mutants to Inactivation by Oxidative Stress in Pancreatic β-Cells

PubMed Central

Cullen, Kirsty S.; Matschinsky, Franz M.; Agius, Loranne; Arden, Catherine

2011-01-01

OBJECTIVE The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells. RESEARCH DESIGN AND METHODS Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non–β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models. RESULTS Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non–β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2. CONCLUSIONS Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells. PMID:22028181

Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen

PubMed Central

Burrell, Matthew; Henderson, Simon J.; Ravnefjord, Anna; Schweikart, Fritz; Fowler, Susan B.; Witt, Susanne; Hansson, Kenny M.; Webster, Carl I.

2016-01-01

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aβ), the main pathological component of Alzheimer’s disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bβ-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions. PMID:27437944

Susceptibility of glucokinase-MODY mutants to inactivation by oxidative stress in pancreatic β-cells.

PubMed

Cullen, Kirsty S; Matschinsky, Franz M; Agius, Loranne; Arden, Catherine

2011-12-01

The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells. Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non-β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models. Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non-β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2. Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells.

Attachment Capability of Antagonistic Yeast Rhodotorula glutinis to Botrytis cinerea Contributes to Biocontrol Efficacy.

PubMed

Li, Boqiang; Peng, Huaimin; Tian, Shiping

2016-01-01

Rhodotorula glutinis as an antagonism show good biocontrol performance against various post-harvest diseases in fruits. In the present study, strong attachment capability of R. glutinis to spores and hyphae of Botrytis cinerea was observed. Further analysis showed that certain protein components on the yeast cell surface played critical role during the interaction between R. glutinis and B. cinerea. The components mainly distributed at the poles of yeast cells and might contain glycosylation modification, as tunicamycin treated yeast cells lost attachment capability to B. cinerea. To investigate contributions of attachment capability of R. glutinis to its biocontrol efficacy, yeast cells were mutagenized with 3% methane-sulfonic acid ethyl ester (EMS), and a mutant CE4 with stable non-attaching phenotype was obtained. No significant difference was found on colony, cell morphology, reproductive ability, and capsule formation between the mutant and wild-type. However, there was a distinct difference in India ink positive staining patterns between the two strains. Moreover, wild-type strain of R. glutinis showed better performance on inhibiting spore germination and mycelial growth of B. cinerea than CE4 strain when yeast cells and B. cinerea were co-cultured in vitro. In biocontrol assay, both wild-type and CE4 strains showed significant biocontrol efficacy against gray mold caused by B. cinerea in apple fruit, whereas, control effect of CE4 strain was lower than that of wild-type. Our findings provided new evidences that attachment capability of R. glutinis to B. cinerea contributed to its biocontrol efficacy.

Attachment Capability of Antagonistic Yeast Rhodotorula glutinis to Botrytis cinerea Contributes to Biocontrol Efficacy

PubMed Central

Li, Boqiang; Peng, Huaimin; Tian, Shiping

2016-01-01

Rhodotorula glutinis as an antagonism show good biocontrol performance against various post-harvest diseases in fruits. In the present study, strong attachment capability of R. glutinis to spores and hyphae of Botrytis cinerea was observed. Further analysis showed that certain protein components on the yeast cell surface played critical role during the interaction between R. glutinis and B. cinerea. The components mainly distributed at the poles of yeast cells and might contain glycosylation modification, as tunicamycin treated yeast cells lost attachment capability to B. cinerea. To investigate contributions of attachment capability of R. glutinis to its biocontrol efficacy, yeast cells were mutagenized with 3% methane-sulfonic acid ethyl ester (EMS), and a mutant CE4 with stable non-attaching phenotype was obtained. No significant difference was found on colony, cell morphology, reproductive ability, and capsule formation between the mutant and wild-type. However, there was a distinct difference in India ink positive staining patterns between the two strains. Moreover, wild-type strain of R. glutinis showed better performance on inhibiting spore germination and mycelial growth of B. cinerea than CE4 strain when yeast cells and B. cinerea were co-cultured in vitro. In biocontrol assay, both wild-type and CE4 strains showed significant biocontrol efficacy against gray mold caused by B. cinerea in apple fruit, whereas, control effect of CE4 strain was lower than that of wild-type. Our findings provided new evidences that attachment capability of R. glutinis to B. cinerea contributed to its biocontrol efficacy. PMID:27199931

The cell adhesion molecule L1 regulates the expression of choline acetyltransferase and the development of septal cholinergic neurons

PubMed Central

Cui, Xuezhi; Weng, Ying-Qi; Frappé, Isabelle; Burgess, Alison; Girão da Cruz, M Teresa; Schachner, Melitta; Aubert, Isabelle

2011-01-01

Mutations in the L1 gene cause severe brain malformations and mental retardation. We investigated the potential roles of L1 in the regulation of choline acetyltransferase (ChAT) and in the development of septal cholinergic neurons, which are known to project to the hippocampus and play key roles in cognitive functions. Using stereological approaches, we detected significantly fewer ChAT-positive cholinergic neurons in the medial septum and vertical limb of the diagonal band of Broca (MS/VDB) of 2-week-old L1-deficient mice compared to wild-type littermates (1644 ± 137 vs. 2051 ± 165, P = 0.038). ChAT protein levels in the septum were 53% lower in 2-week-old L1-deficient mice compared to wild-type littermates. ChAT activity in the septum was significantly reduced in L1-deficient mice compared to wild-type littermates at 1 (34%) and 2 (40%) weeks of age. In vitro, increasing doses of L1-Fc induced ChAT activity in septal neurons with a significant linear trend (*P = 0.0065). At 4 weeks of age in the septum and at all time points investigated in the caudate-putamen (CPu), the number of ChAT-positive neurons and the levels of ChAT activity were not statistically different between L1-deficient mice and wild-type littermates. The total number of cells positive for the neuronal nuclear antigen (NeuN) in the MS/VDB and CPu was not statistically different in L1-deficient mice compared to wild-type littermates, and comparable expression of the cell cycle marker Ki67 was observed. Our results indicate that L1 is required for the timely maturation of septal cholinergic neurons and that L1 promotes the expression and activity of ChAT in septal neurons. PMID:22399087

S1P1 receptor inhibits kidney epithelial mesenchymal transition triggered by ischemia/reperfusion injury via the PI3K/Akt pathway.

PubMed

Wang, Weina; Wang, Aimei; Luo, Guochang; Ma, Fengqiao; Wei, Xiaoming; Bi, Yongyi

2018-06-13

Ischemia/reperfusion (I/R) is a major cause of acute kidney injury (AKI), along with delayed graft function, which can trigger chronic kidney injury by stimulating epithelial to mesenchymal transition (EMT) in the kidney canaliculus. Sphingosine 1-phosphate receptor 1 (S1P1) is a G protein-coupled receptor that is indispensable for vessel homeostasis. This study aimed to investigate the influence of S1P1 on the mechanisms underlying I/R-induced EMT in the kidney using in vivo and in vitro models. Wild-type (WT) and S1P1-overexpressing kidney canaliculus cells were subject to hypoxic conditions followed by reoxygenation in the presence or absence of FTY720-P, a potent S1P1 agonist. In vivo, bilateral arteria renalis in wild-type mice and mice with silenced S1P1 were clamped for 30 min to obtain I/R models. We found that hypoxia/reoxygenation (H/R) significantly enhanced the expressions of EMT biomarkers and down-regulated S1P1 expression in wild-type canaliculus cells. In contrast, FTY720-P treatment or overexpression of S1P1 significantly suppressed EMT in wild-type canaliculus cells. Furthermore, after 48-72 h, a significant upregulation of EMT biomarker expression was triggered by I/R in mice with silenced S1P1, while the expressions of these markers did not change in wild-type mice. A kt activity was increased with H/R-induced EMT, suggesting that the protective influence of FTY720-P was due to its inhibition of PI3K/Akt. Therefore, the results of this study provide evidence that down-regulation of S1P1 expression is essential for the generation and progression of EMT triggered by I/R. S1P1 exhibits a prominent inhibitory effect on kidney I/R-induced EMT in the kidney by affecting the PI3K/Akt pathway.

A quorum sensing-defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems.

PubMed

Moleleki, Lucy Novungayo; Pretorius, Rudolph Gustav; Tanui, Collins Kipngetich; Mosina, Gabolwelwe; Theron, Jacques

2017-01-01

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain. © 2016 BSPP and John Wiley & Sons Ltd.

Bcl-2 protein expression associated with resistance to apoptosis in clear cell adenocarcinomas of the vagina and cervix expressing wild-type p53.

PubMed

Waggoner, S E; Baunoch, D A; Anderson, S A; Leigh, F; Zagaja, V G

1998-09-01

Clear cell adenocarcinomas (CCAs) of the vagina and cervix are rare tumors that often overexpress wild-type p53. In vitro, expression of protooncogene bcl-2 can block p53-mediated apoptosis. The objective of this study was to determine if bcl-2 is expressed in CCAs and whether this expression is associated with inhibition of apoptosis. Twenty-one paraffin-embedded clear cell adenocarcinomas were immunohistochemically stained for bcl-2 (antibody M 887, Dako, Carpinteria, CA) and DNA fragmentation (ApopTag, Oncor, Gaithersburg, MD), a marker for apoptosis. Fifteen tumors were associated with in utero exposure to diethylstilbestrol (DES). Prior p53 gene analysis had indicated the presence of wild-type p53 in each tumor. Human lymphoid tissue containing bcl-2-expressing lymphocytes and DNase I-exposed CCA tissue sections were used as positive controls for the bcl-2 and apoptosis assays, respectively. Expression of bcl-2 and DNA fragmentation was classified (0 to 3+) according to percentage of positive cells and intensity of staining. Expression of bcl-2 was identified in each CCA examined, and was strongly positive (2+ to 3+) in 18 of 21 samples. Despite the presence of wild-type p53, only 4 of 21 tumors showed evidence of apoptosis as assessed through DNA fragmentation. DNA damage leads to increased intracellular p53 levels. Overexpression of p53 induces apoptosis as a means of protecting organisms from the development of malignancy. CCAs of the vagina and cervix, which contain wild-type p53 genes and often overexpress p53 protein, presumably have evolved mechanisms to avoid p53-induced apoptosis. Our observations are consistent with the hypothesis that overexpression of bcl-2 can inhibit p53-mediated apoptosis and suggest a mechanism by which these rare tumors can arise without mutation of the p53 gene.

Regulation of cytokine polarization and T cell recruitment to inflamed paws in mouse collagen-induced arthritis by the chemokine receptor CXCR6.

PubMed

Slauenwhite, Drew; Gebremeskel, Simon; Doucette, Carolyn D; Hoskin, David W; Johnston, Brent

2014-11-01

The chemokine receptor CXCR6 is highly expressed on lymphocytes isolated from the synovium of patients with rheumatoid arthritis, psoriatic arthritis, or juvenile idiopathic arthritis, suggesting that CXCR6 regulates immune cell activation or infiltration into arthritic joints. This study was undertaken to examine the role of CXCR6 in T cell activation and arthritis development. A collagen-induced arthritis model was used to examine arthritis development in wild-type and CXCR6(-/-) mice. CXCR6 expression, lymphocyte accumulation, and intracellular cytokine production were examined by flow cytometry. Collagen-specific antibodies were measured in the serum. Collagen-specific recall responses were examined in vitro via proliferation and cytokine release assays. T cell homing to inflamed joints was examined using competitive adoptive transfer of dye-labeled lymphocytes from wild-type and CXCR6(-/-) mice. The numbers of CXCR6+ T cells were increased in the paws and draining lymph nodes of arthritic mice. The incidence of arthritis, disease severity, extent of T cell accumulation, and levels of collagen-specific IgG2a antibodies were significantly reduced in CXCR6(-/-) mice compared to wild-type mice. T cells from wild-type mice exhibited Th1 (interferon-γ [IFNγ]) polarization in the inguinal lymph nodes following immunization. At disease peak, this shifted to a Th17 (interleukin-17A [IL-17A]) response in the popliteal lymph nodes. T cells in CXCR6(-/-) mice exhibited impaired cytokine polarization, resulting in a decreased frequency and number of IL-17A- and IFNγ-producing cells. Recruitment of activated CXCR6(-/-) mouse T cells to the inflamed paws was impaired compared to recruitment of wild-type mouse T cells. These experiments demonstrate that CXCR6 plays important roles in the pathogenesis of arthritis through its effects on both T cell cytokine polarization and homing of T cells to inflamed joints. Copyright © 2014 by the American College of Rheumatology.

Effects of gintonin-enriched fraction on hippocampal cell proliferation in wild-type mice and an APPswe/PSEN-1 double Tg mouse model of Alzheimer's disease.

PubMed

Kim, Hyeon-Joong; Kim, Dae-Joong; Shin, Eun-Ju; Lee, Byung-Hwan; Choi, Sun-Hye; Hwang, Sung-Hee; Rhim, Hyewhon; Cho, Ik-Hyun; Kim, Hyoung-Chun; Nah, Seung-Yeol

2016-12-01

We previously showed that gintonin, an exogenous lysophosphatidic acid (LPA) receptor ligand, attenuated β-amyloid plaque formation in the cortex and hippocampus, and restored β-amyloid-induced memory dysfunction. Both endogenous LPA and LPA receptors play a key role in embryonic brain development. However, little is known about whether gintonin can induce hippocampal cell proliferation in adult wild-type mice and an APPswe/PSEN-1 double Tg mouse model of Alzheimer's disease (AD). In the present study, we examined the effects of gintonin on the proliferation of hippocampal neural progenitor cells (NPCs) in vitro and its effects on the hippocampal cell proliferation in wild-type mice and a transgenic AD mouse model. Gintonin treatment increased 5-bromo-2'-deoxyuridine (BrdU) incorporation in hippocampal NPCs in a dose- and time-dependent manner. Gintonin (0.3 μg/ml) increased the immunostaining of glial fibrillary acidic protein, NeuN, and LPA1 receptor in hippocampal NPCs. However, the gintonin-induced increase in BrdU incorporation and immunostaining of biomarkers was blocked by an LPA1/3 receptor antagonist and Ca 2+ chelator. Oral administration of the gintonin-enriched fraction (50 and 100 mg/kg) increased hippocampal BrdU incorporation and LPA1/3 receptor expression in adult wild-type and transgenic AD mice. The present study showed that gintonin could increase the number of hippocampal neurons in adult wild-type mice and a transgenic AD mouse model. Our results indicate that gintonin-mediated hippocampal cell proliferation contributes to the gintonin-mediated restorative effect against β-amyloid-induced hippocampal dysfunction. These results support the use of gintonin for the prevention or treatment of neurodegenerative diseases such as AD via promotion of hippocampal neurogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Efficacy of icotinib for advanced non-small cell lung cancer patients with EGFR status identified].

PubMed

Song, Zhengbo; Yu, Xinmin; Cai, Jufen; Shao, Lan; Lin, Baochai; He, Chunxiao; Zhang, Beibei; Zhang, Yiping

2013-03-01

As the first epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in China, icotinib shows promising anticancer activity in vitro and vivo. The phase III clinical study (ICOGEN) showed that icotinib has a good efficacy and tolerability in Chinese patients with advanced non-small cell lung cancer (NSCLC) compared with gefitinib. This retrospective study aims to evaluate the efficacy and tolerability of icotinib monotherapy for advanced NSCLC patients with EGFR mutation and wild-type patients in our hospital. Patients with advanced NSCLC who were treated with icotinib in Zhejiang Cancer Hospital were retrospectively analyzed from August, 2011 to August, 2012. Survival was estimated using Kaplan-Meier analysis and Log-rank tests. The clinical data of 49 patients (13 with wild-type and 36 with EGFR mutation) with NSCLC were enrolled in the current study. The patients' overall objective response rate (ORR) was 58.3% and the disease control rate (DCR) in 36 EGFR mutation patients was 88.9%. The ORR was 7.7% and DCR was 53.8% in the wild-type patients. Median progression-free survival (PFS) with icotinib treatment in EGFR mutation patients was 9.5 months and 2.2 months in wild-type patients (P<0.001). Nineteen patients with EGFR mutation received icotinib as first-line and 17 in further-line treatment. The PFS was 9.5 months in the first-line and 8.5 months for second-line or further-line patients (P=0.41). Median overall survival (OS) in EGFR mutation patients was not reached, but was 12.6 months in wild-type patients. Most of the drug-related adverse events were mild (grade I or II) and reversible with no grade IV toxicity. Icotinib monotherapy showed significant antitumor activity in advanced NSCLC EGFR mutation patients. The toxicity was well tolerated and acceptable.

Protein S is protective in pulmonary fibrosis.

PubMed

Urawa, M; Kobayashi, T; D'Alessandro-Gabazza, C N; Fujimoto, H; Toda, M; Roeen, Z; Hinneh, J A; Yasuma, T; Takei, Y; Taguchi, O; Gabazza, E C

2016-08-01

Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar epithelial cells in vitro. Conclusions These observations suggest clinical relevance and a protective role of protein S in pulmonary fibrosis. © 2016 International Society on Thrombosis and Haemostasis.

PIK3CA oncogenic mutations represent a major mechanism of resistance to trastuzumab in HER2/neu overexpressing uterine serous carcinomas

PubMed Central

Black, Jonathan D; Lopez, Salvatore; Cocco, Emiliano; Bellone, Stefania; Altwerger, Gary; Schwab, Carlton L; English, Diana P; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Santin, Alessandro D

2015-01-01

Objectives: We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Methods: Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Results: Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. Conclusions: Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment. PMID:26325104

PIK3CA oncogenic mutations represent a major mechanism of resistance to trastuzumab in HER2/neu overexpressing uterine serous carcinomas.

PubMed

Black, Jonathan D; Lopez, Salvatore; Cocco, Emiliano; Bellone, Stefania; Altwerger, Gary; Schwab, Carlton L; English, Diana P; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Santin, Alessandro D

2015-09-29

We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment.

Aptamer redesigned tRNA is nonfunctional and degraded in cells

PubMed Central

LEE, DENNIS; MCCLAIN, WILLIAM H.

2004-01-01

An RNA aptamer derived from tRNAGln isolated in vitro and a rationally redesigned tRNAGln were used to address the relationship between structure and function of tRNAGln aminoacylation in Escherichia coli. Two mutant tRNAGln sequences were studied: an aptamer that binds 26-fold tighter to glutaminyl-tRNA synthetase than wild-type tRNAGln in vitro, redesigned in the variable loop, and a mutant with near-normal aminoacylation kinetics for glutamine, redesigned to contain a long variable arm. Both mutants were tested in a tRNAGln knockout strain of E. coli, but neither supported knockout cell growth. It was later found that both mutant tRNAs were present in very low amounts in the cell. These results reveal the difference between in vitro and in vivo studies, demonstrating the complexities of in vivo systems that have not been replicated in vitro. PMID:14681579

Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

PubMed

Maggi, Maristella; Chiarelli, Laurent R; Valentini, Giovanna; Scotti, Claudia

2015-01-01

Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

Engineering of Helicobacter pylori L-Asparaginase: Characterization of Two Functionally Distinct Groups of Mutants

PubMed Central

Maggi, Maristella; Chiarelli, Laurent R.; Valentini, Giovanna; Scotti, Claudia

2015-01-01

Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy. PMID:25664771

Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells.

PubMed

Ge, Y; Li, Z H; Marshall, M S; Broxmeyer, H E; Lu, L

1998-06-01

To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34 cells which are highly enriched for hematopoietic stem/progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34 cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

A co-culture system with an organotypic lung slice and an immortal alveolar macrophage cell line to quantify silica-induced inflammation.

PubMed

Hofmann, Falk; Bläsche, Robert; Kasper, Michael; Barth, Kathrin

2015-01-01

There is growing evidence that amorphous silica nanoparticles cause toxic effects on lung cells in vivo as well as in vitro and induce inflammatory processes. The phagocytosis of silica by alveolar macrophages potentiates these effects. To understand the underlying molecular mechanisms of silica toxicity, we applied a co-culture system including the immortal alveolar epithelial mouse cell line E10 and the macrophage cell line AMJ2-C11. In parallel we exposed precision-cut lung slices (lacking any blood cells as well as residual alveolar macrophages) of wild type and P2rx7-/- mice with or without AMJ2-C11 cells to silica nanoparticles. Exposure of E10 cells as well as slices of wild type mice resulted in an increase of typical alveolar epithelial type 1 cell proteins like T1α, caveolin-1 and -2 and PKC-β1, whereas the co-culture with AMJ2-C11 showed mostly a slightly lesser increase of these proteins. In P2rx7-/- mice most of these proteins were slightly decreased. ELISA analysis of the supernatant of wild type and P2rx7-/- mice precision-cut lung slices showed decreased amounts of IL-6 and TNF-α when incubated with nano-silica. Our findings indicate that alveolar macrophages influence the early inflammation of the lung and also that cell damaging reagents e.g. silica have a smaller impact on P2rx7-/- mice than on wild type mice. The co-culture system with an organotypic lung slice is a useful tool to study the role of alveolar macrophages during lung injury at the organoid level.

Combinations of Adefovir with Nucleoside Analogs Produce Additive Antiviral Effects against Hepatitis B Virus In Vitro

PubMed Central

Delaney, William E.; Yang, Huiling; Miller, Michael D.; Gibbs, Craig S.; Xiong, Shelly

2004-01-01

Combination therapies may be required for long-term management of some patients chronically infected with hepatitis B virus (HBV). Adefovir is a nucleotide analog that has similar activity against wild-type and lamivudine-resistant HBV. In contrast to lamivudine, clinical resistance to the prodrug adefovir dipivoxil emerges infrequently. Based on its clinical efficacy and low frequency of resistance, adefovir dipivoxil may form an important component of combination regimens. We therefore investigated the in vitro antiviral efficacy of combinations of adefovir with other nucleoside analogs (lamivudine, entecavir, emtricitabine [FTC],and telbivudine [L-dT]) and the nucleotide analog tenofovir. Using a novel stable cell line that expresses high levels of wild-type HBV, we assayed the antiviral activity of each drug alone and in combination with adefovir. All two-drug combinations resulted in greater antiviral effects than treatments with single agents and could be characterized as additive by the Bliss independence model. Analysis using the Loewe additivity model indicated that adefovir exerted additive antiviral effects when combined with lamivudine, FTC, or L-dT and moderately synergistic effects when combined with entecavir or tenofovir. There was no evidence of cytotoxicity with any of the drugs when used alone or in combination at the tested doses. PMID:15388423

A Temperature-Independent Cold-Shock Protein Homolog Acts as a Virulence Factor in Xylella fastidiosa.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2016-05-01

Xylella fastidiosa, causal agent of Pierce's disease (PD) of grapevine, is a fastidious organism that requires very specific conditions for replication and plant colonization. Cold temperatures reduce growth and survival of X. fastidiosa both in vitro and in planta. However, little is known regarding physiological responses of X. fastidiosa to temperature changes. Cold-shock proteins (CSP), a family of nucleic acid-binding proteins, act as chaperones facilitating translation at low temperatures. Bacterial genomes often encode multiple CSP, some of which are strongly induced following exposure to cold. Additionally, CSP contribute to the general stress response through mRNA stabilization and posttranscriptional regulation. A putative CSP homolog (Csp1) with RNA-binding activity was identified in X. fastidiosa Stag's Leap. The csp1 gene lacked the long 5' untranslated region characteristic of cold-inducible genes and was expressed in a temperature-independent manner. As compared with the wild type, a deletion mutant of csp1 (∆csp1) had decreased survival rates following cold exposure and salt stress in vitro. The deletion mutant also was significantly less virulent in grapevine, as compared with the wild type, in the absence of cold stress. These results suggest an important function of X. fastidiosa Csp1 in response to cellular stress and during plant colonization.

Loss of Krüppel-like factor 3 (KLF3/BKLF) leads to upregulation of the insulin-sensitizing factor adipolin (FAM132A/CTRP12/C1qdc2).

PubMed

Bell-Anderson, Kim S; Funnell, Alister P; Williams, Helen; Mat Jusoh, Hanapi; Scully, Tiffany; Lim, Wooi F; Burdach, Jon G; Mak, Ka Sin; Knights, Alexander J; Hoy, Andrew J; Nicholas, Hannah R; Sainsbury, Amanda; Turner, Nigel; Pearson, Richard C; Crossley, Merlin

2013-08-01

Krüppel-like factor 3 (KLF3) is a transcriptional regulator that we have shown to be involved in the regulation of adipogenesis in vitro. Here, we report that KLF3-null mice are lean and protected from diet-induced obesity and glucose intolerance. On a chow diet, plasma levels of leptin are decreased, and adiponectin is increased. Despite significant reductions in body weight and adiposity, wild-type and knockout animals show equivalent energy intake, expenditure, and excretion. To investigate the molecular events underlying these observations, we used microarray analysis to compare gene expression in Klf3(+/+) and Klf3(-/-) tissues. We found that mRNA expression of Fam132a, which encodes a newly identified insulin-sensitizing adipokine, adipolin, is significantly upregulated in the absence of KLF3. We confirmed that KLF3 binds the Fam132a promoter in vitro and in vivo and that this leads to repression of promoter activity. Further, plasma adipolin levels were significantly increased in Klf3(-/-) mice compared with wild-type littermates. Boosting levels of adipolin via targeting of KLF3 offers a novel potential therapeutic strategy for the treatment of insulin resistance.

Loss of Krüppel-Like Factor 3 (KLF3/BKLF) Leads to Upregulation of the Insulin-Sensitizing Factor Adipolin (FAM132A/CTRP12/C1qdc2)

PubMed Central

Bell-Anderson, Kim S.; Funnell, Alister P.; Williams, Helen; Mat Jusoh, Hanapi; Scully, Tiffany; Lim, Wooi F.; Burdach, Jon G.; Mak, Ka Sin; Knights, Alexander J.; Hoy, Andrew J.; Nicholas, Hannah R.; Sainsbury, Amanda; Turner, Nigel; Pearson, Richard C.; Crossley, Merlin

2013-01-01

Krüppel-like factor 3 (KLF3) is a transcriptional regulator that we have shown to be involved in the regulation of adipogenesis in vitro. Here, we report that KLF3-null mice are lean and protected from diet-induced obesity and glucose intolerance. On a chow diet, plasma levels of leptin are decreased, and adiponectin is increased. Despite significant reductions in body weight and adiposity, wild-type and knockout animals show equivalent energy intake, expenditure, and excretion. To investigate the molecular events underlying these observations, we used microarray analysis to compare gene expression in Klf3+/+ and Klf3−/− tissues. We found that mRNA expression of Fam132a, which encodes a newly identified insulin-sensitizing adipokine, adipolin, is significantly upregulated in the absence of KLF3. We confirmed that KLF3 binds the Fam132a promoter in vitro and in vivo and that this leads to repression of promoter activity. Further, plasma adipolin levels were significantly increased in Klf3−/− mice compared with wild-type littermates. Boosting levels of adipolin via targeting of KLF3 offers a novel potential therapeutic strategy for the treatment of insulin resistance. PMID:23633521

Effects of icotinib on advanced non-small cell lung cancer with different EGFR phenotypes.

PubMed

Pan, Huiyun; Liu, Rong; Li, Shengjie; Fang, Hui; Wang, Ziwei; Huang, Sheng; Zhou, Jianying

2014-09-01

Icotinib is the first oral epidermal growth factor receptor (EGFR) tyrosine kinase receptor inhibitor, which has been proven to exert significant inhibitory effects on non-small cell lung cancer in vitro. Clinical evidence has showed that the efficacy of Icotinib on retreating advanced non-small cell lung cancer is comparable to Gefitinib. However, different phenotypes of EGFR can affect the therapeutic outcomes of EGFR tyrosine kinase receptor inhibitor. Therefore, our study focused on efficacy and safety of Icotinib in patients with advanced non-small cell lung cancer of different EGPR phenotypes. Clinical data of patients with advanced non-small cell lung cancer who received Icotinib treatment from August, 2011 to May, 2013 were retrospectively analyzed. Kaplan-Meier analysis was used for survival analysis and comparison. 18 wild-type EGFR and 51 mutant type were found in a total of 69 patients. Objective response rate of patients with mutant type EGFR was 54.9 % and disease control rate was 86.3 %. Objective response rate of wild-type patients was 11.1 % (P = 0.0013 vs mutant type), disease control rate was 50.0 % (P = 0.0017). Median progression-free survival (PFS) of mutant type and wild-type patients were 9.7 and 2.6 months, respectively (P < 0.001). Median PFS of exon 19 mutated mutant patients was 11.3 months, mean PFS of exon 21 L858R mutated mutant patients was 8.7 months (P = 0.3145). Median overall survival (OS) of EGFR mutated patients had not reached. OS time of 13 wild-type patients was 12.9 months (P < 0.001). The common adverse reactions of Icotinib included rash, diarrhea, itching skin with occurrence rates of 24.6 % (17/69), 13.0 % (9/69), and 11.6 % (8/69), respectively. Most adverse reactions were grade I-II. Icotinib has great efficacy in EGFR mutated patients, making it an optimal regimen to treat EGFR mutated patients. Furthermore, most of adverse reactions associated with Icotinib treatment were tolerable.

Enhancement of nutritional and bioactive compounds by in vitro culture of wild Fragaria vesca L. vegetative parts.

PubMed

Dias, Maria Inês; Barros, Lillian; Sousa, Maria João; Oliveira, M Beatriz P P; Santos-Buelga, Celestino; Ferreira, Isabel C F R

2017-11-15

In vitro culture emerges as a sustainable way to produce bioactives for further applicability in the food industry. Herein, vegetative parts of Fragaria vesca L. (wild strawberry) obtained by in vitro culture were analyzed regarding nutritional and phytochemical compounds, as well as antioxidant activity. These samples proved to have higher content of protein, polyunsaturated fatty acids, soluble sugars, organic acids (including ascorbic acid) and tocopherols (mainly α-tocopherol) than wild grown F. vesca, as well as containing additional phenolic compounds. The antioxidant activity of hydromethanolic extracts could be correlated with the content of different phenolic groups and other compounds (sugars and organic acids). It was demonstrated that in vitro culture could enhance nutritional and bioactive compounds of Fragaria vesca L. plants, providing a very interesting biotechnological tool for potential food applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multiple transcriptional regulatory domains in the human immunodeficiency virus type 1 long terminal repeat are involved in basal and E1A/E1B-induced promoter activity.

PubMed Central

Kliewer, S; Garcia, J; Pearson, L; Soultanakis, E; Dasgupta, A; Gaynor, R

1989-01-01

The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus E1A and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the E1A and E1B proteins. To determine which regions of the HIV LTR were important for complete E1A/E1B activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. S1 nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivation-responsive element were each required for complete E1A/E1B-mediated activation of the HIV LTR. These same promoter elements were required for both basal and E1A/E1B-induced levels of transcription in in vitro transcription reactions performed with cellular extracts prepared from cells infected with dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. No mutations were found that reduced only E1A/E1B-induced expression without proportionally reducing basal levels of transcription, suggesting that E1A/E1B-mediated induction of the HIV LTR requires multiple promoter elements which are also required for basal transcriptional levels. Unlike activation by the tat protein, there was not a rigid dependence on maintenance of the transactivation-responsive stem base pairing for E1A/E1B-mediated activation either in vivo or in vitro, indicating that activation occurs by a mechanism distinct from that of tat induction. Images PMID:2529378

Oligomerization and chaperone-like activity of Drosophila melanogaster small heat shock protein DmHsp27 and three arginine mutants in the alpha-crystallin domain.

PubMed

Moutaoufik, Mohamed Taha; Morrow, Geneviève; Maaroufi, Halim; Férard, Céline; Finet, Stéphanie; Tanguay, Robert M

2017-07-01

The small Hsp DmHsp27 from Drosophila melanogaster is one of the few small heat shock proteins (sHsps) found within the nucleus. We report that its dimerization is independent of disulfide bond formation and seems to rely on salt bridges. Unlike metazoan sHsps, DmHsp27 forms two populations of oligomers not in equilibrium. Mutations at highly conserved arginine residues in mammalian sHsps have been reported to be associated with protein conformational defects and intracellular aggregation. Independent mutation of three highly conserved arginines (R122, R131, and R135) to glycine in DmHsp27 results in only one population of higher molecular weight form. In vitro, the chaperone-like activity of wild-type DmHsp27 was comparable with that of its two isolated populations and to the single population of the R122G, R131G, and R135G using luciferase as substrate. However, using insulin, the chaperone-like activity of wild-type DmHsp27 was lower than that of R122G and R131G mutants. Altogether, the results characterize wild-type DmHsp27 and its alpha-crystallin domain (ACD) arginine mutants and may give insight into protection mechanism of sHsps.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

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

Zhou, Wenjun; Ercan, Dalia; Chen, Liang

2010-01-12

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potentmore » against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.« less

Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

PubMed Central

El Haddad, Najib; Heathcote, Dean; Moore, Robert; Yang, Sunmi; Azzi, Jamil; Mfarrej, Bechara; Atkinson, Mark; Sayegh, Mohamed H.; Lee, Jeng-Shin; Ashton-Rickardt, Philip G.

2011-01-01

Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases. PMID:21076046

Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia.

PubMed

Bokori-Brown, Monika; Hall, Charlotte A; Vance, Charlotte; Fernandes da Costa, Sérgio P; Savva, Christos G; Naylor, Claire E; Cole, Ambrose R; Basak, Ajit K; Moss, David S; Titball, Richard W

2014-05-13

Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Consequences of long-term oral administration of the mitochondria-targeted antioxidant MitoQ to wild-type mice.

PubMed

Rodriguez-Cuenca, Sergio; Cochemé, Helena M; Logan, Angela; Abakumova, Irina; Prime, Tracy A; Rose, Claudia; Vidal-Puig, Antonio; Smith, Anthony C; Rubinsztein, David C; Fearnley, Ian M; Jones, Bruce A; Pope, Simon; Heales, Simon J R; Lam, Brian Y H; Neogi, Sudeshna Guha; McFarlane, Ian; James, Andrew M; Smith, Robin A J; Murphy, Michael P

2010-01-01

The mitochondria-targeted quinone MitoQ protects mitochondria in animal studies of pathologies in vivo and is being developed as a therapy for humans. However, it is unclear whether the protective action of MitoQ is entirely due to its antioxidant properties, because long-term MitoQ administration may alter whole-body metabolism and gene expression. To address this point, we administered high levels of MitoQ orally to wild-type C57BL/6 mice for up to 28 weeks and investigated the effects on whole-body physiology, metabolism, and gene expression, finding no measurable deleterious effects. In addition, because antioxidants can act as pro-oxidants under certain conditions in vitro, we examined the effects of MitoQ administration on markers of oxidative damage. There were no changes in the expression of mitochondrial or antioxidant genes as assessed by DNA microarray analysis. There were also no increases in oxidative damage to mitochondrial protein, DNA, or cardiolipin, and the activities of mitochondrial enzymes were unchanged. Therefore, MitoQ does not act as a pro-oxidant in vivo. These findings indicate that mitochondria-targeted antioxidants can be safely administered long-term to wild-type mice. Copyright 2009 Elsevier Inc. All rights reserved.

Deletion of Adseverin in Osteoclasts Affects Cell Structure But Not Bone Metabolism.

PubMed

Cao, Yixuan; Wang, Yongqiang; Sprangers, Sara; Picavet, Daisy I; Glogauer, Michael; McCulloch, Christopher A; Everts, Vincent

2017-08-01

Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP + osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm 2 ; p < 0.05), as were nuclear size and the surface area of cytoplasm. The nuclei of adseverin null osteoclasts exhibited more heterochromatin (31 ± 3%) than wild-type cells (8 ± 1%), suggesting that adseverin affects cell differentiation. The data indicate that in healthy, developing tissues, adseverin contributes to the regulation of osteoclast structure but not to bone metabolism in vivo.

Impaired wound healing in mice deficient in a matricellular protein SPARC (osteonectin, BM-40)

PubMed Central

Basu, Amitabha; Kligman, Lorraine H; Samulewicz, Stefan J; Howe, Chin C

2001-01-01

Background SPARC is a matricellular protein involved in cell-matrix interactions. From expression patterns at the wound site and in vitro studies, SPARC has been implicated in the control of wound healing. Here we examined the function of SPARC in cutaneous wound healing using SPARC-null mice and dermal fibroblasts derived from them. Results In large (25 mm) wounds, SPARC-null mice showed a significant delay in healing as compared to wild-type mice (31 days versus 24 days). Granulation tissue formation and extracellular matrix protein production were delayed in small 6 mm SPARC-null wounds initially but were resolved by day 6. In in vitro wound-healing assays, while wild-type primary dermal fibroblasts showed essentially complete wound closure at 11 hours, wound closure of SPARC-null cells was incomplete even at 31 hours. Addition of purified SPARC restored the normal time course of wound closure. Treatment of SPARC-null cells with mitomycin C to analyze cell migration without cell proliferation showed that wound repair remained incomplete after 31 hours. Cell proliferation as measured by 3H-thymidine incorporation and collagen gel contraction by SPARC-null cells were not compromised. Conclusions A significant delay in healing large excisional wounds and setback in granulation tissue formation and extracellular matrix protein production in small wounds establish that SPARC is required for granulation tissue formation during normal repair of skin wounds in mice. A defect in wound closure in vitro indicates that SPARC regulates cell migration. We conclude that SPARC plays a role in wound repair by promoting fibroblast migration and thus granulation tissue formation. PMID:11532190

Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

PubMed Central

Labonté, Eric D.; Howles, Philip N.; Granholm, Norman A.; Rojas, Juan C.; Davies, Joanna P.; Ioannou, Yiannis A.; Hui, David Y.

2007-01-01

Recent studies have documented the importance of Niemann Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1−/− mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1−/− mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1−/− mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1. PMID:17442616

Intact long-type DupA protein in Helicobacter pylori is an ATPase involved in multifunctional biological activities.

PubMed

Wang, Ming-yi; Chen, Cheng; Shao, Chen; Wang, Shao-bo; Wang, Ai-chu; Yang, Ya-chao; Yuan, Xiao-yan; Shao, Shi-he

2015-04-01

The function of intact long-type DupA protein in Helicobacter pylori was analyzed using immunoblotting and molecular biology techniques in the study. After cloning, expression and purification, ATPase activity of DupA protein was detected. Antibody was produced for localization and interaction proteins analysis. The dupA-deleted mutant was generated for adhesion and CagA protein translocation assay, susceptibility to different pH, IL-8 secretion assay, cytotoxicity to MKN-45 cells and proteins-involved apoptosis analysis. DupA protein exhibited an ATPase activity (129.5±17.8 U/mgprot) and located in bacterial membrane, while it did not involve the adhesion and CagA protein delivery of H. pylori. DupA protein involved the urease secretion as the interaction proteins. The wild type strain had a stronger growth in low pH than the dupA-deleted mutant (p < 0.001). IL-8 productions from GES-1 cells infected with the wild type strain were significantly higher than from those with the mutant (p < 0.001). The amounts of vital MKN-45 cells were decreased and the numbers of apoptotic cells were increased with the wild type strain, compared to those with the mutant after 12 h (p < 0.05). The increase of cleaved Caspase-3 and Bax was significantly higher and the decrease of Bcl-2 was more obvious in MKN-45 cells exposed to the wild type strain than that exposed to the mutant after 6 h. We demonstrate that intact long-type DupA protein located in membrane as ATPase is a true virulence factor associated with duodenal ulcer development involving the IL-8 induction and urease secretion, while it inhibits gastric cancer cell growth in vitro by activating the mitochondria-mediated apoptotic pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro.

PubMed

Song, Shaozheng; Ge, Xin; Cheng, Yaobin; Lu, Rui; Zhang, Ting; Yu, Baoli; Ji, Xueqiao; Qi, Zhengqiang; Rong, Yao; Yuan, Yuguo; Cheng, Yong

2016-08-01

The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.

Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank

ERIC Educational Resources Information Center

Ship, Noam J.; Zamble, Deborah B.

2005-01-01

The self directed study of a 3D image of a biomolecule stresses the complex nature of the intra- and intermolecular interactions that come together to define its structure. This is made up of a series of in vitro experiments with a wild-type and mutants forms of human carbonic anhydrase II (hCAII) that examine the structure function relationship…

Accumulation of phenolic compounds in in vitro cultures and wild plants of Lavandula viridis L'Hér and their antioxidant and anti-cholinesterase potential.

PubMed

Costa, Patrícia; Gonçalves, Sandra; Valentão, Patrícia; Andrade, Paula B; Romano, Anabela

2013-07-01

In this study, we evaluated the phenolic profile, antioxidant and anti-cholinesterase potential of different extracts from wild plants and in vitro cultures of Lavandula viridis L'Hér. The HPLC-DAD analysis allowed the identification and quantification of 3-O-caffeoylquinic, 4-O-caffeoylquinic, 5-O-caffeoylquinic and rosmarinic acids, and luteolin and pinocembrin. Water/ethanol extract from in vitro cultures contained the highest amount of the identified phenolic compounds (51652.92 mg/kg). To investigate the antioxidant activity we used Trolox equivalent antioxidant capacity, oxygen radical absorbance capacity, Fe(2+) chelation activity and the inhibition of Fe(2+)-induced lipid peroxidation in mouse brain homogenates (in vitro). Overall, all the extracts from both wild plants and in vitro cultures exhibited ability to scavenge free radicals, to chelate Fe(2+) and to protect against lipid peroxidation. In addition, the extracts from L. viridis were active in inhibiting both acetylcholinesterase and butyrylcholinesterase (Ellman's method). Our findings suggest that L. viridis in vitro cultures represent a promising alternative for the production of active metabolites with antioxidant and anti-cholinesterase activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Multiple adaptive amino acid substitutions increase the virulence of a wild waterfowl-origin reassortant H5N8 avian influenza virus in mice.

PubMed

Yu, Zhijun; Cheng, Kaihui; Sun, Weiyang; Zhang, Xinghai; Xia, Xianzhu; Gao, Yuwei

2018-01-15

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) caused poultry outbreaks in the Republic of Korea in 2014. The novel H5N8 HPAIV has spread to Asia, Europe, and North America and caused great public concern from then on. Here, we generated mouse-adapted variants of a wild waterfowl-origin H5N8 HPAIV to identify adaptive mutants that confer enhanced pathogenicity in mammals. The mouse lethal doses (MLD 50 ) of the mouse-adapted variants were reduced 31623-fold compared to the wild-type (WT) virus. Mouse-adapted variants displayed enhanced replication in vitro and in vivo, and expanded tissue tropism in mice. Sequence analysis revealed four amino acid substitutions in the PB2 (E627K), PA (F35S), HA (R227H), and NA (I462V) proteins. These data suggest that multiple amino acid substitutions collaboratively increase the virulence of a wild bird-origin reassortant H5N8 HPAIV and cause severe disease in mice. Copyright © 2017 Elsevier B.V. All rights reserved.

The role of tumour necrosis factor-α and tumour necrosis factor receptor signalling in inflammation-associated systemic genotoxicity

PubMed Central

Westbrook, Aya M.; Wei, Bo; Hacke, Katrin; Xia, Menghang; Braun, Jonathan; Schiestl, Robert H.

2012-01-01

Chronic inflammatory diseases are characterised by systemically elevated levels of tumour necrosis factor (TNF)-α, a proinflammatory cytokine with pleiotropic downstream effects. We have previously demonstrated increased genotoxicity in peripheral leukocytes and various tissues in models of intestinal inflammation. In the present study, we asked whether TNF-α is sufficient to induce DNA damage systemically, as observed in intestinal inflammation, and whether tumour necrosis factor receptor (TNFR) signalling would be necessary for the resultant genotoxicity. In the wild-type mice, 500 ng per mouse of TNF-α was sufficient to induce DNA damage to multiple cell types and organs 1-h post-administration. Primary splenic T cells manifested TNF-α-induced DNA damage in the absence of other cell types. Furthermore, TNFR1−/−TNFR2−/− mice demonstrated decreased systemic DNA damage in a model of intestinal inflammation and after TNF-α injection versus wild-type mice, indicating the necessity of TNFR signalling. Nuclear factor (NF)-κB inhibitors were also able to decrease damage induced by TNF-α injection in wild-type mice. When TNF-α administration was combined with interleukin (IL)-1β, another proinflammatory cytokine, DNA damage persisted for up to 24 h. When combined with IL-10, an anti-inflammatory cytokine, decreased genotoxicity was observed in vivo and in vitro. TNF-α/TNFR-mediated signalling is therefore sufficient and plays a large role in mediating DNA damage to various cell types, subject to modulation by other cytokines and their mediators. PMID:21980144

InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals.

PubMed

Luo, Yihui; Liu, Yan; Sun, Dexter; Ojcius, David M; Zhao, Jinfang; Lin, Xuai; Wu, Dong; Zhang, Rongguang; Chen, Ming; Li, Lanjuan; Yan, Jie

2011-10-21

Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.

Butyrate-induced apoptotic cascade in colonic carcinoma cells: modulation of the beta-catenin-Tcf pathway and concordance with effects of sulindac and trichostatin A but not curcumin.

PubMed

Bordonaro, M; Mariadason, J M; Aslam, F; Heerdt, B G; Augenlicht, L H

1999-10-01

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.

Mechanism of chloroform-induced renal toxicity: Non-involvement of hepatic cytochrome P450-dependent metabolism

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

Fang Cheng; Behr, Melissa; Xie Fang

2008-02-15

Chloroform causes hepatic and renal toxicity in a number of species. In vitro studies have indicated that chloroform can be metabolized by P450 enzymes in the kidney to nephrotoxic intermediate, although direct in vivo evidence for the role of renal P450 in the nephrotoxicity has not been reported. This study was to determine whether chloroform renal toxicity persists in a mouse model with a liver-specific deletion of the P450 reductase (Cpr) gene (liver-Cpr-null). Chloroform-induced renal toxicity and chloroform tissue levels were compared between the liver-Cpr-null and wild-type mice at 24 h following differing doses of chloroform. At a chloroform dosemore » of 150 mg/kg, the levels of blood urea nitrogen (BUN) were five times higher in the exposed group than in the vehicle-treated one for the liver-Cpr-null mice, but they were only slightly higher in the exposed group than in the vehicle-treated group for the wild-type mice. Severe lesions were found in the kidney of the liver-Cpr-null mice, while only mild lesions were found in the wild-type mice. At a chloroform dose of 300 mg/kg, severe kidney lesions were observed in both strains, yet the BUN levels were still higher in the liver-Cpr-null than in the wild-type mice. Higher chloroform levels were found in the tissues of the liver-Cpr-null mice. These findings indicated that loss of hepatic P450-dependent chloroform metabolism does not protect against chloroform-induced renal toxicity, suggesting that renal P450 enzymes play an essential role in chloroform renal toxicity.« less

Haemophilus influenzae OxyR: Characterization of Its Regulation, Regulon and Role in Fitness

PubMed Central

Whitby, Paul W.; Morton, Daniel J.; VanWagoner, Timothy M.; Seale, Thomas W.; Cole, Brett K.; Mussa, Huda J.; McGhee, Phillip A.; Bauer, Chee Yoon S.; Springer, Jennifer M.; Stull, Terrence L.

2012-01-01

To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a crucial role for OxyR in bacterial fitness. PMID:23226321

Uncoupling the Effects of Abscisic Acid on Plant Growth and Water Relations. Analysis of sto1/nced3, an Abscisic Acid-Deficient but Salt Stress-Tolerant Mutant in Arabidopsis1

PubMed Central

Ruggiero, Bruno; Koiwa, Hisashi; Manabe, Yuzuki; Quist, Tanya M.; Inan, Gunsu; Saccardo, Franco; Joly, Robert J.; Hasegawa, Paul M.; Bressan, Ray A.; Maggio, Albino

2004-01-01

We have identified a T-DNA insertion mutation of Arabidopsis (ecotype C24), named sto1 (salt tolerant), that results in enhanced germination on both ionic (NaCl) and nonionic (sorbitol) hyperosmotic media. sto1 plants were more tolerant in vitro than wild type to Na+ and K+ both for germination and subsequent growth but were hypersensitive to Li+. Postgermination growth of the sto1 plants on sorbitol was not improved. Analysis of the amino acid sequence revealed that STO1 encodes a 9-cis-epoxicarotenoid dioxygenase (similar to 9-cis-epoxicarotenoid dioxygenase GB:AAF26356 [Phaseolus vulgaris] and to NCED3 GB:AB020817 [Arabidopsis]), a key enzyme in the abscisic acid (ABA) biosynthetic pathway. STO1 transcript abundance was substantially reduced in mutant plants. Mutant sto1 plants were unable to accumulate ABA following a hyperosmotic stress, although their basal ABA level was only moderately altered. Either complementation of the sto1 with the native gene from the wild-type genome or supplementation of ABA to the growth medium restored the wild-type phenotype. Improved growth of sto1 mutant plants on NaCl, but not sorbitol, medium was associated with a reduction in both NaCl-induced expression of the ICK1 gene and ethylene accumulation. Osmotic adjustment of sto1 plants was substantially reduced compared to wild-type plants under conditions where sto1 plants grew faster. The sto1 mutation has revealed that reduced ABA can lead to more rapid growth during hyperionic stress by a signal pathway that apparently is at least partially independent of signals that mediate nonionic osmotic responses. PMID:15466233

Fimbria-Encoding Gene yadC Has a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity.

PubMed

Verma, Renu; Rojas, Thaís Cabrera Galvão; Maluta, Renato Pariz; Leite, Janaína Luisa; da Silva, Livia Pilatti Mendes; Nakazato, Gerson; Dias da Silveira, Wanderley

2016-01-01

The extraintestinal pathogen termed avian pathogenic Escherichia coli (APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07. In vitro, the transcription level of yadC was upregulated at 41°C and downregulated at 22°C. The yadC expression in vivo was more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadC strain presented a slightly decreased ability to adhere to HeLa cells with or without the d-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed that fimH was downregulated (P < 0.05) and csgA and ecpA were slightly upregulated in the mutant strain, showing that yadC modulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadC strain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P < 0.05). Motility assays in soft agar demonstrated that the ΔyadC strain was less motile than the wild type (P < 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadC strain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Impaired dacarbazine activation and 7-ethoxyresorufin deethylation in vitro by polymorphic variants of CYP1A1 and CYP1A2: implications for cancer therapy.

PubMed

Lewis, Benjamin C; Korprasertthaworn, Porntipa; Miners, John O

2016-10-01

To extend our understanding of how interindividual variability mediates the efficacy of cancer treatment. The kinetics of dacarbazine (DTIC) N-demethylation by the most frequent polymorphic variants of CYP1A1 (T461N, I462V) and CYP1A2 (F186L, D348N, I386F, R431W, R456H) were characterized, along with kinetic parameters for the O-deethylation of the prototypic CYP1A substrate 7-ethoxyresorufin, using recombinant protein expression and high-performance liquid chromatographic techniques. A reduction of ∼30% in the catalytic efficiencies (measured as in-vitro intrinsic clearance, CLint) was observed for DTIC N-demethylation by the two CYP1A1 variants relative to wild type. Although a modest increase in the CLint value for DTIC N-demethylation was observed for the CYP1A2 D348N variant relative to the wild type, the CLint for the F186L variant was reduced and the I386F, R431W, and R456H variants all showed loss of catalytic function. Comparison of the kinetic data for DTIC N-demethylation and 7-ethoxyresorufin O-deethylation indicated that alterations in the kinetic parameters (Km, Vmax, CLint) observed with each of the CYP1A1 and CYP1A2 polymorphic variants were substrate dependent. These data indicate that cancer patients treated with DTIC who possess any of the CYP1A1-T461N and I462V variants or the CYP1A2-F186L, D348N, I386F, R431W, and R456H variants are likely to have decreased prodrug activation, and hence may respond less favorably to DTIC treatment compared with individuals with wild-type CYP1A alleles.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice.

PubMed

Bozue, Joel A; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K; Toothman, Ronald G; Dankmeyer, Jennifer L; Klimko, Christopher P; Wilhelmsen, Catherine L; Raymond, Jolynn W; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

2016-01-01

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice

PubMed Central

Bozue, Joel A.; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K.; Toothman, Ronald G.; Dankmeyer, Jennifer L.; Klimko, Christopher P.; Wilhelmsen, Catherine L.; Raymond, Jolynn W.; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

2016-01-01

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders. PMID:26955620

Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*

PubMed Central

G. Lavoie, Elise; Dranoff, Jonathan A.

2017-01-01

Liver myofibroblasts are specialized effector cells that drive hepatic fibrosis, a hallmark process of chronic liver diseases, leading to progressive scar formation and organ failure. Liver myofibroblasts are increasingly recognized as heterogeneous with regards to their origin, phenotype, and functions. For instance, liver myofibroblasts express cell markers that are universally represented such as, ItgαV and Pdgfrβ, or restricted to a given subpopulation such as, Lrat exclusively expressed in hepatic stellate cells, and Gpm6a in mesothelial cells. To study liver myofibroblasts in vitro, we have previously generated and characterized a SV40-immortalized polyclonal rat activated portal fibroblast cell line called RGF-N2 expressing multiple mesothelin mRNA transcripts. Mesothelin, a cell-surface molecule expressed in normal mesothelial cells and overexpressed in several cancers such as, mesothelioma and cholangiocarcinoma, was recently identified as a key regulator of portal myofibroblast proliferation, and fibrosis progression in the setting of chronic cholestatic liver disease. Here, we identify novel mesothelin splice variants expressed in rat activated portal fibroblasts. RGF-N2 portal fibroblast cDNA was used as template for insertion of hemagglutinin tag consensus sequence into the complete open reading frame of rat mesothelin variant coding sequences by extension PCR. Purified amplicons were subsequently cloned into an expression vector for in vitro translation and transfection in monkey COS7 fibroblasts, before characterization of fusion proteins by immunoblot and immunofluorescence. We show that rat activated portal fibroblasts, hepatic stellate cells, and cholangiocarcinoma cells express wild-type mesothelin and additional splice variants, while mouse activated hepatic stellate cells appear to only express wild-type mesothelin. Notably, rat mesothelin splice variants differ from the wild-type isoform by their protein properties and cellular distribution in transfected COS7 fibroblasts. We conclude that mesothelin is a marker of activated murine liver myofibroblasts. Mesothelin gene expression and regulation may be critical in liver myofibroblasts functions and fibrosis progression. PMID:28898276

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

Hervey, IV, William Judson; Khalsa-Moyers, Gurusahai K; Lankford, Patricia K

Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed bymore » liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid-encoding strategies for bait protein expression. This approach has the potential for enabling discovery of protein-protein interactions among the growing number of sequenced microbial species without the need for development of chromosomal insertion systems.« less

Virulence Effects and Signaling Partners Modulated by Brucella melitensis Light-sensing Histidine Kinase

NASA Astrophysics Data System (ADS)

Gourley, Christopher R.

The facultative intracellular pathogen Brucella melitensis utilizes diverse virulence factors. A Brucella light sensing histidine kinase can influence in vitro virulence of the bacteria during intracellular infection. First, we demonstrated that the B. melitensis light sensing kinase (BM-LOV-HK) affects virulence in an IRF-1-/- mouse model of infection. Infection with a Δ BM-LOV-HK strain resulted in less bacterial colonization of IRF-1-/- spleens and extended survivorship compared to mice infected with wild type B. melitensis 16M. Second, using PCR arrays, we observed less expression of innate and adaptive immune system activation markers in ΔBM-LOV-HK infected mouse spleens than wild type B. melitensis 16M infected mouse spleens 6 days after infection. Third, we demonstrated by microarray analysis of B. melitensis that deletion of BM-LOV-HK alters bacterial gene expression. Downregulation of genes involved in control of the general stress response system included the alternative sigma factor RpoE1 and its anti-anti sigma factor PhyR. Conversely, genes involved in flagella production, quorum sensing, and the type IV secretion system (VirB operon) were upregulated in the Δ BM-LOV-HK strain compared to the wild type B. melitensis 16M. Analysis of genes differentially regulated in Δ BM-LOV-HK versus the wild type strain indicated an overlap of 110 genes with data from previous quorum sensing regulator studies of Δ vjbR and/ΔblxR(babR) strains. Also, several predicted RpoE1 binding sites located upstream of genes were differentially regulated in the ΔBM-LOV-HK strain. Our results suggest BM-LOV-HK is important for in vivo Brucella virulence, and reveals that BM-LOV-HK directly or indirect regulates members of the Brucella quorum sensing, type IV secretion, and general stress systems.

Mutating the converter-relay interface of Drosophila myosin perturbs ATPase activity, actin motility, myofibril stability and flight ability.

PubMed

Kronert, William A; Melkani, Girish C; Melkani, Anju; Bernstein, Sanford I

2010-05-21

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase (V(max)) by approximately 60% compared to wild-type myosin, but there is no change in apparent actin affinity (K(m)). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by approximately 15% or approximately 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by approximately 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional "cracking" of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle. (c) 2010 Elsevier Ltd. All rights reserved.

Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase: In Vivo and In Vitro Studies of Enzymatic Activity and Substrate Specificity

PubMed Central

Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu

2013-01-01

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780

Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

PubMed

Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J; Gottesman, Susan; Woodson, Sarah A

2015-11-06

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.

PubMed Central

Kirby, S L; Cook, D N; Walton, W; Smithies, O

1996-01-01

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells. Images Fig. 3 PMID:8790342

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function.

PubMed

Sommese, Ruth F; Sung, Jongmin; Nag, Suman; Sutton, Shirley; Deacon, John C; Choe, Elizabeth; Leinwand, Leslie A; Ruppel, Kathleen; Spudich, James A

2013-07-30

Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

Pigmented-MDCK (P-MDCK) Cell Line with Tunable Melanin Expression: An In Vitro Model for the Outer Blood-Retinal-Barrier

PubMed Central

Kadam, Rajendra S.; Scheinman, Robert. I.; Kompella, Uday B.

2013-01-01

Purpose Retinal pigment epithelium, which forms the outer blood-retinal-barrier, is a critical barrier for transport of drugs to the retina. The purpose of this study was to develop a pigmented MDCK (P-MDCK) cell line as a rapidly established in vitro model for the outer blood-retinal-barrier to assess the influence of melanin pigment on solute permeability. Methods A melanin synthesizing P-MDCK cell line was developed by lentiviral transduction of human tyrosinase and p-protein genes in MDCK (NBL-2) cells. Melanin content, tyrosinase activity (conversion of L-dopa to dopachrome), and transepithelial electrical resistance (TEER) were measured. Expression of tyrosinase protein and p-protein in P-MDCK cells was confirmed by confocal microscopy. Effect of L-tyrosine (0 to 2 mM) in culture medium on melanin synthesis in P-MDCK cells was evaluated. Cell uptake and transepithelial transport of pigment-binding chloroquine (Log D = 1.59) and a negative control salicylic acid (Log D = −1.14) were investigated. Results P-MDCK cells expressed tyrosinase and p-protein. Tyrosinase activity was 4.5 fold higher in P-MDCK cells as compared to wild-type MDCK cells. The transepithelial electrical resistance stabilized by day 4 in both cell types, with the TEER being 871 ± 30 and 876 ± 53 Ω.cm2 for P-MDCK and wild-type cells, respectively. Melanin content in P-MDCK cells depended on the concentration of L-tyrosine in culture medium, and increased from 3 to 54 µg/mg protein with an increase in L-tyrosine content from 0 to 2 mM. When the cells were grown in 2 mM L-tyrosine, uptake of chloroquine was 2.3 fold higher and the transepithelial transport was 2.2 fold lower in P-MDCK cells when compared to wild-type MDCK cells. No significant difference was observed for both cell uptake and transport of salicylic acid. Conclusions We developed a P-MDCK cell line with tunable melanin synthesis as a rapidly developing surrogate for retinal pigment epithelium. PMID:23003570

Role of Metal Ions on the Activity of Mycobacterium tuberculosis Pyrazinamidase

PubMed Central

Sheen, Patricia; Ferrer, Patricia; Gilman, Robert H.; Christiansen, Gina; Moreno-Román, Paola; Gutiérrez, Andrés H.; Sotelo, Jun; Evangelista, Wilfredo; Fuentes, Patricia; Rueda, Daniel; Flores, Myra; Olivera, Paula; Solis, José; Pesaresi, Alessandro; Lamba, Doriano; Zimic, Mirko

2012-01-01

Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co2+, Mn2+, and Zn2+ restored pyrazinamidase activity, only Co2+ enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu2+, Fe2+, Fe3+, and Mg2+ did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn2+. However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn. PMID:22764307

Proximal tubular secretion of creatinine by organic cation transporter OCT2 in cancer patients.

PubMed

Ciarimboli, Giuliano; Lancaster, Cynthia S; Schlatter, Eberhard; Franke, Ryan M; Sprowl, Jason A; Pavenstädt, Hermann; Massmann, Vivian; Guckel, Denise; Mathijssen, Ron H J; Yang, Wenjian; Pui, Ching-Hon; Relling, Mary V; Herrmann, Edwin; Sparreboom, Alex

2012-02-15

Knowledge of transporters responsible for the renal secretion of creatinine is key to a proper interpretation of serum creatinine and/or creatinine clearance as markers of renal function in cancer patients receiving chemotherapeutic agents. Creatinine transport was studied in transfected HEK293 cells in vitro and in wild-type mice and age-matched organic cation transporter 1 and 2-deficient [Oct1/2(-/-)] mice ex vivo and in vivo. Clinical pharmacogenetic and transport inhibition studies were done in two separate cohorts of cancer patients. Compared with wild-type mice, creatinine clearance was significantly impaired in Oct1/2(-/-) mice. Furthermore, creatinine inhibited organic cation transport in freshly isolated proximal tubules from wild-type mice and humans, but not in those from Oct1/2(-/-) mice. In a genetic association analysis (n = 590), several polymorphisms around the OCT2/SLC22A2 gene locus, including rs2504954 (P = 0.000873), were significantly associated with age-adjusted creatinine levels. Furthermore, in cancer patients (n = 68), the OCT2 substrate cisplatin caused an acute elevation of serum creatinine (P = 0.0083), consistent with inhibition of an elimination pathway. Collectively, this study shows that OCT2 plays a decisive role in the renal secretion of creatinine. This process can be inhibited by OCT2 substrates, which impair the usefulness of creatinine as a marker of renal function. ©2012 AACR.

Estrogen modulates mesenchyme-epidermis interactions in the adult nipple

PubMed Central

Wu, Hsing-Jung; Oh, Ji Won; Spandau, Dan F.; Tholpady, Sunil; Diaz, Jesus; Schroeder, Laura J.; Offutt, Carlos D.; Glick, Adam B.; Plikus, Maksim V.; Koyama, Sachiko

2017-01-01

Maintenance of specialized epidermis requires signals from the underlying mesenchyme; however, the specific pathways involved remain to be identified. By recombining cells from the ventral skin of the K14-PTHrP transgenic mice [which overexpress parathyroid hormone-related protein (PTHrP) in their developing epidermis and mammary glands] with those from wild type, we show that transgenic stroma is sufficient to reprogram wild-type keratinocytes into nipple-like epidermis. To identify candidate nipple-specific signaling factors, we compared gene expression signatures of sorted Pdgfrα-positive ventral K14-PTHrP and wild-type fibroblasts, identifying differentially expressed transcripts that are involved in WNT, HGF, TGFβ, IGF, BMP, FGF and estrogen signaling. Considering that some of the growth factor pathways are targets for estrogen regulation, we examined the upstream role of this hormone in maintaining the nipple. Ablation of estrogen signaling through ovariectomy produced nipples with abnormally thin epidermis, and we identified TGFβ as a negatively regulated target of estrogen signaling. Estrogen treatment represses Tgfβ1 at the transcript and protein levels in K14-PTHrP fibroblasts in vitro, while ovariectomy increases Tgfb1 levels in K14-PTHrP ventral skin. Moreover, ectopic delivery of Tgfβ1 protein into nipple connective tissue reduced epidermal proliferation. Taken together, these results show that specialized nipple epidermis is maintained by estrogen-induced repression of TGFβ signaling in the local fibroblasts. PMID:28289136

Attenuated activation of macrophage TLR9 by DNA from virulent mycobacteria.

PubMed

Kiemer, Alexandra K; Senaratne, Ryan H; Hoppstädter, Jessica; Diesel, Britta; Riley, Lee W; Tabeta, Koichi; Bauer, Stefan; Beutler, Bruce; Zuraw, Bruce L

2009-01-01

Alveolar macrophages are the first line of host defence against mycobacteria, but an insufficient host response allows survival of bacteria within macrophages. We aimed to investigate the role of Toll-like receptor 9 (TLR9) activation in macrophage defence against mycobacteria. Human in vitro differentiated macrophages as well as human and mouse alveolar macrophages showed TLR9 mRNA and protein expression. The cells were markedly activated by DNA isolated from attenuated mycobacterial strains (H37Ra and Mycobacterium bovis BCG) as assessed by measuring cytokine expression by real-time PCR, whereas synthetic phosphorothioate-modified oligonucleotides had a much lower potency to activate human macrophages. Intracellular replication of H37Ra was higher in macrophages isolated from TLR9-deficient mice than in macrophages from wild-type mice, whereas H37Rv showed equal survival in cells from wild-type or mutant mice. Increased bacterial survival in mouse macrophages was accompanied by altered cytokine production as determined by Luminex bead assays. In vivo infection experiments also showed differential cytokine production in TLR9-deficient mice compared to wild-type animals. Both human monocyte-derived macrophages as well as human alveolar macrophages showed reduced activation upon treatment with DNA isolated from bacteria from virulent (M. bovis and H37Rv) compared to attenuated mycobacteria. We suggest attenuated TLR9 activation contributes to the insufficient host response against virulent mycobacteria. Copyright 2008 S. Karger AG, Basel.

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status.

PubMed

Granja, Sara; Marchiq, Ibtissam; Le Floch, Renaud; Moura, Conceição Souto; Baltazar, Fátima; Pouysségur, Jacques

2015-03-30

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via 'Zinc-Finger Nucleases'. The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

The R403Q Myosin Mutation Implicated in Familial Hypertrophic Cardiomyopathy Causes Disorder at the Actomyosin Interface

PubMed Central

Volkmann, Niels; Lui, HongJun; Hazelwood, Larnele; Trybus, Kathleen M.; Lowey, Susan; Hanein, Dorit

2007-01-01

Background Mutations in virtually all of the proteins comprising the cardiac muscle sarcomere have been implicated in causing Familial Hypertrophic Cardiomyopathy (FHC). Mutations in the β-myosin heavy chain (MHC) remain among the most common causes of FHC, with the widely studied R403Q mutation resulting in an especially severe clinical prognosis. In vitro functional studies of cardiac myosin containing the R403Q mutation have revealed significant changes in enzymatic and mechanical properties compared to wild-type myosin. It has been proposed that these molecular changes must trigger events that ultimately lead to the clinical phenotype. Principal Findings Here we examine the structural consequences of the R403Q mutation in a recombinant smooth muscle myosin subfragment (S1), whose kinetic features have much in common with slow β-MHC. We obtained three-dimensional reconstructions of wild-type and R403Q smooth muscle S1 bound to actin filaments in the presence (ADP) and absence (apo) of nucleotide by electron cryomicroscopy and image analysis. We observed that the mutant S1 was attached to actin at highly variable angles compared to wild-type reconstructions, suggesting a severe disruption of the actin-myosin interaction at the interface. Significance These results provide structural evidence that disarray at the molecular level may be linked to the histopathological myocyte disarray characteristic of the diseased state. PMID:17987111

Thinking outside the "bug": a unique assay to measure intracellular drug penetration in gram-negative bacteria.

PubMed

Zhou, Ying; Joubran, Camil; Miller-Vedam, Lakshmi; Isabella, Vincent; Nayar, Asha; Tentarelli, Sharon; Miller, Alita

2015-04-07

Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5-50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

Cylindromatosis mediates neuronal cell death in vitro and in vivo.

PubMed

Ganjam, Goutham K; Terpolilli, Nicole Angela; Diemert, Sebastian; Eisenbach, Ina; Hoffmann, Lena; Reuther, Christina; Herden, Christiane; Roth, Joachim; Plesnila, Nikolaus; Culmsee, Carsten

2018-01-19

The tumor-suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme and key regulator of cell proliferation and inflammation. A genome-wide siRNA screen linked CYLD to receptor interacting protein-1 (RIP1) kinase-mediated necroptosis; however, the exact mechanisms of CYLD-mediated cell death remain unknown. Therefore, we investigated the precise role of CYLD in models of neuronal cell death in vitro and evaluated whether CYLD deletion affects brain injury in vivo. In vitro, downregulation of CYLD increased RIP1 ubiquitination, prevented RIP1/RIP3 complex formation, and protected neuronal cells from oxidative death. Similar protective effects were achieved by siRNA silencing of RIP1 or RIP3 or by pharmacological inhibition of RIP1 with necrostatin-1. In vivo, CYLD knockout mice were protected from trauma-induced brain damage compared to wild-type littermate controls. These findings unravel the mechanisms of CYLD-mediated cell death signaling in damaged neurons in vitro and suggest a cell death-mediating role of CYLD in vivo.

Expression of Haemophilus ducreyi Collagen Binding Outer Membrane Protein NcaA Is Required for Virulence in Swine and Human Challenge Models of Chancroid

PubMed Central

Fulcher, Robert A.; Cole, Leah E.; Janowicz, Diane M.; Toffer, Kristen L.; Fortney, Kate R.; Katz, Barry P.; Orndorff, Paul E.; Spinola, Stanley M.; Kawula, Thomas H.

2006-01-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection. PMID:16622201

Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

PubMed

Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

2006-05-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

Carboetomidate: A Pyrrole Analogue of Etomidate Designed Not To Suppress Adrenocortical Function

PubMed Central

Cotten, Joseph F.; Forman, Stuart A.; Laha, Joydev K.; Cuny, Gregory D.; Husain, S. Shaukat; Miller, Keith W.; Nguyen, Hieu H.; Kelly, Elizabeth W.; Stewart, Deirdre; Liu, Aiping; Raines, Douglas E.

2010-01-01

Background Etomidate is a sedative-hypnotic that is often used in critically ill patients because it provides superior hemodynamic stability. However it also binds with high affinity to 11β-hydroxylase, potently suppressing synthesis of steroids by the adrenal gland that are necessary for survival. We report the results of studies to define the pharmacology of (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), a pyrrole analogue of etomidate specifically designed not to bind with high affinity to 11β-hydroxylase. Methods The hypnotic potency of carboetomidate was defined in tadpoles and rats using loss of righting reflex assays. Its ability to enhance wild-type α1β2γ2L and etomidate-insensitive mutant α1β2(M286W)γ2L human γ-aminobutyric acid type A receptor activities was assessed using electrophysiological techniques. Its potency for inhibiting in vitro cortisol synthesis was defined using a human adrenocortical cell assay. Its effects on in vivo hemodynamic and adrenocortical function were defined in rats. Results Carboetomidate was a potent hypnotic in tadpoles and rats. It increased currents mediated by wild-type, but not etomidate-insensitive mutant γ-aminobutyric acid type A receptors. Carboetomidate was three orders of magnitude less potent an inhibitor of in vitro cortisol synthesis by adrenocortical cells than was etomidate. In rats, carboetomidate caused minimal hemodynamic changes and did not suppress adrenocortical function at hypnotic doses. Conclusions Carboetomidate is an etomidate analogue that retains many of etomidate’s beneficial properties, but is dramatically less potent as an inhibitor of adrenocortical steroid synthesis. Carboetomidate is a promising new sedative-hypnotic for potential use in critically ill patients in whom adrenocortical suppression is undesirable. PMID:20179500

Delayed in vitro development of Up states but normal network plasticity in Fragile X circuits.

PubMed

Motanis, Helen; Buonomano, Dean

2015-09-01

A broad range of neurophysiological phenotypes have been reported since the generation of the first mouse model of Fragile X syndrome (FXS). However, it remains unclear which phenotypes are causally related to the cognitive deficits associated with FXS. Indeed, because many of these phenotypes are known to be modulated by experience, a confounding factor in the interpretation of many studies is whether some phenotypes are an indirect consequence of abnormal development and experience. To help diminish this confound we first conducted an in vitro developmental study of spontaneous neural dynamics in cortical organotypic cultures. A significant developmental increase in network activity and Up states was observed in both wild-type and Fmr1(-/y) circuits, along with a specific developmental delay in the emergence of Up states in knockout circuits. To determine whether Up state regulation is generally impaired in FXS circuits, we examined Up state plasticity using chronic optogenetic stimulation. Wild-type and Fmr1(-/y) stimulated circuits exhibited a significant decrease in overall spontaneous activity including Up state frequency; however, no significant effect of genotype was observed. These results demonstrate that developmental delays characteristic of FXS are recapitulated during in vitro development, and that Up state abnormalities are probably a direct consequence of the disease, and not an indirect consequence of abnormal experience. However, the fact that Fmr1(-/y) circuits exhibited normal homeostatic modulation of Up states suggests that these plasticity mechanisms are largely intact, and that some of the previously reported plasticity deficits could reflect abnormal experience or the engagement of compensatory mechanisms. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Loss of Bmal1 decreases oocyte fertilization, early embryo development and implantation potential in female mice.

PubMed

Xu, Jian; Li, Yan; Wang, Yizi; Xu, Yanwen; Zhou, Canquan

2016-10-01

Biological clock genes expressed in reproductive tissues play important roles in maintaining the normal functions of reproductive system. However, disruption of female circadian rhythm on oocyte fertilization, preimplantation embryo development and blastocyst implantation potential is still unclear. In this study, ovulation, in vivo and in vitro oocyte fertilization, embryo development, implantation and intracellular reactive oxygen species (ROS) levels in ovary and oviduct were studied in female Bmal1+/+ and Bmal1-/- mice. The number of naturally ovulated oocyte in Bmal1-/- mice decreased (5.2 ± 0.8 vs 7.8 ± 0.8, P < 0.001), with an increasing abnormal oocyte ratio (20.4 ± 3.5 vs 11.7 ± 2.0%, P = 0.001) after superovulation. Significantly lower fertilization rate and obtained blastocyst number were observed in Bmal1-/- female mice either mated with wild-type in vivo or fertilized by sperm from wild-type male mice in vitro (all P < 0.05). Interestingly, in vitro fertilization rate of oocytes derived from Bmal1-/- increased significantly compared with in vivo study (P < 0.01). After transferring blastocysts derived from Bmal1+/+ and Bmal1-/- female mice to pseudopregnant mice, the implantation sites of the latter decreased 5 days later (8.0 ± 0.8 vs 5.3 ± 1.0, P = 0.005). The intracellular ROS levels in the ovary on proestrus day and in the oviduct on metestrus day increased significantly in Bmal1-/- mice compared with that of Bmal1+/+ mice. Deletion of the core biological clock gene Bmal1 significantly decreases oocyte fertilization rate, early embryo development and implantation potential in female mice, and these may be possibly caused by excess ROS levels generated in ovary and oviduct.

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

PubMed

Chen, Jun; Miller, Marina; Unno, Hirotoshi; Rosenthal, Peter; Sanderson, Michael J; Broide, David H

2017-09-07

Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Candida albicans ISW2 Regulates Chlamydospore Suspensor Cell Formation and Virulence In Vivo in a Mouse Model of Disseminated Candidiasis

PubMed Central

Lionakis, Michail S.; Nickerson, Kenneth W.

2016-01-01

Formation of chlamydospores by Candida albicans was an established medical diagnostic test to confirm candidiasis before the molecular era. However, the functional role and pathological relevance of this in vitro morphological transition to pathogenesis in vivo remain unclear. We compared the physical properties of in vitro-induced chlamydospores with those of large C. albicans cells purified by density gradient centrifugation from Candida-infected mouse kidneys. The morphological and physical properties of these cells in kidneys of mice infected intravenously with wild type C. albicans confirmed that chlamydospores can form in infected kidneys. A previously reported chlamydospore-null Δisw2/Δisw2 mutant was used to investigate its role in virulence and chlamydospore induction. Virulence of the Δisw2/Δisw2 mutant strain was reduced 3.4-fold compared to wild type C. albicans or the ISW2 reconstituted strain. Altered host inflammatory reactions to the null mutant further indicate that ISW2 is a virulence factor in C. albicans. ISW2 deletion abolished chlamydospore formation within infected mouse kidneys, whereas the reconstituted strain restored chlamydospore formation in kidneys. Under chlamydospore inducing conditions in vitro, deletion of ISW2 significantly delayed chlamydospore formation, and those late induced chlamydospores lacked associated suspensor cells while attaching laterally to hyphae via novel spore-hypha septa. Our findings establish the induction of chlamydospores by C. albicans during mouse kidney colonization. Our results indicate that ISW2 is not strictly required for chlamydospores formation but is necessary for suspensor cell formation. The importance of ISW2 in chlamydospore morphogenesis and virulence may lead to additional insights into morphological differentiation and pathogenesis of C. albicans in the host microenvironment. PMID:27727302

The flagella of F18ab Escherichia coli is a virulence factor that contributes to infection in a IPEC-J2 cell model in vitro.

PubMed

Duan, Qiangde; Zhou, Mingxu; Zhu, Xiaofang; Bao, Wenbin; Wu, Shenglong; Ruan, Xiaosai; Zhang, Weiping; Yang, Yang; Zhu, Jun; Zhu, Guoqiang

2012-11-09

Bacterial flagella contribute to pathogen virulence; however, the role of flagella in the pathogenesis of F18ab E. coli-mediated swine edema disease (ED) is not currently known. We therefore evaluated the role of flagella in F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production using an in vitro cell infection model approach with gene-deletion mutant and complemented bacterial strains. We demonstrated that the flagellin-deficient fliC mutant had a marked decrease in the ability to adhere to and invade porcine epithelial IPEC-J2 cells. Surprisingly, there was no difference in adhesion between the F18 fimbriae-deficient ΔfedA mutant and its parent strain. In addition, both the ΔfedA and double ΔfliCΔfedA mutants exhibited an increased ability to invade IPEC-J2 cells compared to the wild-type strain, although this may be due to increased expression of other adhesins following the loss of F18ab fimbriae and flagella. Compared to the wild-type strain, the ΔfliC mutant showed significantly reduced ability to form biofilm, whereas the ΔfedA mutant increased biofilm formation. Although ΔfliC, ΔfedA, and ΔfliCΔfedA mutants had a reduced ability to stimulate IL-8 production from infected Caco-2 cells, the ΔfliC mutant impaired this ability to a greater extent than the ΔfedA mutant. The results from this study clearly demonstrate that flagella are required for efficient F18ab E. coli adhesion, invasion, biofilm formation, and IL-8 production in vitro. Copyright © 2012 Elsevier B.V. All rights reserved.

In vitro characterization of Multi-Drug Resistant HIV-1 Isolates from a Recently Infected Patient Associated with Dual Tropism and Rapid Disease Progression

PubMed Central

Mohri, Hiroshi; Markowitz, Martin

2013-01-01

Objective: Multi-drug resistant (MDR)-HIV-1 variants are thought to be less fit than wild type virus. In 2005 we reported a case of transmitted MDR-HIV-1 infection associated with dual tropism and rapid clinical progression. Here, we report the in vitro characterization of the virus isolates. Methods: Replication characteristics of bulk and clonal isolates from this case (MDR-1) were examined and compared with these to a panel of transmitted MDR and wild type viruses (MDR-2~4, WT-1, 2). Results: Infectivity and frequency of infectious virion of propagated isolates were high in MDR-1 biological clones (mean titer, 3.5×105 TCID50/ml; mean frequency of infectious virion, 1/2,444) and its bulk isolate (3.2×106TCID50/ml; 1/301), as compared to the other biological clones (7.3×103TCID50/ml; 1/21,320). Up-slope (log10p24/ml/d) of viral replication in PBMC culture was much higher in MDR-1 clones (1.30±0.30: mean±SD) than those of MDR-2~4 (0.75±0.08) or WT-1, -2 clones (0.82±0.03). The bulk isolate and dual tropic biological clones from MDR-1 depleted CD4+ T cells very rapidly in vitro compared to the other viruses tested. Conclusion: These findings support the hypothesis that multi-drug resistant HIV-1 can effectively evolve and compensate to not only retain high level replication but exhibit virulence associated with rapid disease progression. PMID:18645523

Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire

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

Magari, Masaki; Kanehiro, Yuichi; Todo, Kagefumi

Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell linemore » DT40-SW{Delta}C, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SW{Delta}C cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SW{Delta}C cells, although the protein might be highly susceptible to degradation. In DT40-SW{Delta}C cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SW{Delta}C cells may be useful for constructing Ab libraries for efficient screening of mAbs in vitro.« less

The RET p.G533C mutation confers predisposition to multiple endocrine neoplasia type 2A in a Brazilian kindred and is able to induce a malignant phenotype in vitro and in vivo.

PubMed

Oliveira, Mariana N L; Hemerly, Jefferson P; Bastos, André U; Tamanaha, Rosana; Latini, Flavia R M; Camacho, Cléber P; Impellizzeri, Anelise; Maciel, Rui M B; Cerutti, Janete M

2011-09-01

We have previously described a p.G533C substitution in the rearranged during transfection (RET) oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming potential of RET p.G533C mutation. Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C-carriers in this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma. Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis, hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they were absent in the DNA isolated from blood. Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.

Computational modeling of the bHLH domain of the transcription factor TWIST1 and R118C, S144R and K145E mutants

PubMed Central

2012-01-01

Background Human TWIST1 is a highly conserved member of the regulatory basic helix-loop-helix (bHLH) transcription factors. TWIST1 forms homo- or heterodimers with E-box proteins, such as E2A (isoforms E12 and E47), MYOD and HAND2. Haploinsufficiency germ-line mutations of the twist1 gene in humans are the main cause of Saethre-Chotzen syndrome (SCS), which is characterized by limb abnormalities and premature fusion of cranial sutures. Because of the importance of TWIST1 in the regulation of embryonic development and its relationship with SCS, along with the lack of an experimentally solved 3D structure, we performed comparative modeling for the TWIST1 bHLH region arranged into wild-type homodimers and heterodimers with E47. In addition, three mutations that promote DNA binding failure (R118C, S144R and K145E) were studied on the TWIST1 monomer. We also explored the behavior of the mutant forms in aqueous solution using molecular dynamics (MD) simulations, focusing on the structural changes of the wild-type versus mutant dimers. Results The solvent-accessible surface area of the homodimers was smaller on wild-type dimers, which indicates that the cleft between the monomers remained more open on the mutant homodimers. RMSD and RMSF analyses indicated that mutated dimers presented values that were higher than those for the wild-type dimers. For a more careful investigation, the monomer was subdivided into four regions: basic, helix I, loop and helix II. The basic domain presented a higher flexibility in all of the parameters that were analyzed, and the mutant dimer basic domains presented values that were higher than the wild-type dimers. The essential dynamic analysis also indicated a higher collective motion for the basic domain. Conclusions Our results suggest the mutations studied turned the dimers into more unstable structures with a wider cleft, which may be a reason for the loss of DNA binding capacity observed for in vitro circumstances. PMID:22839202

Mutations of the KISS1 Gene in Disorders of Puberty

PubMed Central

Silveira, L. G.; Noel, S. D.; Silveira-Neto, A. P.; Abreu, A. P.; Brito, V. N.; Santos, M. G.; Bianco, S. D. C.; Kuohung, W.; Xu, S.; Gryngarten, M.; Escobar, M. E.; Arnhold, I. J. P.; Mendonca, B. B.; Kaiser, U. B.; Latronico, A. C.

2010-01-01

Context: Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP). Objective: Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH. Patients: Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development. Methods: The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells. Results: Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group. Conclusion: Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype. PMID:20237166

Mutations of the KISS1 gene in disorders of puberty.

PubMed

Silveira, L G; Noel, S D; Silveira-Neto, A P; Abreu, A P; Brito, V N; Santos, M G; Bianco, S D C; Kuohung, W; Xu, S; Gryngarten, M; Escobar, M E; Arnhold, I J P; Mendonca, B B; Kaiser, U B; Latronico, A C

2010-05-01

Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP). Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH. Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development. The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells. Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group. Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype.

Staurosporine scaffold-based rational discovery of the wild-type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog-sensitive kinase technology.

PubMed

Song, Xiaoyun; Liu, Xingcai; Ding, Xi

2017-04-01

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first-line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small-molecule kinase inhibitors in EGFR-targeted chemotherapy. Previously, the reversible pan-kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR T790M ) over wild-type kinase (EGFR WT ), suggesting that the staurosporine scaffold is potentially to develop the wild-type sparing reversible inhibitors of EGFR T790M . Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold-based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico-in vitro analog-sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild-type sparing inhibitors UCN-01, UCN-02, AFN941, and SB-218078 with high or moderate selectivity of 30-, 45-, 5-, and 8-fold for EGFR T790M over EGFR WT , respectively, which are comparable with or even better than that of the parent compound staurosporine (24-fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN-02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding. A hydroxyl group of UCN-02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN-02 than staurosporine for mutant over wild-type kinase. Copyright © 2016 John Wiley & Sons, Ltd.

Antiangiogenic effects and mechanisms of trans-ethyl p-methoxycinnamate from Kaempferia galanga L.

PubMed

He, Zhi-Heng; Yue, Grace Gar-Lee; Lau, Clara Bik-San; Ge, Wei; But, Paul Pui-Hay

2012-11-14

Kaempferia galanga L. (Zingiberaceae) is an aromatic herb and a popular spice used as a condiment in Asian cuisine. The ethanol extract of the dried plant and its successive four subfractions were investigated on zebrafish model by quantitative endogenous alkaline phosphatase assay. Both n-hexane and ethyl acetate fractions had antiangiogenic activity, and two major active components (trans-ethyl p-methoxycinnamate and kaempferol) showed potent antiangiogenic effects on wild-type zebrafish. Because of its much stronger effect and no antiangiogenic activity reported, trans-ethyl p-methoxycinnamate was further investigated for its action mechanism. It dose dependently inhibited vessel formation on both wild- and Tg(fli1a:EGFP)y1-type zebrafish embryos. The semiquantitative reverse transcription polymerase chain reaction assay suggested that trans-ethyl p-methoxycinnamate affects multiple molecular targets related to angiogenesis. In vitro, it specifically inhibited the migration and tube formation of human umbilical vein endothelial cells. In vivo, it could block bFGF-induced vessel formation on Matrigel plug assay.

Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase.

PubMed

Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

2017-07-01

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

Transcriptome and proteome analysis of Salmonella enterica serovar Typhimurium systemic infection of wild type and immune-deficient mice

PubMed Central

Oshota, Olusegun; Fookes, Maria; Schreiber, Fernanda; Chaudhuri, Roy R.; Yu, Lu; Clare, Simon; Choudhary, Jyoti; Thomson, Nicholas R.; Lio, Pietro

2017-01-01

Salmonella enterica are a threat to public health. Current vaccines are not fully effective. The ability to grow in infected tissues within phagocytes is required for S. enterica virulence in systemic disease. As the infection progresses the bacteria are exposed to a complex host immune response. Consequently, in order to continue growing in the tissues, S. enterica requires the coordinated regulation of fitness genes. Bacterial gene regulation has so far been investigated largely using exposure to artificial environmental conditions or to in vitro cultured cells, and little information is available on how S. enterica adapts in vivo to sustain cell division and survival. We have studied the transcriptome, proteome and metabolic flux of Salmonella, and the transcriptome of the host during infection of wild type C57BL/6 and immune-deficient gp91-/-phox mice. Our analyses advance the understanding of how S. enterica and the host behaves during infection to a more sophisticated level than has previously been reported. PMID:28796780

Differential Activity of the Oral Glucan Synthase Inhibitor SCY-078 against Wild-Type and Echinocandin-Resistant Strains of Candida Species.

PubMed

Pfaller, Michael A; Messer, Shawn A; Rhomberg, Paul R; Borroto-Esoda, Katyna; Castanheira, Mariana

2017-08-01

SCY-078 (formerly MK-3118) is a novel orally active inhibitor of fungal β-(1,3)-glucan synthase (GS). SCY-078 is a derivative of enfumafungin and is structurally distinct from the echinocandin class of antifungal agents. We evaluated the in vitro activity of this compound against wild-type (WT) and echinocandin-resistant isolates containing mutations in the FKS genes of Candida spp. Against 36 Candida spp. FKS mutants tested, 30 (83.3%) were non-WT to 1 or more echinocandins, and only 9 (25.0%) were non-WT (MIC, >WT-upper limit) to SCY-078. Among C. glabrata isolates carrying FKS alterations, 84.0% were non-WT to the echinocandins versus only 24.0% for SCY-078. In contrast to the echinocandin comparators, the activity of SCY-078 was minimally affected by the presence of FKS mutations, suggesting that this agent is useful in the treatment of Candida infections due to echinocandin-resistant strains. Copyright © 2017 American Society for Microbiology.

Matrilysin (Matrix Metalloproteinase-7) Mediates E-Cadherin Ectodomain Shedding in Injured Lung Epithelium

PubMed Central

McGuire, John K.; Li, Qinglang; Parks, William C.

2003-01-01

Matrilysin (matrix metalloproteinase-7) is highly expressed in lungs of patients with pulmonary fibrosis and other conditions associated with airway and alveolar injury. Although matrilysin is required for closure of epithelial wounds ex vivo, the mechanism of its action in repair is unknown. We demonstrate that matrilysin mediates shedding of E-cadherin ectodomain from injured lung epithelium both in vitro and in vivo. In alveolar-like epithelial cells, transfection of activated matrilysin resulted in shedding of E-cadherin and accelerated cell migration. In vivo, matrilysin co-localized with E-cadherin at the basolateral surfaces of migrating tracheal epithelium, and the reorganization of cell-cell junctions seen in wild-type injured tissue was absent in matrilysin-null samples. E-cadherin ectodomain was shed into the bronchoalveolar lavage fluid of bleomycin-injured wild-type mice, but was not shed in matrilysin-null mice. These findings identify E-cadherin as a novel substrate for matrilysin and indicate that shedding of E-cadherin ectodomain is required for epithelial repair. PMID:12759241

Recombinant Marburg viruses containing mutations in the IID region of VP35 prevent inhibition of Host immune responses.

PubMed

Albariño, César G; Wiggleton Guerrero, Lisa; Spengler, Jessica R; Uebelhoer, Luke S; Chakrabarti, Ayan K; Nichol, Stuart T; Towner, Jonathan S

2015-02-01

Previous in vitro studies have demonstrated that Ebola and Marburg virus (EBOV and MARV) VP35 antagonize the host cell immune response. Moreover, specific mutations in the IFN inhibitory domain (IID) of EBOV and MARV VP35 that abrogate their interaction with virus-derived dsRNA, lack the ability to inhibit the host immune response. To investigate the role of MARV VP35 in the context of infectious virus, we used our reverse genetics system to generate two recombinant MARVs carrying specific mutations in the IID region of VP35. Our data show that wild-type and mutant viruses grow to similar titers in interferon deficient cells, but exhibit attenuated growth in interferon-competent cells. Furthermore, in contrast to wild-type virus, both MARV mutants were unable to inhibit expression of various antiviral genes. The MARV VP35 mutants exhibit similar phenotypes to those previously described for EBOV, suggesting the existence of a shared immune-modulatory strategy between filoviruses. Published by Elsevier Inc.

Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models.

PubMed

Ng, Samuel Y; Yoshida, Noriaki; Christie, Amanda L; Ghandi, Mahmoud; Dharia, Neekesh V; Dempster, Joshua; Murakami, Mark; Shigemori, Kay; Morrow, Sara N; Van Scoyk, Alexandria; Cordero, Nicolas A; Stevenson, Kristen E; Puligandla, Maneka; Haas, Brian; Lo, Christopher; Meyers, Robin; Gao, Galen; Cherniack, Andrew; Louissaint, Abner; Nardi, Valentina; Thorner, Aaron R; Long, Henry; Qiu, Xintao; Morgan, Elizabeth A; Dorfman, David M; Fiore, Danilo; Jang, Julie; Epstein, Alan L; Dogan, Ahmet; Zhang, Yanming; Horwitz, Steven M; Jacobsen, Eric D; Santiago, Solimar; Ren, Jian-Guo; Guerlavais, Vincent; Annis, D Allen; Aivado, Manuel; Saleh, Mansoor N; Mehta, Amitkumar; Tsherniak, Aviad; Root, David; Vazquez, Francisca; Hahn, William C; Inghirami, Giorgio; Aster, Jon C; Weinstock, David M; Koch, Raphael

2018-05-22

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.

Rescue of neonatal cardiac dysfunction in mice by administration of cardiac progenitor cells in utero

PubMed Central

Liu, Xiaoli; Hall, Sean R. R.; Wang, Zhihong; Huang, He; Ghanta, Sailaja; Di Sante, Moises; Leri, Annarosa; Anversa, Piero; Perrella, Mark A.

2015-01-01

Striated preferentially expressed gene (Speg) is a member of the myosin light chain kinase family. We previously showed that disruption of the Speg gene locus in mice leads to a dilated cardiomyopathy with immature-appearing cardiomyocytes. Here we show that cardiomyopathy of Speg−/− mice arises as a consequence of defects in cardiac progenitor cell (CPC) function, and that neonatal cardiac dysfunction can be rescued by in utero injections of wild-type CPCs into Speg−/− foetal hearts. CPCs harvested from Speg−/− mice display defects in clone formation, growth and differentiation into cardiomyocytes in vitro, which are associated with cardiac dysfunction in vivo. In utero administration of wild-type CPCs into the hearts of Speg−/− mice results in CPC engraftment, differentiation and myocardial maturation, which rescues Speg−/− mice from neonatal heart failure and increases the number of live births by fivefold. We propose that in utero administration of CPCs may have future implications for treatment of neonatal heart diseases. PMID:26593099

Virulence characteristics of extraintestinal pathogenic Escherichia coli deletion of gene encoding the outer membrane protein X.

PubMed

Meng, Xianrong; Liu, Xueling; Zhang, Liyuan; Hou, Bo; Li, Binyou; Tan, Chen; Li, Zili; Zhou, Rui; Li, Shaowen

2016-09-01

Outer membrane protein X (OmpX) and its homologues have been proposed to contribute to the virulence in various bacterial species. But, their role in virulence of extraintestinal pathogenic Escherichia coli (ExPEC) is yet to be determined. This study evaluates the role of OmpX in ExPEC virulence in vitro and in vivo using a clinical strain PPECC42 of porcine origin. The ompX deletion mutant exhibited increased swimming motility and decreased adhesion to, and invasion of pulmonary epithelial A549 cell, compared to the wild-type strain. A mild increase in LD50 and distinct decrease in bacterial load in such organs as heart, liver, spleen, lung and kidney were observed in mice infected with the ompX mutant. Complementation of the complete ompX gene in trans restored the virulence of mutant strain to the level of wild-type strain. Our results reveal that OmpX contributes to ExPEC virulence, but may be not an indispensable virulence determinant.

Interleukin-12 (IL-12)-driven alloimmune responses in vitro and in vivo: requirement for beta1 subunit of the IL-12 receptor.

PubMed

Piccotti, J R; Li, K; Chan, S Y; Eichwald, E J; Bishop, D K

1999-06-15

Interleukin-12 (IL-12) mediates its biologic activities via binding high-affinity receptors on T and natural killer cells. Although emphasis has been placed on the requirement for IL-12Rbeta2 in IL-12 bioactivity, the role of IL-12Rbeta1 is less well defined. The current study evaluated the effects of exogenous IL-12 on alloantigen-specific immune responses and determined the requirement for IL-12Rbeta1 in IL-12-mediated alloimmunity. The mouse heterotopic cardiac transplant model was employed to evaluate the effects of IL-12 on alloantigen-specific immune responses in vivo. In addition, IFN-gamma production in mixed lymphocyte cultures (MLC) supplemented with IL-12 was measured to assess the effects of IL-12 on Th1 function in vitro. Mice deficient in IL-12Rbeta1 (IL-12Rbeta1-/-) were used to determine the requirement for this receptor component in IL-12-driven alloimmune responses. Addition of IL-12 to MLC consisting of wild-type splenocytes enhanced alloantigen-specific proliferative responses and Th1 development. In contrast, IL-12 did not alter these in vitro immune parameters in IL-12Rbeta1-/- MLC. Treatment of wild-type cardiac allograft recipients with IL-12 resulted in high concentrations of serum interferon-gamma (IFN-gamma) and a 10-fold increase in IFN-gamma production by recipient splenocytes after restimulation in vitro. However, this fulminate Th1 response did not accelerate allograft rejection. Importantly, IL-12 had no effect on serum IFN-gamma or in vivo priming of Thl in IL-12Rbeta1-/- recipients. Finally, administration of IL-12 to WT allograft recipients resulted in a bimodal alloantibody response: antibody production was suppressed at high doses of IL-12, and enhanced at lower doses. IL-12 markedly enhances alloantigen-specific immune function; however, these exaggerated Th1-driven responses do not culminate in accelerated allograft rejection. Further, these data indicate that IL-12Rbeta1 is essential for the enhancement of both in vitro and in vivo alloimmune responses by exogenous IL-12.

Type 1 fimbriae contribute to catheter-associated urinary tract infections caused by Escherichia coli.

PubMed

Reisner, Andreas; Maierl, Mario; Jörger, Michael; Krause, Robert; Berger, Daniela; Haid, Andrea; Tesic, Dijana; Zechner, Ellen L

2014-03-01

Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI), which represent the most frequent nosocomial infections. Knowledge of genetic factors for catheter colonization is limited, since their role has not been assessed using physicochemical conditions prevailing in a catheterized human bladder. The current study aimed to combine data from a dynamic catheterized bladder model in vitro with in vivo expression analysis for understanding molecular factors relevant for CAUTI caused by Escherichia coli. By application of the in vitro model that mirrors the physicochemical environment during human infection, we found that an E. coli K-12 mutant defective in type 1 fimbriae, but not isogenic mutants lacking flagella or antigen 43, was outcompeted by the wild-type strain during prolonged catheter colonization. The importance of type 1 fimbriae for catheter colonization was verified using a fimA mutant of uropathogenic E. coli strain CFT073 with human and artificial urine. Orientation of the invertible element (IE) controlling type 1 fimbrial expression in bacterial populations harvested from the colonized catheterized bladder in vitro suggested that the vast majority of catheter-colonizing cells (up to 88%) express type 1 fimbriae. Analysis of IE orientation in E. coli populations harvested from patient catheters revealed that a median level of ∼73% of cells from nine samples have switched on type 1 fimbrial expression. This study supports the utility of the dynamic catheterized bladder model for analyzing catheter colonization factors and highlights a role for type 1 fimbriae during CAUTI.

A gene capable of blocking apoptosis can substitute for the herpes simplex virus type 1 latency-associated transcript gene and restore wild-type reactivation levels.

PubMed

Perng, Guey-Chuen; Maguen, Barak; Jin, Ling; Mott, Kevin R; Osorio, Nelson; Slanina, Susan M; Yukht, Ada; Ghiasi, Homayon; Nesburn, Anthony B; Inman, Melissa; Henderson, Gail; Jones, Clinton; Wechsler, Steven L

2002-02-01

After ocular herpes simplex virus type 1 (HSV-1) infection, the virus travels up axons and establishes a lifelong latent infection in neurons of the trigeminal ganglia. LAT (latency-associated transcript), the only known viral gene abundantly transcribed during HSV-1 neuronal latency, is required for high levels of reactivation. The LAT function responsible for this reactivation phenotype is not known. Recently, we showed that LAT can block programmed cell death (apoptosis) in neurons of the trigeminal ganglion in vivo and in tissue culture cells in vitro (G.-C. Perng et al., Science 287:1500-1503, 2000; M. Inman et al., J. Virol. 75:3636-3646, 2001). Consequently, we proposed that this antiapoptosis function may be a key to the mechanism by which LAT enhances reactivation. To study this further, we constructed a recombinant HSV-1 virus in which the HSV-1 LAT gene was replaced by an alternate antiapoptosis gene. We used the bovine herpes virus 1 (BHV-1) latency-related (LR) gene, which was previously shown to have antiapoptosis activity, for this purpose. The resulting chimeric virus, designated CJLAT, contains two complete copies of the BHV-1 LR gene (one in each viral long repeat) in place of the normal two copies of the HSV-1 LAT, on an otherwise wild-type HSV-1 strain McKrae genomic background. We report here that in both rabbits and mice reactivation of CJLAT was significantly greater than the LAT null mutant dLAT2903 (P < 0.0004 and P = 0.001, respectively) and was at least as efficient as wild-type McKrae. This strongly suggests that a BHV-1 LR gene function was able to efficiently substitute for an HSV-1 LAT gene function involved in reactivation. Although replication of CJLAT in rabbits and mice was similar to that of wild-type McKrae, CJLAT killed more mice during acute infection and caused more corneal scarring in latently infected rabbits. This suggested that the BHV-1 LR gene and the HSV-1 LAT gene are not functionally identical. However, LR and LAT both have antiapoptosis activity. These studies therefore strongly support the hypothesis that replacing LAT with an antiapoptosis gene restores the wild-type reactivation phenotype to a LAT null mutant of HSV-1 McKrae.

Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins.

PubMed Central

Bayer, A S; Ramos, M D; Menzies, B E; Yeaman, M R; Shen, A J; Cheung, A L

1997-01-01

Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis. PMID:9353046

Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.

PubMed

Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S

2015-06-01

We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors.

Gas6 Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.

PubMed

Laurance, Sandrine; Bertin, François-René; Ebrahimian, Talin; Kassim, Yusra; Rys, Ryan N; Lehoux, Stéphanie; Lemarié, Catherine A; Blostein, Mark D

2017-07-01

Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl 3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6 -/- mice contain less inflammatory (CCR2 hi CX 3 CR1 lo ) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2 hi CX 3 CR1 lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2 hi CX 3 CR1 lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis. © 2017 American Heart Association, Inc.

Regulation of Mammary Stem Cell Quiescence via Post-Translational Modification of DeltaNp63alpha

DTIC Science & Technology

2012-12-01

This document is the Annual Summary Report on the training grant awarded to Andrew DeCastro entitled Regulation of Mammary Stem Cell Quiescence via...screen) mediated phosphorylation of deltaNPdelta3 on stem cell behavior and mitotic activity. Task 1 aims to determine the effects of wild-type, phospho...ablative and phospho-mimetic alleles of deltaNP63delta phosphorylation on stem cell behavior in vitro. Thus far, we demonstrate that stem cell enriched

Electrotransformation of highly DNA-restrictive corynebacteria with synthetic DNA.

PubMed

Ankri, S; Reyes, O; Leblon, G

1996-01-01

Highly DNA-restrictive Corynebacteria can be transformed with DNA made in vitro by PCR amplification of a sequence that contains the replication origin of pBL1, a plasmid common to many Corynebacteria. In all strains examined, the transformation efficiencies of PCR-synthetized DNA equal or improve the performances of heterologous DNA extracted from wild-type and dam(-)-dcm-strains of Escherichia coli. The transformation efficiencies obtained with PCR-made DNA may be high enough to permit its general application to experiments of gene integration.

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

Jeon, En Hee; Pak, Jung Hun; Kim, Mi Jin

Highlights: Black-Right-Pointing-Pointer We isolated a novel E2 ubiquitin-conjugating enzyme from leaves of wild rice plants. Black-Right-Pointing-Pointer The OgUBC1 was highly expressed in leaves treated with SA and UV-B radiation. Black-Right-Pointing-Pointer The recombinant OgUBC1 has an enzymatic activity of E2 in vitro. Black-Right-Pointing-Pointer The OgUBC1 could protect disruption of plant cells by UV-B radiation. Black-Right-Pointing-Pointer OgUBC1 confers disease resistance and UV-B tolerance in transgenic Arabidopsis plants. -- Abstract: A previously unidentified gene encoding ubiquitin-conjugating enzyme was isolated from leaves of wild rice plant treated with wounding and microbe-associated molecular patterns. The OgUBC1 gene was composed of 148 amino acids and containedmore » a typical active site and 21 ubiquitin thioester intermediate interaction residues and 4 E3 interaction residues. Both exogenous application of salicylic acid and UV-B irradiation triggered expression of OgUBC1 in leaves of wild rice. Recombinant OgUBC1 proteins bound to ubiquitins in vitro, proposing that the protein might act as E2 enzyme in planta. Heterologous expression of the OgUBC1 in Arabidopsis thaliana protected plants from cellular damage caused by an excess of UV-B radiation. A stable expression of chalcone synthase gene was detected in leaves of OgUBC1-expressing Arabidopsis, resulting in producing higher amounts of anthocyanin than those in wild-type Col-0 plants. Additionally, both pathogenesis-related gene1 and 5 were transcribed in the transgenic Arabidopsis in the absence of pathogen infection. The OgUBC1-expressing plants were resistant to the infection of Botrytis cinerea. Taken together, we suggested that the OgUBC1 is involved in ubiquitination process important for cellular response against biotic and abiotic stresses in plants.« less

Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress

PubMed Central

Havaux, Michel; Ksas, Brigitte; Szewczyk, Agnieszka; Rumeau, Dominique; Franck, Fabrice; Caffarri, Stefano; Triantaphylidès, Christian

2009-01-01

Background Vitamin B6 is a collective term for a group of six interconvertible compounds: pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives. Vitamin B6 plays essential roles as a cofactor in a range of biochemical reactions. In addition, vitamin B6 is able to quench reactive oxygen species in vitro, and exogenously applied vitamin B6 protects plant cells against cell death induced by singlet oxygen (1O2). These results raise the important question as to whether plants employ vitamin B6 as an antioxidant to protect themselves against reactive oxygen species. Results The pdx1.3 mutation affects the vitamin B6 biosynthesis enzyme, pyridoxal synthase (PDX1), and leads to a reduction of the vitamin B6 concentration in Arabidopsis thaliana leaves. Although leaves of the pdx1.3 Arabidopsis mutant contained less chlorophyll than wild-type leaves, we found that vitamin B6 deficiency did not significantly impact photosynthetic performance or shoot and root growth. Chlorophyll loss was associated with an increase in the chlorophyll a/b ratio and a selective decrease in the abundance of several PSII antenna proteins (Lhcb1/2, Lhcb6). These changes were strongly dependent on light intensity, with high light amplifying the difference between pdx1.3 and the wild type. When leaf discs were exposed to exogenous 1O2, lipid peroxidation in pdx1.3 was increased relative to the wild type; this effect was not observed with superoxide or hydrogen peroxide. When leaf discs or whole plants were exposed to excess light energy, 1O2-mediated lipid peroxidation was enhanced in leaves of the pdx1.3 mutant relative to the wild type. High light also caused an increased level of 1O2 in vitamin B6-deficient leaves. Combining the pdx1.3 mutation with mutations affecting the level of 'classical' quenchers of 1O2 (zeaxanthin, tocopherols) resulted in a highly photosensitive phenotype. Conclusion This study demonstrates that vitamin B6 has a function in the in vivo antioxidant defense of plants. Thus, the antioxidant activity of vitamin B6 inferred from in vitro studies is confirmed in planta. Together with the finding that chloroplasts contain vitamin B6 compounds, the data show that vitamin B6 functions as a photoprotector that limits 1O2 accumulation in high light and prevents 1O2-mediated oxidative damage. PMID:19903353

Effect of inactivation of the global oxidative stress regulator oxyR on the colonization ability of Escherichia coli O1:K1:H7 in a mouse model of ascending urinary tract infection.

PubMed

Johnson, James R; Clabots, Connie; Rosen, Henry

2006-01-01

To survive within the host urinary tract, Escherichia coli strains that cause urinary tract infection (UTI) presumably must overcome powerful oxidant stresses, including the oxygen-dependent killing mechanisms of neutrophils. Accordingly, we assessed the global oxygen stress regulator OxyR of Escherichia coli as a possible virulence factor in UTI by determining the impact of oxyR inactivation on experimental urovirulence in CBA/J and C57BL (both wild-type and p47(phox-/-)) mice. The oxyR and oxyS genes of wild-type E. coli strain Ec1a (O1:K1:H7) were replaced with a kanamycin resistance cassette to produce an oxyRS mutant. During in vitro growth in broth or human urine, the oxyRS mutant exhibited the same log-phase growth rate (broth) and plateau density (broth and urine) as Ec1a, despite its prolonged lag phase (broth) or initial decrease in concentration (urine). The mutant, and oxyRS mutants of other wild-type ExPEC strains, exhibited significantly increased in vitro susceptibility to inhibition by H(2)O(2), which, like the altered growth kinetics observed with oxyRS inactivation, were reversed by restoration of oxyR on a multiple-copy-number plasmid. In CBA/J mice, Ec1a significantly outcompeted its oxyRS mutant (by >1 log(10)) in urine, bladder, and kidney cultures harvested 48 h after perurethral inoculation of mice, whereas an oxyR-complemented mutant exhibited equal or greater colonizing ability than that of the parent. Although C57BL mice were less susceptible to experimental UTI than CBA/J mice, wild-type and p47(phox-/-) C57BL mice were similarly susceptible, and the oxyR mutant of Ec1a was similarly attenuated in C57BL mice, regardless of the p47(phox) genotype, as in CBA/J mice. Within the E. coli Reference collection, 94% of strains were positive for oxyR. These findings fulfill the second and third of Koch's molecular postulates for oxyR as a candidate virulence-facilitating factor in E. coli and indicate that oxyR is a broadly prevalent potential target for future preventive interventions against UTI due to E. coli. They also suggest that neutrophil phagocyte oxidase is not critical for defense against E. coli UTI and that the major oxidative stresses against which OxyR protects E. coli within the host milieu are not phagocyte derived.

Structural Stability of Human Fibroblast Growth Factor-1 Is Essential for Protective Effects Against Radiation-Induced Intestinal Damage

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

Nakayama, Fumiaki, E-mail: f_naka@nirs.go.jp; Umeda, Sachiko; Yasuda, Takeshi

2013-02-01

Purpose: Human fibroblast growth factor-1 (FGF1) has radioprotective effects on the intestine, although its structural instability limits its potential for practical use. Several stable FGF1 mutants were created increasing stability in the order, wild-type FGF1, single mutants (Q40P, S47I, and H93G), Q40P/S47I, and Q40P/S47I/H93G. This study evaluated the contribution of the structural stability of FGF1 to its radioprotective effect. Methods and Materials: Each FGF1 mutant was administered intraperitoneally to BALB/c mice in the absence of heparin 24 h before or after total body irradiation (TBI) with {gamma}-rays at 8-12 Gy. Several radioprotective effects were examined in the jejunum. Results: Q40P/S47I/H93Gmore » could activate all subtypes of FGF receptors in vitro much more strongly than the wild-type without endogenous or exogenous heparin. Preirradiation treatment with Q40P/S47I/H93G significantly increased crypt survival more than wild-type FGF1 after TBI at 10 or 12 Gy, and postirradiation treatment with Q40P/S47I/H93G was effective in promoting crypt survival after TBI at 10, 11, or 12 Gy. In addition, crypt cell proliferation, crypt depth, and epithelial differentiation were significantly promoted by postirradiation treatment with Q40P/S47I/H93G. The level of stability of FGF1 mutants correlated with their mitogenic activities in vitro in the absence of heparin; however, preirradiation treatment with the mutants increased the crypt number to almost the same level as Q40P/S47I/H93G. When given 24 h after TBI at 10 Gy, all FGF1 mutants increased crypt survival more than wild-type FGF1, and Q40P/S47I/H93G had the strongest mitogenic effects in intestinal epithelial cells after radiation damage. Moreover, Q40P/S47I/H93G prolonged mouse survival after TBI because of the repair of intestinal damage. Conclusion: These findings suggest that the structural stability of FGF1 can contribute to the enhancement of protective effects against radiation-induced intestinal damage. Therefore, Q40P/S47I/H93G is pharmacologically one of the most promising candidates for clinical applications for radiation-induced gastrointestinal syndrome.« less

LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis

PubMed Central

Hannan, Thomas J.; Mysorekar, Indira U.; Chen, Swaine L.; Walker, Jennifer N.; Jones, Jennifer M.; Pinkner, Jerome S.; Hultgren, Scott J.; Seed, Patrick C.

2013-01-01

Summary Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity-associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili-mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI IIUTI89 disrupts the adjacent leuX tRNA locus. Loss of wild-type leuX-encoded tRNA5Leu significantly delayed, but did not eliminate, FimB recombinase-mediated phase variation of type 1 pili. FimX, an additional FimB-like, leuX-independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non-piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI IIUTI89 genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI IIUTI89 during the establishment of acute cystitis is maintenance of wild-type leuX, and not PAI IIUTI89 gene content. PMID:18036139

Effect of Oseltamivir Carboxylate Consumption on Emergence of Drug-Resistant H5N2 Avian Influenza Virus in Mallard Ducks

PubMed Central

Achenbach, Jenna E.

2013-01-01

Oseltamivir carboxylate (OC) has been detected in environmental waters at various levels during recent influenza seasons in humans, reflecting levels of usage and stability of this drug. In consideration of the role of waterfowl as hosts for influenza viruses that may contribute to human infections, we evaluated the effect of consumption of low doses of OC on development of oseltamivir-resistant influenza virus mutants in mallard ducks (Anas platyrhynchos) infected with two different low-pathogenic (LP) H5N2 avian influenza viruses (AIV). We detected development of virus variants carrying a known molecular marker of oseltamivir resistance (neuraminidase E119V) in 4 out of 6 mallards infected with A/Mallard/Minnesota/182742/1998 (H5N2) and exposed to 1,000 ng/liter OC. The mutation first appeared as a minor population on days 5 to 6 and was the dominant genotype on days 6 to 8. Oseltamivir-resistant mutations were not detected in virus from ducks not exposed to the drug or in ducks infected with a second strain of virus and similarly exposed to OC. Virus isolates carrying the E119V mutation displayed in vitro replication kinetics similar to those of the wild-type virus, but in vivo, the E119V virus rapidly reverted back to wild type in the absence of OC, and only the wild-type parental strain was transmitted to contact ducks. These results indicate that consumption by wild waterfowl of OC in drinking water may promote selection of the E119V resistance mutation in some strains of H5N2 AIV that could contribute to viruses infecting human populations. PMID:23459475

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B.

PubMed

Fukasawa, Kayoko M; Hirose, Junzo; Hata, Toshiyuki; Ono, Yukio

2006-09-26

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.

Bioactive Compounds in Wild, In vitro Obtained, Ex vitro Adapted, and Acclimated Plants of Centaurea davidovii (Asteraceae).

PubMed

Trendafilova, Antoaneta; Jadranin, Milka; Gorgorov, Rossen; Stanilova, Marina

2015-06-01

In vitro cultures were initiated from a single seed of Centaurea davidovii. Whole plantlets were regenerated and cultivated for several months on agar-solidified nutrient media differing by their composition: basal MS medium, MS medium supplemented with plant growth regulators, and liquid MS medium. Plantlets were ex vitro adapted and successfully acclimated to open-air conditions; flowering was observed in some individuals in the first summer, and mass flowering during the second summer. The contents of the total flavonoids and the total phenolic compounds were determined spectrophotometrically in the leaves of the in vitro plantlets cultured on different media, and then compared with those in the leaves of the wild plants and in the leaves of the acclimated plants of the field plot. The sesquiterpene lactone 8α-(5'-hydroxyangeloyl)-salonitenolide was determined by HPLC in leaf samples of C. davidovii wild plants, in vitro obtained plantlets and ex vitro acclimated plants in the greenhouse and on the experimental field plot. The composition of the nutrient medium influenced the contents of all studied bioactive substances. The highest concentrations of all tested secondary metabolites were detected in the leaves of the acclimated plants during mass flowering, the content of the lactone reaching 56.2 mg/g DW, which was several times more than in the other leaf samples. The obtained results revealed both the effectiveness of biotechnological methods for propagation and conservation of rare and endangered plant species, and the possibility to use C. davidovii plants ex vitro acclimated to field conditions as a source of secondary metabolites with potential biological activity.

Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I.

PubMed

Wang, Penghua; Arjona, Alvaro; Zhang, Yue; Sultana, Hameeda; Dai, Jianfeng; Yang, Long; LeBlanc, Philippe M; Doiron, Karine; Saleh, Maya; Fikrig, Erol

2010-10-01

Caspase-12 has been shown to negatively modulate inflammasome signaling during bacterial infection. Its function in viral immunity, however, has not been characterized. We now report an important role for caspase-12 in controlling viral infection via the pattern-recognition receptor RIG-I. After challenge with West Nile virus (WNV), caspase-12-deficient mice had greater mortality, higher viral burden and defective type I interferon response compared with those of challenged wild-type mice. In vitro studies of primary neurons and mouse embryonic fibroblasts showed that caspase-12 positively modulated the production of type I interferon by regulating E3 ubiquitin ligase TRIM25-mediated ubiquitination of RIG-I, a critical signaling event for the type I interferon response to WNV and other important viral pathogens.

In vitro propagation of Paphiopedilum orchids.

PubMed

Zeng, Songjun; Huang, Weichang; Wu, Kunlin; Zhang, Jianxia; da Silva, Jaime A Teixeira; Duan, Jun

2016-01-01

Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation in vitro is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated via asymbiotic seed germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols for ex vitro-derived explants.

Protein kinase D1 is essential for bone acquisition during pubertal growth.

PubMed

Ford, Jeffery J; Yeh, Lee-Chuan C; Schmidgal, Eric C; Thompson, Jason F; Adamo, Martin L; Lee, John C

2013-11-01

Bone formation and maintenance represents the summation of the balance of local and endocrine hormonal stimuli within a complex organ. Protein kinase D (PKD) is a member of the Ca(2+)/calmodulin-dependent kinase superfamily of serine/threonine kinases and has been described as the crossroads for the bone morphogenetic protein (BMP)-IGF-I signaling axis, which plays a major role in bone formation. The current study exploits the PKD1-deficient mouse model to examine the role of PKD in vivo in the skeleton. Dual-energy x-ray absorptiometry scan analysis of male and female pubescent mice demonstrated significantly decreased bone mineral density in the whole body and femoral bone compartments of PKD1 (+/-) mice, compared with their wild-type littermates. The body weight, nasal-anal length, and percentage body fat of the mice were not significantly different from their wild-type littermates. Cultured bone marrow stromal cells from PKD1 (+/-) mice demonstrated lower alkaline phosphatase activity in early differentiating osteoblasts and decreased mineralized nodule formation in mature osteoblasts. Quantitative RT-PCR analysis of osteoblast differentiation markers and osteoclast markers exhibited lower levels of expression in PKD1 (+/-) male mice than wild type. In female mice, however, only markers of osteoblast differentiation were reduced. PKD1 (+/-) mice also demonstrated a profound reduction in mRNA expression levels of BMP type II receptor and IGF-I receptor and in BMP-7 responsiveness in vitro. Together these data suggest that in mice, PKD1 action contributes to the regulation of osteoblastogenesis by altering gene expression with gender-specific effects on osteoclastogenesis, subsequently affecting skeletal matrix acquisition during puberty.

Direct Action of Endothelin-1 on Podocytes Promotes Diabetic Glomerulosclerosis

PubMed Central

Lenoir, Olivia; Milon, Marine; Virsolvy, Anne; Hénique, Carole; Schmitt, Alain; Massé, Jean-Marc; Kotelevtsev, Yuri; Yanagisawa, Masashi; Webb, David J.; Richard, Sylvain

2014-01-01

The endothelin system has emerged as a novel target for the treatment of diabetic nephropathy. Endothelin-1 promotes mesangial cell proliferation and sclerosis. However, no direct pathogenic effect of endothelin-1 on podocytes has been shown in vivo and endothelin-1 signaling in podocytes has not been investigated. This study investigated endothelin effects in podocytes during experimental diabetic nephropathy. Stimulation of primary mouse podocytes with endothelin-1 elicited rapid calcium transients mediated by endothelin type A receptors (ETARs) and endothelin type B receptors (ETBRs). We then generated mice with a podocyte-specific double deletion of ETAR and ETBR (NPHS2-Cre×Ednralox/lox×Ednrblox/lox [Pod-ETRKO]). In vitro, treatment with endothelin-1 increased total β-catenin and phospho-NF-κB expression in wild-type glomeruli, but this effect was attenuated in Pod-ETRKO glomeruli. After streptozotocin injection to induce diabetes, wild-type mice developed mild diabetic nephropathy with microalbuminuria, mesangial matrix expansion, glomerular basement membrane thickening, and podocyte loss, whereas Pod-ETRKO mice presented less albuminuria and were completely protected from glomerulosclerosis and podocyte loss, even when uninephrectomized. Moreover, glomeruli from normal and diabetic Pod-ETRKO mice expressed substantially less total β-catenin and phospho-NF-κB compared with glomeruli from counterpart wild-type mice. This evidence suggests that endothelin-1 drives development of glomerulosclerosis and podocyte loss through direct activation of endothelin receptors and NF-κB and β-catenin pathways in podocytes. Notably, both the expression and function of the ETBR subtype were found to be important. Furthermore, these results indicate that activation of the endothelin-1 pathways selectively in podocytes mediates pathophysiologic crosstalk that influences mesangial architecture and sclerosis. PMID:24722437

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

PubMed

Wang, Yongzhi; Zhang, Su; Luo, Lingtao; Norström, Eva; Braun, Oscar Ö; Mörgelin, Matthias; Thorlacius, Henrik

2018-02-01

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis. © 2017 Wiley Periodicals, Inc.

The Na+ /H+ antiporter Nhx1 controls vacuolar fusion indispensible for life cycles in vitro and in vivo in a fungal insect pathogen.

PubMed

Zhu, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

2016-11-01

The sole Na + /H + antiporter Nhx1 has been generally unexplored in filamentous fungi. We characterized Nhx1 in the entomopathogenic fungus Beauveria bassiana. An eGFP-tagged Nhx1 fusion accumulated in small punctuate structures, presumably endosomal and trans-Golgi network compartments, between septum and tubular vacuole of each wild-type cell stained with a vacuole-specific dye. Deletion of nhx1 resulted in significant acidification and severe fusion defect in vacuoles, which were fragmented and distinct from large or tubular wild-type vacuoles. The deletion also caused a drastic reduction in aerial conidiation or submerged blastospore production and more severe defect in vegetative growth than in conidial germination. The Δnhx1 mutant became more sensitive to high osmolarity, heat shock and several metal ions during growth but its conidia showed increased UV-B tolerance. Intriguingly, Δnhx1 was unable to infect a model insect through cuticle penetration or intrahaemocoel injection because it produced much less biomass and cuticle-degrading enzymes in a minimal broth and failed to form blastospores in the insect haemolymph. All changes were completely or largely restored by targeted nhx1 complementation. Our results provide novel insight into an indispensability of Nhx1 for not only vacuolar fusion but also life cycles in vitro and in vivo in B. bassiana. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Urea transporter UT-B deletion induces DNA damage and apoptosis in mouse bladder urothelium.

PubMed

Dong, Zixun; Ran, Jianhua; Zhou, Hong; Chen, Jihui; Lei, Tianluo; Wang, Weiling; Sun, Yi; Lin, Guiting; Bankir, Lise; Yang, Baoxue

2013-01-01

Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders.

Losartan and captopril treatment rescue normal thrombus formation in microfibril associated glycoprotein-1 (MAGP1) deficient mice.

PubMed

Vassequi-Silva, Tallita; Pereira, Danielle Sousa; Nery Diez, Ana Cláudia C; Braga, Guilherme G; Godoy, Juliana A; Mendes, Camila B; Dos Santos, Leonardo; Krieger, José E; Antunes, Edson; Costa, Fábio T M; Vicente, Cristina P; Werneck, Claudio C

2016-02-01

MAGP1 is a glycoprotein present in the elastic fibers and is a part of the microfibrils components. MAGP1 interacts with von Willebrand factor and the active form of TGF-β and BMP. In mice lacking MAGP1, thrombus formation is delayed, increasing the occlusion time of carotid artery despite presenting normal blood coagulation in vitro. MAGP1-containing microfibrils may play a role in hemostasis and thrombosis. In this work, we evaluated the function of MAGP1 and its relation to TGF-β in the arterial thrombosis process. We analyzed thrombus formation time in wild type and MAGP1-deficient mice comparing Rose Bengal and Ferric Chloride induced arterial lesion. The potential participation of TGF-β in this process was accessed when we treated both wild type and MAGP1-deficient mice with losartan (an antihypertensive drug that decreases TGF-β activity) or captopril (an angiotensin converting enzyme inhibitor that was used as a control antihypertensive drug). Besides, we evaluated thrombus embolization and the gelatinolytic activity in the arterial walls in vitro and ex vivo. Losartan and captopril were able to recover the thrombus formation time without changing blood pressure, activated partial thromboplastin time (aPTT), PT (prothrombin time), platelet aggregation and adhesion, but decreased gelatinase activity. Our results suggest that both treatments are effective in the prevention of the sub-endothelial ECM degradation, allowing the recovery of normal thrombus formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Role of interstitial cells of Cajal in the generation and modulation of motor activity induced by cholinergic neurotransmission in the stomach.

PubMed

Zhang, R-X; Wang, X-Y; Chen, D; Huizinga, J D

2011-09-01

Interstitial cells of Cajal (ICC) are intimately linked to the enteric nervous system and a better understanding of the interactions between the two systems is going to advance our understanding of gut motor control. The objective of the present study was to investigate the role of ICC in the generation of gastric motor activity induced by cholinergic neurotransmission. Gastric motor activity was evoked through activation of intrinsic cholinergic neural activity, in in vitro muscle strips by electrical field stimulation, in the in vitro whole stomach by distension and in vivo by fluoroscopy after gavaging the stomach with barium sulfate. The cholinergic activity was assessed as that component of the effect of the stimulus that was sensitive to atropine. These experiments were carried out in wild-type and Ws/Ws rats that have few intramuscular ICC (ICC-IM) in the stomach. Under all three experimental conditions, cholinergic activity was prominent in both wild-type and W mutant rats providing evidence against the hypothesis that cholinergic neurotransmission to smooth muscle is primarily mediated by ICC-IM. Strong cholinergic activity in Ws/Ws rats was not due to upregulation of muscarinic receptors in ICC but possibly in smooth muscle of the antrum. Pacemaker ICC play a prominent role in the expression of motor activity induced by cholinergic activity and our data suggest that cholinergic neurotransmission to ICC affects the pacemaker frequency. © 2011 Blackwell Publishing Ltd.

Desert hedgehog is a mediator of demyelination in compression neuropathies.

PubMed

Jung, James; Frump, Derek; Su, Jared; Wang, Weiping; Mozaffar, Tahseen; Gupta, Ranjan

2015-09-01

The secreted protein desert hedgehog (dhh) controls the formation of the nerve perineurium during development and is a key component of Schwann cells that ensures peripheral nerve survival. We postulated that dhh may play a critical role in maintaining myelination and investigated its role in demyelination-induced compression neuropathies by using a post-natal model of a chronic nerve injury in wildtype and dhh(-/-) mice. We evaluated demyelination using electrophysiological, morphological, and molecular approaches. dhh transcripts and protein are down-regulated early after injury in wild-type mice, suggesting an intimate relationship between the hedgehog pathway and demyelination. In dhh(-/-) mice, nerve injury induced more prominent and severe demyelination relative to their wild-type counterparts, suggesting a protective role of dhh. Alterations in nerve fiber characteristics included significant decreases in nerve conduction velocity, increased myelin debris, and substantial decreases in internodal length. Furthermore, in vitro studies showed that dhh blockade via either adenovirus-mediated (shRNA) or pharmacological inhibition both resulted in severe demyelination, which could be rescued by exogenous Dhh. Exogenous Dhh was protective against this demyelination and maintained myelination at baseline levels in a custom in vitro bioreactor to applied biophysical forces to myelinated DRG/Schwann cell co-cultures. Together, these results demonstrate a pivotal role for dhh in maintaining myelination. Furthermore, dhh signaling reveals a potential target for therapeutic intervention to prevent and treat demyelination of peripheral nerves in compression neuropathies. Copyright © 2015 Elsevier Inc. All rights reserved.

NFκB-Induced Periostin Activates Integrin-β3 Signaling to Promote Renal Injury in GN

PubMed Central

Prakoura, Niki; Kavvadas, Panagiotis; Kormann, Raphaёl; Dussaule, Jean-Claude; Chadjichristos, Christos E.

2017-01-01

De novo expression in the kidney of periostin, a protein involved in odontogenesis and osteogenesis, has been suggested as a biomarker of renal disease. In this study, we investigated the mechanism(s) of induction and the role of periostin in renal disease. Using a combination of bioinformatics, reporter assay, and chromatin immunoprecipitation analyses, we found that NFκB and other proinflammatory transcription factors induce periostin expression in vitro and that binding of these factors on the periostin promoter is enriched in glomeruli during experimental GN. Mice lacking expression of periostin displayed preserved renal function and structure during GN. Furthermore, delayed administration of periostin antisense oligonucleotides in wild-type animals with GN reversed already established proteinuria, diminished tissue inflammation, and improved renal structure. Lack of periostin expression also blunted the de novo renal expression of integrin-β3 and phosphorylation of focal adhesion kinase and AKT, known mediators of integrin-β3 signaling that affect cell motility and survival, observed during GN in wild-type animals. In vitro, recombinant periostin increased the expression of integrin-β3 and the concomitant phosphorylation of focal adhesion kinase and AKT in podocytes. Notably, periostin and integrin-β3 were highly colocalized in biopsy specimens from patients with inflammatory GN. These results demonstrate that interplay between periostin and renal inflammation orchestrates inflammatory and fibrotic responses, driving podocyte damage through downstream activation of integrin-β3 signaling. Targeting periostin may be a novel therapeutic strategy for treating CKD. PMID:27920156

ABCC6 does not transport vitamin K3-glutathione conjugate from the liver: relevance to pathomechanisms of pseudoxanthoma elasticum.

PubMed

Fülöp, Krisztina; Jiang, Qiujie; Wetering, Koen V D; Pomozi, Viola; Szabó, Pál T; Arányi, Tamás; Sarkadi, Balázs; Borst, Piet; Uitto, Jouni; Váradi, András

2011-11-25

Vitamin K is a cofactor required for gamma-glutamyl carboxylation of several proteins regulating blood clotting, bone formation and soft tissue mineralization. Vitamin K3 is an important intermediate during conversion of the dietary vitamin K1 to the most abundant vitamin K2 form. It has been suggested that ABCC6 may have a role in transporting vitamin K or its derivatives from the liver to the periphery. This activity is missing in pseudoxanthoma elasticum, a genetic disorder caused by mutations in ABCC6 characterized by abnormal soft tissue mineralization. Here we examined the efflux of the glutathione conjugate of vitamin K3 (VK3GS) from the liver in wild type and Abcc6(-/-) mice, and in transport assays in vitro. We found in liver perfusion experiments that VK3GS is secreted into the inferior vena cava, but we observed no significant difference between wild type and Abcc6(-/-) animals. We overexpressed the human ABCC6 transporter in Sf9 insect and MDCKII cells and assayed its vitamin K3-conjugate transport activity in vitro. We found no measurable transport of VK3GS by ABCC6, whereas ABCC1 transported this compound at high rate in these assays. These results show that VK3GS is not the essential metabolite transported by ABCC6 from the liver and preventing the symptoms of pseudoxanthoma elasticum. Copyright © 2011 Elsevier Inc. All rights reserved.

Urea Transporter UT-B Deletion Induces DNA Damage and Apoptosis in Mouse Bladder Urothelium

PubMed Central

Zhou, Hong; Chen, Jihui; Lei, Tianluo; Wang, Weiling; Sun, Yi; Lin, Guiting; Bankir, Lise; Yang, Baoxue

2013-01-01

Background Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. Methodology/Principal Findings Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. Conclusions/Significance UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders. PMID:24204711

Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro

PubMed Central

Kievit, Forrest M.; Wang, Freddy Y.; Fang, Chen; Mok, Hyejung; Wang, Kui; Silber, John R.; Ellenbogen, Richard G.; Zhang, Miqin

2011-01-01

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR. PMID:21277920

Biochemical constituents of a wild strain of Schizophyllum commune isolated from Achanakmar-Amarkantak Biosphere Reserve (ABR), India.

PubMed

Tripathi, Arpita Mani; Tiwary, Bhupendra N

2013-08-01

A wild strain of Schizophyllum commune (MTCC 9670) isolated from Achanakmar-Amarkantak Biosphere Reserve of Central India was evaluated for the production of bioactive compounds. The chemical constituents of wild and in vitro grown cultures were compared. Under optimized conditions, different organic and aqueous extracts from mycelia and fruiting bodies were used to extract chemical components from the cultures grown in vitro. The gas chromatography combined wih mass spectrometry analysis of extracts identified two phenolic compounds, namely Phenyl benzoate (C13H10O2) and 4-(phenyl methoxy) phenol (C13H12O2) in the ethanolic extract of in vitro grown fruiting bodies and one antibacterial compound Pyrrolo (1, 2-a) piperazine-3, 6-dione (C7H10O2N2) in the methanolic extract of mycelia. High-performance liquid chromatography analysis revealed that the gallic acid and L-ascorbic acid were identifiable antioxidant components in the extracts possessing high free radical scavenging activity. The findings suggest that the wild strain of S. commune may serve as the source of novel bioactive compounds with effective antimicrobial and antioxidant activities.

Human MAMLD1 Gene Variations Seem Not Sufficient to Explain a 46,XY DSD Phenotype.

PubMed

Camats, Núria; Fernández-Cancio, Mónica; Audí, Laura; Mullis, Primus E; Moreno, Francisca; González Casado, Isabel; López-Siguero, Juan Pedro; Corripio, Raquel; Bermúdez de la Vega, José Antonio; Blanco, José Antonio; Flück, Christa E

2015-01-01

MAMLD1 is thought to cause disordered sex development in 46,XY patients. But its role is controversial because some MAMLD1 variants are also detected in normal individuals, several MAMLD1 mutations have wild-type activity in functional tests, and the male Mamld1-knockout mouse has normal genitalia and reproduction. Our aim was to search for MAMLD1 variations in 108 46,XY patients with disordered sex development, and to test them functionally. We detected MAMDL1 variations and compared SNP frequencies in controls and patients. We tested MAMLD1 transcriptional activity on promoters involved in sex development and assessed the effect of MAMLD1 on androgen production. MAMLD1 expression in normal steroid-producing tissues and mutant MAMLD1 protein expression were also assessed. Nine MAMLD1 mutations (7 novel) were characterized. In vitro, most MAMLD1 variants acted similarly to wild type. Only the L210X mutation showed loss of function in all tests. We detected no effect of wild-type or MAMLD1 variants on CYP17A1 enzyme activity in our cell experiments, and Western blots revealed no significant differences for MAMLD1 protein expression. MAMLD1 was expressed in human adult testes and adrenals. In conclusion, our data support the notion that MAMLD1 sequence variations may not suffice to explain the phenotype in carriers and that MAMLD1 may also have a role in adult life.

Human MAMLD1 Gene Variations Seem Not Sufficient to Explain a 46,XY DSD Phenotype

PubMed Central

Audí, Laura; Mullis, Primus E.; Moreno, Francisca; González Casado, Isabel; López-Siguero, Juan Pedro; Corripio, Raquel; Bermúdez de la Vega, José Antonio; Blanco, José Antonio; Flück, Christa E.

2015-01-01

MAMLD1 is thought to cause disordered sex development in 46,XY patients. But its role is controversial because some MAMLD1 variants are also detected in normal individuals, several MAMLD1 mutations have wild-type activity in functional tests, and the male Mamld1-knockout mouse has normal genitalia and reproduction. Our aim was to search for MAMLD1 variations in 108 46,XY patients with disordered sex development, and to test them functionally. We detected MAMDL1 variations and compared SNP frequencies in controls and patients. We tested MAMLD1 transcriptional activity on promoters involved in sex development and assessed the effect of MAMLD1 on androgen production. MAMLD1 expression in normal steroid-producing tissues and mutant MAMLD1 protein expression were also assessed. Nine MAMLD1 mutations (7 novel) were characterized. In vitro, most MAMLD1 variants acted similarly to wild type. Only the L210X mutation showed loss of function in all tests. We detected no effect of wild-type or MAMLD1 variants on CYP17A1 enzyme activity in our cell experiments, and Western blots revealed no significant differences for MAMLD1 protein expression. MAMLD1 was expressed in human adult testes and adrenals. In conclusion, our data support the notion that MAMLD1 sequence variations may not suffice to explain the phenotype in carriers and that MAMLD1 may also have a role in adult life. PMID:26580071

Aleglitazar, a dual peroxisome proliferator-activated receptor-α and -γ agonist, protects cardiomyocytes against the adverse effects of hyperglycaemia.

PubMed

Chen, Yan; Chen, Hongmei; Birnbaum, Yochai; Nanhwan, Manjyot K; Bajaj, Mandeep; Ye, Yumei; Qian, Jinqiao

2017-03-01

To assess the effects of Aleglitazar on hyperglycaemia-induced apoptosis. We incubated human cardiomyocytes, cardiomyocytes from cardiac-specific peroxisome proliferator-activated receptor-γ knockout or wild-type mice in normoglycaemic or hyperglycaemic conditions (glucose 25 mM). Cells were treated with different concentrations of Aleglitazar for 48 h. We measured viability, apoptosis, caspase-3 activity, cytochrome-C release, total antioxidant capacity and reactive oxygen species formation in the treated cardiomyocytes. Human cardiomyocytes were transfected with short interfering RNA against peroxisome proliferator-activated receptor-α or peroxisome proliferator-activated receptor-γ. Aleglitazar attenuated hyperglycaemia-induced apoptosis, caspase-3 activity and cytochrome-C release and increased viability in human cardiomyocyte, cardiomyocytes from cardiac-specific peroxisome proliferator-activated receptor-γ knockout and wild-type mice. Hyperglycaemia reduced the antioxidant capacity and Aleglitazar significantly blunted this effect. Hyperglycaemia-induced reactive oxygen species production was attenuated by Aleglitazar in both human cardiomyocyte and wild-type mice cardiomyocytes. Aleglitazar improved cell viability in cells exposed to hyperglycaemia. The protective effect was partially blocked by short interfering RNA against peroxisome proliferator-activated receptor-α alone and short interfering RNA against peroxisome proliferator-activated receptor-γ alone and completely blocked by short interfering RNA to both peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ. Aleglitazar protects cardiomyocytes against hyperglycaemia-induced apoptosis by combined activation of both peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ in a short-term vitro model.

Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis

PubMed Central

Zimbler, Daniel L.; Eddy, Justin L.; Schroeder, Jay A.

2015-01-01

Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague. PMID:26553463

SLC26A9-mediated chloride secretion prevents mucus obstruction in airway inflammation

PubMed Central

Anagnostopoulou, Pinelopi; Riederer, Brigitte; Duerr, Julia; Michel, Sven; Binia, Aristea; Agrawal, Raman; Liu, Xuemei; Kalitzki, Katrin; Xiao, Fang; Chen, Mingmin; Schatterny, Jolanthe; Hartmann, Dorothee; Thum, Thomas; Kabesch, Michael; Soleimani, Manoocher; Seidler, Ursula; Mall, Marcus A.

2012-01-01

Asthma is a chronic condition with unknown pathogenesis, and recent evidence suggests that enhanced airway epithelial chloride (Cl–) secretion plays a role in the disease. However, the molecular mechanism underlying Cl– secretion and its relevance in asthma pathophysiology remain unknown. To determine the role of the solute carrier family 26, member 9 (SLC26A9) Cl– channel in asthma, we induced Th2-mediated inflammation via IL-13 treatment in wild-type and Slc26a9-deficient mice and compared the effects on airway ion transport, morphology, and mucus content. We found that IL-13 treatment increased Cl– secretion in the airways of wild-type but not Slc26a9-deficient mice. While IL-13–induced mucus overproduction was similar in both strains, treated Slc26a9-deficient mice exhibited airway mucus obstruction, which did not occur in wild-type controls. In a study involving healthy children and asthmatics, a polymorphism in the 3′ UTR of SLC26A9 that reduced protein expression in vitro was associated with asthma. Our data demonstrate that the SLC26A9 Cl– channel is activated in airway inflammation and suggest that SLC26A9-mediated Cl– secretion is essential for preventing airway obstruction in allergic airway disease. These results indicate that SLC26A9 may serve as a therapeutic target for airway diseases associated with mucus plugging. PMID:22945630

Pressure-Accelerated Dissociation of Amyloid Fibrils in Wild-Type Hen Lysozyme

PubMed Central

Shah, Buddha R.; Maeno, Akihiro; Matsuo, Hiroshi; Tachibana, Hideki; Akasaka, Kazuyuki

2012-01-01

The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml−1 fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (kobs = 0.00193 min−1 at 0.1 MPa, 0.0348 min−1 at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV0‡ = −32.9 ± 1.7 ml mol(monomer)−1 and the activation compressibility Δκ‡ = −0.0075 ± 0.0006 ml mol(monomer)−1 bar−1 for the dissociation reaction. The negative ΔV0‡ and Δκ‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril. PMID:22225805

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status

PubMed Central

Floch, Renaud Le; Moura, Conceição Souto

2015-01-01

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function. To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via ‘Zinc-Finger Nucleases’. The three homozygous BSG−/− cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG−/− cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin. PMID:25894929

In Vivo, Villin Is Required for Ca2+-Dependent F-Actin Disruption in Intestinal Brush Borders

PubMed Central

Ferrary, Evelyne; Cohen-Tannoudji, Michel; Pehau-Arnaudet, Gérard; Lapillonne, Alexandre; Athman, Rafika; Ruiz, Tereza; Boulouha, Lilia; El Marjou, Fatima; Doye, Anne; Fontaine, Jean-Jacques; Antony, Claude; Babinet, Charles; Louvard, Daniel; Jaisser, Frédéric; Robine, Sylvie

1999-01-01

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca2+-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca2+ differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca2+, whereas Ca2+ had no effect in villin-null isolates. Moreover, increase in intracellular Ca2+ by serosal carbachol or mucosal Ca2+ ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 ± 9.6%, compared with wild-type mice, 70 ± 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury. PMID:10459016

Wild chrysanthemum extract prevents UVB radiation-induced acute cell death and photoaging.

PubMed

Sun, Sujiao; Jiang, Ping; Su, Weiting; Xiang, Yang; Li, Jian; Zeng, Lin; Yang, Shuangjuan

2016-03-01

Wild chrysanthemum (Chrysanthemum indicum L.) is traditionally used in folk medicine as an anti-inflammatory agent. It is also used in the southwest plateau region of China to prevent ultraviolet-induced skin damage. However, the role and mechanism by which wild chrysanthemum prevents UV-induced skin damage and photoaging have never been investigated in vitro. In the present study, we found that aqueous extracts from wild chrysanthemum strongly reduced high-dose UVB-induced acute cell death of human immortalized keratinocytic HaCat cells. Wild chrysanthemum extract was also demonstrated to reduce low-dose UVB-induced expression of the photoaging-related matrix metalloproteinases MMP-2 and MMP-9. The ROS level elevated by UVB irradiation was strongly attenuated by wild chrysanthemum extract. Further study revealed that wild chrysanthemum extract reduced UVB-triggered ERK1/2 and p38 MAPK phosphorylation and their protective role, which is partially dependent on inhibiting p38 activation. These results suggest that wild chrysanthemum extract can protect the skin from UVB-induced acute skin damage and photoaging by reducing the intracellular reactive oxygen species (ROS) level and inhibiting p38 MAPK phosphorylation. The present study confirmed the protective role of wild chrysanthemum against UV-induced skin disorders in vitro and indicated the possible mechanism. Further study to identify the active components in wild chrysanthemum extract would be useful for developing new drugs for preventing and treating skin diseases, including skin cancer and photoaging, induced by UV irradiation.

Nicotinamidase/pyrazinamidase of Mycobacterium tuberculosis forms homo-dimers stabilized by disulfide bonds

PubMed Central

Rueda, Daniel; Sheen, Patricia; Gilman, Robert H.; Bueno, Carlos; Santos, Marco; Pando-Robles, Victoria; Batista, Cesar V.; Zimic, Mirko

2014-01-01

Recombinant wild-pyrazinamidase from H37Rv M. tuberculosis was analyzed by gel electrophoresis under differential reducing conditions to evaluate its quaternary structure. PZAse was fractionated by size exclusion chromatography under non-reducing conditions. PZAse activity was measured and mass spectrometry analysis was performed to determine the identity of proteins by de novo sequencing and to determine the presence of disulfide bonds. This study confirmed that M. tuberculosis wild type PZAse was able to form homo-dimers in vitro. Homo-dimers showed a slightly lower specific PZAse activity compared to monomeric PZAse. PZAse dimers were dissociated into monomers in response to reducing conditions. Mass spectrometry analysis confirmed the existence of disulfide bonds (C72-C138 and C138-C138) stabilizing the quaternary structure of the PZAse homo-dimer. PMID:25199451

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

DOE PAGES

Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; ...

2016-03-23

α 1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based onmore » biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography. PLOS« less

Attenuation of Replication-Competent Adenovirus Serotype 26 Vaccines by Vectorization

PubMed Central

Maxfield, Lori F.; Abbink, Peter; Stephenson, Kathryn E.; Borducchi, Erica N.; Ng'ang'a, David; Kirilova, Marinela M.; Paulino, Noelix; Boyd, Michael; Shabram, Paul; Ruan, Qian; Patel, Mayank

2015-01-01

Replication-competent adenovirus (rcAd)-based vaccine vectors may theoretically provide immunological advantages over replication-incompetent Ad vectors, but they also raise additional potential clinical and regulatory issues. We produced replication-competent Ad serotype 26 (rcAd26) vectors by adding the E1 region back into a replication-incompetent Ad26 vector backbone with the E3 or E3/E4 regions deleted. We assessed the effect of vectorization on the replicative capacity of the rcAd26 vaccines. Attenuation occurred in a stepwise fashion, with E3 deletion, E4 deletion, and human immunodeficiency virus type 1 (HIV-1) envelope (Env) gene insertion all contributing to reduced replicative capacity compared to that with the wild-type Ad26 vector. The rcAd26 vector with E3 and E4 deleted and containing the Env transgene exhibited 2.7- to 4.4-log-lower replicative capacity than that of the wild-type Ad26 in vitro. This rcAd26 vector is currently being evaluated in a phase 1 clinical trial. Attenuation as a result of vectorization and transgene insertion has implications for the clinical development of replication-competent vaccine vectors. PMID:26376928

Attenuation of Replication-Competent Adenovirus Serotype 26 Vaccines by Vectorization.

PubMed

Maxfield, Lori F; Abbink, Peter; Stephenson, Kathryn E; Borducchi, Erica N; Ng'ang'a, David; Kirilova, Marinela M; Paulino, Noelix; Boyd, Michael; Shabram, Paul; Ruan, Qian; Patel, Mayank; Barouch, Dan H

2015-11-01

Replication-competent adenovirus (rcAd)-based vaccine vectors may theoretically provide immunological advantages over replication-incompetent Ad vectors, but they also raise additional potential clinical and regulatory issues. We produced replication-competent Ad serotype 26 (rcAd26) vectors by adding the E1 region back into a replication-incompetent Ad26 vector backbone with the E3 or E3/E4 regions deleted. We assessed the effect of vectorization on the replicative capacity of the rcAd26 vaccines. Attenuation occurred in a stepwise fashion, with E3 deletion, E4 deletion, and human immunodeficiency virus type 1 (HIV-1) envelope (Env) gene insertion all contributing to reduced replicative capacity compared to that with the wild-type Ad26 vector. The rcAd26 vector with E3 and E4 deleted and containing the Env transgene exhibited 2.7- to 4.4-log-lower replicative capacity than that of the wild-type Ad26 in vitro. This rcAd26 vector is currently being evaluated in a phase 1 clinical trial. Attenuation as a result of vectorization and transgene insertion has implications for the clinical development of replication-competent vaccine vectors. Copyright © 2015, Maxfield et al.

DREAM regulates BDNF-dependent spinal sensitization

PubMed Central

2010-01-01

Background The transcriptional repressor DREAM (downstream regulatory element antagonist modulator) controls the expression of prodynorphin and has been involved in the modulation of endogenous responses to pain. To investigate the role of DREAM in central mechanisms of pain sensitization, we used a line of transgenic mice (L1) overexpressing a Ca2+- and cAMP-insensitive DREAM mutant in spinal cord and dorsal root ganglia. Results L1 DREAM transgenic mice showed reduced expression in the spinal cord of several genes related to pain, including prodynorphin and BDNF (brain-derived neurotrophic factor) and a state of basal hyperalgesia without change in A-type currents. Peripheral inflammation produced enhancement of spinal reflexes and increased expression of BDNF in wild type but not in DREAM transgenic mice. The enhancement of the spinal reflexes was reproduced in vitro by persistent electrical stimulation of C-fibers in wild type but not in transgenic mice. Exposure to exogenous BDNF produced a long-term enhancement of dorsal root-ventral root responses in transgenic mice. Conclusions Our results indicate that endogenous BDNF is involved in spinal sensitization following inflammation and that blockade of BDNF induction in DREAM transgenic mice underlies the failure to develop spinal sensitization. PMID:21167062

Mutation at Tyrosine in AMLRY (GILRY Like) Motif of Yeast eRF1 on Nonsense Codons Suppression and Binding Affinity to eRF3

PubMed Central

Akhmaloka; Susilowati, Prima Endang; Subandi; Madayanti, Fida

2008-01-01

Termination translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized yeast eRF1 mutant at position 410 (correspond to 415 human eRF1) from tyrosine to serine residue resulting eRF1(Y410S). The mutations did not affect the viability and temperature sensitivity of the cell. The stop codons suppression of the mutant was analyzed in vivo using PGK-stop codon-LACZ gene fusion and showed that the suppression of the mutant was significantly increased in all of codon terminations. The suppression on UAG codon was the highest increased among the stop codons by comparing the suppression of the wild type respectively. In vitro interaction between eRF1 (mutant and wild type) to eRF3 were carried out using eRF1-(His)6 and eRF1(Y410S)-(His)6 expressed in Escherichia coli and indigenous Saccharomyces cerevisiae eRF3. The results showed that the binding affinity of eRF1(Y410S) to eRF3 was decreased up to 20% of the wild type binding affinity. Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1(Y410S) has no significant different with the wild type. However, substitution of tyrosine to serine triggered the structural change on the other motif of C-terminal domain of eRF1. The data suggested that increasing stop codon suppression and decreasing of the binding affinity of eRF1(Y410S) were probably due to the slight modification on the structure of the C-terminal domain. PMID:18463713

Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents

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

Higgins, Larry G.; Kelleher, Michael O.; Eggleston, Ian M.

2009-06-15

Sulforaphane can stimulate cellular adaptation to redox stressors through transcription factor Nrf2. Using mouse embryonic fibroblasts (MEFs) as a model, we show herein that the normal homeostatic level of glutathione in Nrf2{sup -/-} MEFs was only 20% of that in their wild-type counterparts. Furthermore, the rate of glutathione synthesis following its acute depletion upon treatment with 3 {mu}mol/l sulforaphane was very substantially lower in Nrf2{sup -/-} MEFs than in wild-type cells, and the rebound leading to a {approx} 1.9-fold increase in glutathione that occurred 12-24 h after Nrf2{sup +/+} MEFs were treated with sulforaphane was not observed in Nrf2{sup -/-}more » fibroblasts. Wild-type MEFs that had been pre-treated for 24 h with 3 {mu}mol/l sulforaphane exhibited between 1.4- and 3.2-fold resistance against thiol-reactive electrophiles, including isothiocyanates, {alpha},{beta}-unsaturated carbonyl compounds (e.g. acrolein), aryl halides and alkene epoxides. Pre-treatment of Nrf2{sup +/+} MEFs with sulforaphane also protected against hydroperoxides (e.g. cumene hydroperoxide, CuOOH), free radical-generating compounds (e.g. menadione), and genotoxic electrophiles (e.g. chlorambucil). By contrast, Nrf2{sup -/-} MEFs were typically {approx} 50% less tolerant of these agents than wild-type fibroblasts, and sulforaphane pre-treatment did not protect the mutant cells against xenobiotics. To test whether Nrf2-mediated up-regulation of glutathione represents the major cytoprotective mechanism stimulated by sulforaphane, 5 {mu}mol/l buthionine sulfoximine (BSO) was used to inhibit glutathione synthesis. In Nrf2{sup +/+} MEFs pre-treated with sulforaphane, BSO diminished intrinsic resistance and abolished inducible resistance to acrolein, CuOOH and chlorambucil, but not menadione. Thus Nrf2-dependent up-regulation of GSH is the principal mechanism by which sulforaphane pre-treatment induced resistance to acrolein, CuOOH and chlorambucil, but not menadione.« less

Molecular Modeling and Structural Stability of Wild-Type and Mutant CYP51 from Leishmania major: In Vitro and In Silico Analysis of a Laboratory Strain.

PubMed

Keighobadi, Masoud; Emami, Saeed; Lagzian, Milad; Fakhar, Mahdi; Rafiei, Alireza; Valadan, Reza

2018-03-19

Cutaneous leishmaniasis is a neglected tropical disease and a major public health in the most countries. Leishmania major is the most common cause of cutaneous leishmaniasis. In the Leishmania parasites, sterol 14α-demethylase (CYP51), which is involved in the biosynthesis of sterols, has been identified as an attractive target for development of new therapeutic agents. In this study, the sequence and structure of CYP51 in a laboratory strain (MRHO/IR/75/ER) of L. major were determined and compared to the wild-type strain. The results showed 19 mutations including seven non-synonymous and 12 synonymous ones in the CYP51 sequence of strain MRHO/IR/75/ER. Importantly, an arginine to lysine substitution at position of 474 resulted in destabilization of CYP51 (ΔΔG = 1.17 kcal/mol) in the laboratory strain; however, when the overall effects of all substitutions were evaluated by 100 ns molecular dynamics simulation, the final structure did not show any significant changes ( p -value < 0.05) in stability parameter of the strain MRHO/IR/75/ER compared to the wild-type protein. The energy level for the CYP51 of wild-type and MRHO/IR/75/ER strain were -40,027.1 and -39,706.48 Kcal/mol respectively. The overall Root-mean-square deviation (RMSD) deviation between two proteins was less than 1 Å throughout the simulation and Root-mean-square fluctuation (RMSF) plot also showed no substantial differences between amino acids fluctuation of the both protein. The results also showed that, these mutations were located on the protein periphery that neither interferes with protein folding nor with substrate/inhibitor binding. Therefore, L. major strain MRHO/IR/75/ER is suggested as a suitable laboratory model for studying biological role of CYP51 and inhibitory effects of sterol 14α-demethylase inhibitors.

Increased sensitivity to radiochemotherapy in IDH1 mutant glioblastoma as demonstrated by serial quantitative MR volumetry

PubMed Central

Tran, Anh N.; Lai, Albert; Li, Sichen; Pope, Whitney B.; Teixeira, Stephanie; Harris, Robert J.; Woodworth, Davis C.; Nghiemphu, Phioanh L.; Cloughesy, Timothy F.; Ellingson, Benjamin M.

2014-01-01

Background Isocitrate dehydrogenase 1 (IDH1) mutations have been linked to favorable outcomes in patients with glioblastoma multiforme (GBM). Recent in vitro experiments suggest that IDH1 mutation sensitizes tumors to radiation damage. We hypothesized that radiographic treatment response would be significantly different between IDH1 mutant versus wild-type GBMs after radiotherapy (RT) and concurrent temozolomide (TMZ). Methods A total of 39 newly diagnosed GBM patients with known IDH1 mutational status (10 IDH1 mutants), who followed standard therapy and had regular post-contrast T1W (T1+C) and T2W/ fluid-attenuated inversion recovery (FLAIR) images in the 6-month period after starting RT, were enrolled. The volume of contrast-enhancing and FLAIR hyperintensity were calculated from each scan. Linear and polynomial regression techniques were used to estimate the rate of change and temporal patterns in tumor volumes. Results IDH1 mutant GBMs demonstrated a favorable response to RT/TMZ in the study period, as demonstrated by 10 of 10 mutants showing radiographic response (decreasing VT1+C), compared with 13 of 29 wild-types (P < .001). During the study period, VT1+C and VFLAIR changed at −3.6% per week and +0.6% per week in IDH1 mutant tumors, respectively, as compared with +0.8% per week and +5.2% per week in IDH1 wild-type tumors (P = .0076 and P = .0118, respectively). Amongst the radiographic responders, IDH1 mutant GBMs still demonstrated significant progression-free and overall survival benefit. Aggregated tumor kinetics by group showed significant lower rate in IDH1 mutant GBMs in specific periods: >105 days for VFLAIR and 95–120 and >150 days for VT1+C from starting RT/TMZ. Conclusions The current study supports the hypothesis that IDH1 mutant GBMs are more sensitive to radiochemotherapy than IDH1 wild-type GBMs. PMID:24305712

Introducing a Rigid Loop Structure from Deer into Mouse Prion Protein Increases Its Propensity for Misfolding In Vitro

PubMed Central

Kyle, Leah M.; John, Theodore R.; Schätzl, Hermann M.; Lewis, Randolph V.

2013-01-01

Prion diseases are fatal neurodegenerative disorders characterized by misfolding of the cellular prion protein (PrPc) into the disease-associated isoform (PrPSc) that has increased β-sheet content and partial resistance to proteolytic digestion. Prion diseases from different mammalian species have varying propensities for transmission upon exposure of an uninfected host to the infectious agent. Chronic Wasting Disease (CWD) is a highly transmissible prion disease that affects free ranging and farmed populations of cervids including deer, elk and moose, as well as other mammals in experimental settings. The molecular mechanisms allowing CWD to maintain comparatively high transmission rates have not been determined. Previous work has identified a unique structural feature in cervid PrP, a rigid loop between β-sheet 2 and α-helix 2 on the surface of the protein. This study was designed to test the hypothesis that the rigid loop has a direct influence on the misfolding process. The rigid loop was introduced into murine PrP as the result of two amino acid substitutions: S170N and N174T. Wild-type and rigid loop murine PrP were expressed in E. coli and purified. Misfolding propensity was compared for the two proteins using biochemical techniques and cell free misfolding and conversion systems. Murine PrP with a rigid loop misfolded in cell free systems with greater propensity than wild type murine PrP. In a lipid-based conversion assay, rigid loop PrP converted to a PK resistant, aggregated isoform at lower concentrations than wild-type PrP. Using both proteins as substrates in real time quaking-induced conversion, rigid loop PrP adopted a misfolded isoform more readily than wild type PrP. Taken together, these findings may help explain the high transmission rates observed for CWD within cervids. PMID:23825561

Role of ATP-binding cassette and solute carrier transporters in erlotinib CNS penetration and intracellular accumulation.

PubMed

Elmeliegy, Mohamed A; Carcaboso, Angel M; Tagen, Michael; Bai, Feng; Stewart, Clinton F

2011-01-01

To study the role of drug transporters in central nervous system (CNS) penetration and cellular accumulation of erlotinib and its metabolite, OSI-420. After oral erlotinib administration to wild-type and ATP-binding cassette (ABC) transporter-knockout mice (Mdr1a/b(-/-), Abcg2(-/-), Mdr1a/b(-/-)Abcg2(-/-), and Abcc4(-/-)), plasma was collected and brain extracellular fluid (ECF) was sampled using intracerebral microdialysis. A pharmacokinetic model was fit to erlotinib and OSI-420 concentration-time data, and brain penetration (P(Brain)) was estimated by the ratio of ECF-to-unbound plasma area under concentration-time curves. Intracellular accumulation of erlotinib was assessed in cells overexpressing human ABC transporters or SLC22A solute carriers. P(Brain) in wild-type mice was 0.27 ± 0.11 and 0.07 ± 0.02 (mean ± SD) for erlotinib and OSI-420, respectively. Erlotinib and OSI-420 P(Brain) in Abcg2(-/-) and Mdr1a/b(-/-)Abcg2(-/-) mice were significantly higher than in wild-type mice. Mdr1a/b(-/-) mice showed similar brain ECF penetration as wild-type mice (0.49 ± 0.37 and 0.04 ± 0.02 for erlotinib and OSI-420, respectively). In vitro, erlotinib and OSI-420 accumulation was significantly lower in cells overexpressing breast cancer resistance protein (BCRP) than in control cells. Only OSI-420, not erlotinib, showed lower accumulation in cells overexpressing P-glycoprotein (P-gp) than in control cells. The P-gp/BCRP inhibitor elacridar increased erlotinib and OSI-420 accumulation in BCRP-overexpressing cells. Erlotinib uptake was higher in OAT3- and OCT2-transfected cells than in empty vector control cells. Abcg2 is the main efflux transporter preventing erlotinib and OSI-420 penetration in mouse brain. Erlotinib and OSI-420 are substrates for SLC22A family members OAT3 and OCT2. Our findings provide a mechanistic basis for erlotinib CNS penetration, cellular uptake, and efflux mechanisms. ©2010 AACR.

Mutation of the Maturase Lipoprotein Attenuates the Virulence of Streptococcus equi to a Greater Extent than Does Loss of General Lipoprotein Lipidation▿

PubMed Central

Hamilton, Andrea; Robinson, Carl; Sutcliffe, Iain C.; Slater, Josh; Maskell, Duncan J.; Davis-Poynter, Nick; Smith, Ken; Waller, Andrew; Harrington, Dean J.

2006-01-01

Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (ΔprtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δlgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and Δlgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δlgt190-685 mutant did still exhibit signs of disease. In contrast, only the ΔprtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δlgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted. PMID:17015455

MGE-derived nNOS+ interneurons promote fear acquisition in nNOS-/- mice.

PubMed

Zhang, Lin; Yuan, Hong-Jin; Cao, Bo; Kong, Cheng-Cheng; Yuan, Fang; Li, Jun; Ni, Huan-Yu; Wu, Hai-Yin; Chang, Lei; Liu, Yan; Luo, Chun-Xia

2017-12-02

Neuronal nitric oxide synthase (nNOS) 1 , mainly responsible for NO release in central nervous system (CNS) 2 , plays a significant role in multiple physiological functions. However, the function of nNOS + interneurons in fear learning has not been much explored. Here we focused on the medial ganglionic eminences (MGE) 3 -derived nNOS + interneurons in fear learning. To determine the origin of nNOS + interneurons, we cultured neurons in vitro from MGE, cortex, lateral ganglionic eminence (LGE) 4 , caudal ganglionic eminences (CGE) 5 and preoptic area (POA) 6 . The results showed that MGE contained the most abundant precursors of nNOS + interneurons. Moreover, donor cells from E12.5 embryos demonstrated the highest positive rate of nNOS + interneurons compared with other embryonic periods (E11.5, E12, E13, E13.5 and E14). Additionally, these cells from E12.5 embryos showed long axonal and abundant dendritic arbors after 10 days culture, indicating the capability to disperse and integrate in host neural circuits after transplantation. To investigate the role of MGE-derived nNOS + interneurons in fear learning, donor MGE cells were transplanted into dentate gyrus (DG) 7 of nNOS knock-out (nNOS -/- ) or wild-type mice. Results showed that the transplantation of MGE cells promoted the acquisition of nNOS -/- but not the wild-type mice, suggesting the importance of nNOS + neurons in fear acquisition. Moreover, we transplanted MGE cells from nNOS -/- mice or wild-type mice into DG of the nNOS -/- mice and found that only MGE cells from wild-type mice but not the nNOS -/- mice rescued the deficit in acquisition of the nNOS -/- mice, further confirming the positive role of nNOS + neurons in fear learning. Copyright © 2017 Elsevier Inc. All rights reserved.

Total Raltegravir Concentrations in Cerebrospinal Fluid Exceed the 50-Percent Inhibitory Concentration for Wild-Type HIV-1▿

PubMed Central

Croteau, David; Letendre, Scott; Best, Brookie M.; Ellis, Ronald J.; Breidinger, Sheila; Clifford, David; Collier, Ann; Gelman, Benjamin; Marra, Christina; Mbeo, Gilbert; McCutchan, Allen; Morgello, Susan; Simpson, David; Way, Lauren; Vaida, Florin; Ueland, Susan; Capparelli, Edmund; Grant, Igor

2010-01-01

HIV-associated neurocognitive disorders continue to be common. Antiretrovirals that achieve higher concentrations in cerebrospinal fluid (CSF) are associated with better control of HIV and improved cognition. The objective of this study was to measure total raltegravir (RAL) concentrations in CSF and to compare them with matched concentrations in plasma and in vitro inhibitory concentrations. Eighteen subjects with HIV-1 infection were enrolled based on the use of RAL-containing regimens and the availability of CSF and matched plasma samples. RAL was measured in 21 CSF and plasma pairs by liquid chromatography-tandem mass spectrometry, and HIV RNA was detected by reverse transcription-PCR (RT-PCR). RAL concentrations were compared to the 50% inhibitory concentration (IC50) for wild-type HIV-1 (3.2 ng/ml). Volunteers were predominantly middle-aged white men with AIDS and without hepatitis C virus (HCV) coinfection. The median concurrent CD4+ cell count was 276/μl, and 28% of CD4+ cell counts were below 200/μl. HIV RNA was detectable in 38% of plasma specimens and 4% of CSF specimens. RAL was present in all CSF specimens, with a median total concentration of 14.5 ng/ml. The median concentration in plasma was 260.9 ng/ml, with a median CSF-to-plasma ratio of 0.058. Concentrations in CSF correlated with those in with plasma (r2, 0.24; P, 0.02) but not with the postdose sampling time (P, >0.50). RAL concentrations in CSF exceeded the IC50 for wild-type HIV in all specimens by a median of 4.5-fold. RAL is present in CSF and reaches sufficiently high concentrations to inhibit wild-type HIV in all individuals. As a component of effective antiretroviral regimens or as the main antiretroviral, RAL likely contributes to the control of HIV replication in the nervous system. PMID:20876368

Enhanced p53 gene transfer to human ovarian cancer cells using the cationic nonviral vector, DDC.

PubMed

Kim, Chong-Kook; Choi, Eun-Jeong; Choi, Sung-Hee; Park, Jeong-Sook; Haider, Khawaja Hasnain; Ahn, Woong Shick

2003-08-01

Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo. DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription-polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed. The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group. The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its effective induction of apoptosis and tumor growth inhibition.

Mathematical modeling of mutant transferrin-CRM107 molecular conjugates for cancer therapy.

PubMed

Yoon, Dennis J; Chen, Kevin Y; Lopes, André M; Pan, April A; Shiloach, Joseph; Mason, Anne B; Kamei, Daniel T

2017-03-07

The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro assessment of 39 CYP2C9 variants found in the Chinese population on the metabolism of the model substrate fluoxetine and a summary of their effects on other substrates.

PubMed

Ji, Y; Chen, S; Zhao, L; Pan, P; Wang, L; Cai, J; Dai, D; Hu, G; Cai, J P; Huang, H

2015-06-01

Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme that is responsible for clearing approximately 15% of clinically important drugs. The objective of this study was to assess the catalytic characteristics of 39 CYP2C9 isoforms found in the Chinese population and their effects on the metabolism of the model substrate fluoxetine in vitro. Baculovirus-mediated expressing system was used to highly express wild-type and the 38 CYP2C9 allelic variants in insect cell microsomes. Then, the enzymatic characteristics of each variant were evaluated using fluoxetine as the substrate. Reactions were performed at 37 °C with the insect microsomes and 10-200 μm fluoxetine for 60 min. After termination, the products were precipitated and used for signal collection by UPLC-MS/MS. Of the 39 tested CYP2C9 isoforms, only four variants (CYP2C9*3, CYP2C9*27, CYP2C9*34 and CYP2C9*37) exhibited similar relative clearance values to that of the wild-type CYP2C9*1. Moreover, five variants (CYP2C9*14, CYP2C9*36, CYP2C9*45, CYP2C9*48 and CYP2C9*55) showed a higher intrinsic clearance value than the wild-type protein, whereas the remaining 29 CYP2C9 isoforms exhibited significantly decreased clearance values (from 6·23% to 87·74%) compared to CYP2C9*1. In addition, 28 CYP2C9 isoforms including CYP2C9*3 exhibited a trend towards substrate inhibition for fluoxetine. This study provides the most comprehensive data on the enzymatic activities associated with all reported CYP2C9 variants in the Chinese population with regard to the widely used antidepressant drug, fluoxetine. Our data indicate that more attention should be paid to subjects carrying the corresponding infrequent CYP2C9 alleles when administering fluoxetine in the clinic. © 2015 John Wiley & Sons Ltd.

Determination of Anti-Adeno-Associated Virus Vector Neutralizing Antibody Titer with an In Vitro Reporter System

PubMed Central

Meliani, Amine; Leborgne, Christian; Triffault, Sabrina; Jeanson-Leh, Laurence; Veron, Philippe

2015-01-01

Abstract Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors. PMID:25819687

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

PubMed Central

Dubreuil, Patrice; Letard, Sébastien; Ciufolini, Marco; Gros, Laurent; Humbert, Martine; Castéran, Nathalie; Borge, Laurence; Hajem, Bérengère; Lermet, Anne; Sippl, Wolfgang; Voisset, Edwige; Arock, Michel; Auclair, Christian; Leventhal, Phillip S.; Mansfield, Colin D.; Moussy, Alain; Hermine, Olivier

2009-01-01

Background The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity. PMID:19789626

iNOS-Derived Nitric Oxide Stimulates Osteoclast Activity and Alveolar Bone Loss in Ligature-Induced Periodontitis in Rats

PubMed Central

Herrera, Bruno S.; Martins-Porto, Rodrigo; Maia-Dantas, Aline; Campi, Paula; Spolidorio, Luis C.; Costa, Soraia K.P.; Van Dyke, Thomas E.; Gyurko, Robert; Muscara, Marcelo N.

2012-01-01

Background Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long-term iNOS inhibition, and the differentiation and activity of OC from iNOS-knockout (KO) mice in vitro. Methods Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild-type C57BL/6 mice. OC were counted 6 days later after tartrate-resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks. Results Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature-induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild-type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts. Conclusion Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity. PMID:21417589

Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo*

PubMed Central

Dislich, Bastian; Wohlrab, Felix; Bachhuber, Teresa; Müller, Stephan A.; Kuhn, Peer-Hendrik; Hogl, Sebastian; Meyer-Luehmann, Melanie; Lichtenthaler, Stefan F.

2015-01-01

Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1−/− and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1−/− and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1−/− mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors. PMID:26139848

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

PubMed

Kang, Yoon-Suk; Kirby, James E

2017-05-01

We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus , B. melitensis , and B. suis , B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila , we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection. Copyright © 2017 American Society for Microbiology.

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

PubMed Central

Santofimia-Castaño, Patricia; Rizzuti, Bruno; Pey, Ángel L.; Soubeyran, Philippe; Vidal, Miguel; Urrutia, Raúl; Iovanna, Juan L.; Neira, José L.

2017-01-01

Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 μM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins. PMID:28720707

Bacteroidaceae in Thromboembolic Disease: Effects of Cell Wall Components on Blood Coagulation In Vivo and In Vitro

PubMed Central

Bjornson, H. S.; Hill, E. O.

1973-01-01

The effects of Bacteroides sp., Fusobacterium mortiferum, Bacteroides fragilis, and Sphaerophorus necrophorus on various parameters of blood coagulation in vivo and in vitro were determined and compared to the coagulation effects of Escherichia coli and Salmonella minnesota, wild type and R595. Intravenous injection of washed cells, culture filtrate, lipopolysaccharide, or lipid A of the anaerobic gram-negative microorganisms into mice resulted in acceleration of coagulation. Lipopolysaccharide and lipid A of the anaerobic microorganisms had no apparent effect on circulating platelets in mice or rabbits and did not cause aggregation of human platelets in vitro. Washed cells, lipopolysaccharide, and lipid A of Bacteroides sp. and F. mortiferum also significantly accelerated the clotting time of recalcified platelet poor normal human plasma and C6-deficient rabbit plasma. Lipid A, but not lipopolysaccharide, of E. coli and washed cells of S. minnesota R595 accelerated coagulation by a similar mechanism. These results indicated that Bacteroides sp. and F. mortiferum can accelerate blood coagulation in vivo and in vitro by a mechanism which does not involve platelets or terminal components of complement. PMID:4594118

The combination effect of auxin and cytokinin on in vitro callus formation of Physalis angulata L. - A medicinal plant

NASA Astrophysics Data System (ADS)

Mastuti, Retno; Munawarti, Aminatun; Firdiana, Elok Rifqi

2017-11-01

Physalis angulata L. (Ciplukan) is one member of Solanaceae that has a potential as herbal medicine. This plant grows wild in the crop fields, forest edges, etc. However, ciplukan is increasingly difficult to find recently. In vitro callus is an alternative source to produce secondary metabolite production as well as to regenerate plants through indirect organogenesis. This study aims to identify the response of hypocotyl explants on in vitro callus formation induced by a combination of auxin and cytokinins. Two types of cytokinins, Kinetin and BAP (0.5 ppm) were combined with three types of auxin, i.e. 2.4-D, IBA and IAA, at three concentrations 0.5, 1.0 and 1.5 ppm. In all combinations of cytokinin and auxin, 50-100% of hypocotyl explants derived from in vitro seedling were able to produce callus either in a compact or watery friable texture. In MS medium supplemented with 2.4-D, callus FW (fresh weight) began to decline in the fourth week after culture. Callus FW that increased until 5 weeks of culture was obtained in medium IAA 0.5 + Kin 0.5, IBA 1.0 + Kin 0.5 and IBA 1 + BA 0.5. Almost all calli induced on a medium + Kinetin also produced roots. While medium + BAP was able to induce shoots regeneration.

D-enantiomeric peptides that eradicate wild-type and multi-drug resistant biofilms and protect against lethal Pseudomonas aeruginosa infections

PubMed Central

de la Fuente-Núñez, César; Reffuveille, Fany; Mansour, Sarah C.; Reckseidler-Zenteno, Shauna L.; Hernández, Diego; Brackman, Gilles; Coenye, Tom; Hancock, Robert E.W.

2015-01-01

SUMMARY In many infections, bacteria form surface-associated communities known as biofilms that are substantially more resistant to antibiotics than their planktonic counterparts. Based on the design features of active anti-biofilm peptides, we made a series of related 12-amino acid L-, D- and retro-inverso derivatives. Specific D-enantiomeric peptides were the most potent at inhibiting biofilm development and eradicating pre-formed biofilms of seven species of wild-type and multiply antibiotic resistant Gram-negative pathogens. Moreover, these peptides showed strong synergy with conventional antibiotics, reducing the antibiotic concentrations required for complete biofilm inhibition by up to 64-fold. As shown previously for 1018, these D-amino acid peptides targeted the intracellular stringent response signal (p)ppGpp. The most potent peptides DJK-5 and DJK-6 protected invertebrates from lethal P. aeruginosa infections, and were considerably more active than a previously described L-amino acid peptide 1018. Thus, the protease resistant peptides produced here were more effective both in vitro and in vivo. PMID:25699603

let-7 Contributes to Diabetic Retinopathy but Represses Pathological Ocular Angiogenesis

PubMed Central

Zhou, Qinbo; Frost, Robert J. A.; Anderson, Chastain; Zhao, Fangkun; Ma, Jing; Yu, Bo

2017-01-01

ABSTRACT The in vivo function of microRNAs (miRs) in diabetic retinopathy (DR) and age-related macular degeneration (AMD) remains unclear. We report here that let-7 family members are expressed in retinal and choroidal endothelial cells (ECs). In ECs, overexpression of let-7 by adenovirus represses EC proliferation, migration, and networking in vitro, whereas inhibition of the let-7 family with a locked nucleic acid (LNA)–anti-miR has the opposite effect. Mechanistically, silencing of the let-7 target HMGA2 gene mimics the phenotype of let-7 overexpression in ECs. let-7 transgenic (let-7-Tg) mice show features of nonproliferative DR, including tortuous retinal vessels and defective pericyte coverage. However, these mice develop significantly less choroidal neovascularization (CNV) compared to wild-type controls after laser injury. Consistently, silencing of let-7 in the eye increased laser-induced CNV in wild-type mice. Together, our data establish a causative role of let-7 in nonproliferative diabetic retinopathy and a repressive function of let-7 in pathological angiogenesis, suggesting distinct implications of let-7 in the pathogenesis of DR and AMD. PMID:28584193

Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.

PubMed

Armstrong, Jane L; Hill, David S; McKee, Christopher S; Hernandez-Tiedra, Sonia; Lorente, Mar; Lopez-Valero, Israel; Eleni Anagnostou, Maria; Babatunde, Fiyinfoluwa; Corazzari, Marco; Redfern, Christopher P F; Velasco, Guillermo; Lovat, Penny E

2015-06-01

Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain <10%. Novel treatment strategies are therefore urgently required, particularly for patients bearing BRAF/NRAS wild-type tumors. Targeting autophagy is a means to promote cancer cell death in chemotherapy-resistant tumors, and the aim of this study was to test the hypothesis that cannabinoids promote autophagy-dependent apoptosis in melanoma. Treatment with Δ(9)-Tetrahydrocannabinol (THC) resulted in the activation of autophagy, loss of cell viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.

Paradoxical Abatement of Striatal Dopaminergic Transmission by Cocaine and Methylphenidate*

PubMed Central

Federici, Mauro; Latagliata, Emanuele Claudio; Ledonne, Ada; Rizzo, Francesca R.; Feligioni, Marco; Sulzer, Dave; Dunn, Matthew; Sames, Dalibor; Gu, Howard; Nisticò, Robert; Puglisi-Allegra, Stefano; Mercuri, Nicola B.

2014-01-01

We combined in vitro amperometric, optical analysis of fluorescent false neurotransmitters and microdialysis techniques to unveil that cocaine and methylphenidate induced a marked depression of the synaptic release of dopamine (DA) in mouse striatum. In contrast to the classical dopamine transporter (DAT)-dependent enhancement of the dopaminergic signal observed at concentrations of cocaine lower than 3 μm, the inhibitory effect of cocaine was found at concentrations higher than 3 μm. The paradoxical inhibitory effect of cocaine and methylphenidate was associated with a decrease in synapsin phosphorylation. Interestingly, a cocaine-induced depression of DA release was only present in cocaine-insensitive animals (DAT-CI). Similar effects of cocaine were produced by methylphenidate in both wild-type and DAT-CI mice. On the other hand, nomifensine only enhanced the dopaminergic signal either in wild-type or in DAT-CI mice. Overall, these results indicate that cocaine and methylphenidate can increase or decrease DA neurotransmission by blocking reuptake and reducing the exocytotic release, respectively. The biphasic reshaping of DA neurotransmission could contribute to different behavioral effects of psychostimulants, including the calming ones, in attention deficit hyperactivity disorder. PMID:24280216

Circular RNA hsa_circ_0008344 regulates glioblastoma cell proliferation, migration, invasion, and apoptosis.

PubMed

Zhou, Jinxu; Wang, Hongxiang; Chu, Junsheng; Huang, Qilin; Li, Guangxu; Yan, Yong; Xu, Tao; Chen, Juxiang; Wang, Yuhai

2018-04-24

Recent studies have found circular RNAs (circRNAs) involved in the biological process of cancers. However, little is known about their functional roles in glioblastoma. Human circRNA microarray analysis was performed to screen the expression profile of circRNAs in IDH1 wild-type glioblastoma tissue. The expression of hsa_circ_0008344 in glioblastoma and normal brain samples was quantified by qRT-PCR. Functional experiments were performed to investigate the biological functions of hsa_circ_0008344, including MTT assay, colony formation assay, transwell assay, and cell apoptosis assay. CircRNA microarray revealed a total of 417 abnormally expressed circRNAs (>1.5-fold, P < .05) in glioblastoma tissue compared with the adjacent normal brain. Hsa_circ_0008344, among the top differentially expressed circRNAs, was significantly upregulated in IDH1 wild-type glioblastoma. Further in vitro studies showed that knockdown of hsa_circ_0008344 suppressed glioblastoma cell proliferation, colony formation, migration, and invasion, but increased cell apoptotic rate. Hsa_circ_0008344 is upregulated in glioblastoma and may contribute to the progression of this malignancy. © 2018 Wiley Periodicals, Inc.

Metalloproteinase pregnancy-associated plasma protein A is a critical growth regulatory factor during fetal development.

PubMed

Conover, Cheryl A; Bale, Laurie K; Overgaard, Michael T; Johnstone, Edward W; Laursen, Ulla H; Füchtbauer, Ernst-Martin; Oxvig, Claus; van Deursen, Jan

2004-03-01

Pregnancy-associated plasma protein A (PAPPA) is a metzincin superfamily metalloproteinase in the insulin-like growth factor (IGF) system. PAPPA increases IGF bioavailability and mitogenic effectiveness in vitro through regulated cleavage of IGF-binding protein 4 (IGFBP4). To determine its function in vivo, we generated PAPPA-null mice by gene targeting. Mice homozygous for targeted disruption of the PAPPA gene were viable but 60% the size of wild-type littermates at birth. The impact of the mutation was exerted during the early embryonic period prior to organogenesis, resulting in proportional dwarfism. PAPPA, IGF2 and IGFBP4 transcripts co-localized in wild-type embryos, and expression of IGF2 and IGFBP4 mRNA was not altered in PAPPA-deficient embryos. However, IGFBP4 proteolytic activity was completely lacking in fibroblasts derived from PAPPA-deficient embryos, and IGFBP4 effectively inhibited IGF-stimulated mitogenesis in these cells. These results provide the first direct evidence that PAPPA is an essential growth regulatory factor in vivo, and suggest a novel mechanism for regulated IGF bioavailability during early fetal development.

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model.

PubMed

Szentpáli-Gavallér, Katalin; Lim, Stephanie M; Dencső, László; Bányai, Krisztián; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E E; Bakonyi, Tamás; Bálint, Ádám

2016-02-19

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.

Designed Reduction of Streptococcus pneumoniae Pathogenicity via Synthetic Changes in Virulence Factor Codon-pair Bias

PubMed Central

Coleman, J. Robert; Papamichail, Dimitris; Yano, Masahide; García-Suárez, María del Mar

2011-01-01

In this study, we used a previously described method of controlling gene expression with computer-based gene design and de novo DNA synthesis to attenuate the virulence of Streptococcus pneumoniae. We produced 2 S. pneumoniae serotype 3 (SP3) strains in which the pneumolysin gene (ply) was recoded with underrepresented codon pairs while retaining its amino acid sequence and determined their ply expression and pneumolysin production in vitro and their virulence in a mouse pulmonary infection model. Expression of ply and production of pneumolysin of the recoded SP3 strains were decreased, and the recoded SP3 strains were less virulent in mice than the wild-type SP3 strain or a Δply SP3 strain. Further studies showed that the least virulent recoded strain induced a markedly reduced inflammatory response in the lungs compared with the wild-type or Δply strain. These findings suggest that reducing pneumococcal virulence gene expression by altering codon-pair bias could hold promise for rational design of live-attenuated pneumococcal vaccines. PMID:21343143

The repeating nucleotide sequence in the repetitive mitochondrial DNA from a "low-density" petite mutant of yeast.

PubMed Central

Van Kreijl, C F; Bos, J L

1977-01-01

The repeating nucleotide sequence of 68 base pairs in the mtDNA from an ethidium-induced cytoplasmic petite mutant of yeast has been determined. For sequence analysis specifically primed and terminated RNA copies, obtained by in vitro transcription of the separated strands, were use. The sequence consists of 66 consecutive AT base pairs flanked by two GC pairs and comprises nearly all of the mutant mitochondrial genome. The sequence, moreover, also represents the first part of wild-type mtDNA sequence so far. Images PMID:198740

A mouse model for studying viscerotropic disease caused by yellow fever virus infection.

PubMed

Meier, Kathryn C; Gardner, Christina L; Khoretonenko, Mikhail V; Klimstra, William B; Ryman, Kate D

2009-10-01

Mosquito-borne yellow fever virus (YFV) causes highly lethal, viscerotropic disease in humans and non-human primates. Despite the availability of efficacious live-attenuated vaccine strains, 17D-204 and 17DD, derived by serial passage of pathogenic YFV strain Asibi, YFV continues to pose a significant threat to human health. Neither the disease caused by wild-type YFV, nor the molecular determinants of vaccine attenuation and immunogenicity, have been well characterized, in large part due to the lack of a small animal model for viscerotropic YFV infection. Here, we describe a small animal model for wild-type YFV that manifests clinical disease representative of that seen in primates without adaptation of the virus to the host, which was required for the current hamster YF model. Investigation of the role of type I interferon (IFN-alpha/beta) in protection of mice from viscerotropic YFV infection revealed that mice deficient in the IFN-alpha/beta receptor (A129) or the STAT1 signaling molecule (STAT129) were highly susceptible to infection and disease, succumbing within 6-7 days. Importantly, these animals developed viscerotropic disease reminiscent of human YF, instead of the encephalitic signs typically observed in mice. Rapid viremic dissemination and extensive replication in visceral organs, spleen and liver, was associated with severe pathologies in these tissues and dramatically elevated MCP-1 and IL-6 levels, suggestive of a cytokine storm. In striking contrast, infection of A129 and STAT129 mice with the 17D-204 vaccine virus was subclinical, similar to immunization in humans. Although, like wild-type YFV, 17D-204 virus amplified within regional lymph nodes and seeded a serum viremia in A129 mice, infection of visceral organs was rarely established and rapidly cleared, possibly by type II IFN-dependent mechanisms. The ability to establish systemic infection and cause viscerotropic disease in A129 mice correlated with infectivity for A129-derived, but not WT129-derived, macrophages and dendritic cells in vitro, suggesting a role for these cells in YFV pathogenesis. We conclude that the ability of wild-type YFV to evade and/or disable components of the IFN-alpha/beta response may be primate-specific such that infection of mice with a functional IFN-alpha/beta antiviral response is attenuated. Consequently, subcutaneous YFV infection of A129 mice represents a biologically relevant model for studying viscerotropic infection and disease development following wild-type virus inoculation, as well as mechanisms of 17D-204 vaccine attenuation, without a requirement for adaptation of the virus.

Characterization of adenoviral transduction profile in prostate cancer cells and normal prostate tissue.

PubMed

Ai, Jianzhong; Tai, Phillip W L; Lu, Yi; Li, Jia; Ma, Hong; Su, Qin; Wei, Qiang; Li, Hong; Gao, Guangping

2017-09-01

Prostate diseases are common in males worldwide with high morbidity. Gene therapy is an attractive therapeutic strategy for prostate diseases, however, it is currently underdeveloped. As well known, adeno virus (Ad) is the most widely used gene therapy vector. The aims of this study are to explore transduction efficiency of Ad in prostate cancer cells and normal prostate tissue, thus further providing guidance for future prostate pathophysiological studies and therapeutic development of prostate diseases. We produced Ad expressing enhanced green fluorescence protein (EGFP), and characterized the transduction efficiency of Ad in both human and mouse prostate cancer cell lines in vitro, as well as prostate tumor xenograft, and wild-type mouse prostate tissue in vivo. Ad transduction efficiency was determined by EGFP fluorescence using microscopy and flow cytometry. Cell type-specific transduction was examined by immunofluorescence staining of cell markers. Our data showed that Ad efficiently transduced human and mouse prostate cancer cells in vitro in a dose dependent manner. Following intratumoral and intraprostate injection, Ad could efficiently transduce prostate tumor xenograft and the major prostatic cell types in vivo, respectively. Our findings suggest that Ad can efficiently transduce prostate tumor cells in vitro as well as xenograft and normal prostate tissue in vivo, and further indicate that Ad could be a potentially powerful toolbox for future gene therapy of prostate diseases. © 2017 Wiley Periodicals, Inc.

In vitro pathogenicity assay for the ergot fungus Claviceps purpurea.

PubMed

Scheffer, Jan; Tudzynski, Paul

2006-04-01

The pathogenic development of the biotrophic ergot fungus Claviceps purpurea is strictly limited to the ovary of grasses. Early colonization stages occur within a defined spatio-temporal course of events, including the directed growth to the vascular tissue for nutrient supply. To characterize mutant strains with putative defects in pathogenicity, the close observation of the infection pathway is therefore indispensable. Here, we describe the establishment of a new pathogenicity assay, based on the in vitro cultivation of isolated rye ovaries. The pathogenic development of a wild-type strain of C. purpurea was compared with the infection of mature rye flowers on whole plants. Up to the sixth day post inoculation, the route of infection within the isolated ovaries was maintained and temporally equal to that seen in mature flowers. Therefore, the in vitro pathogenicity assay is an effective alternative to the whole-plant infection tests, and suitable for detailed infection studies and screening high numbers of mutants for defects in early pathogenesis.

Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro.

PubMed

Lambrecht, Jennifer A; Browne, Beth Ann; Downs, Diana M

2010-11-05

The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.

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

PubMed

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

2012-10-01

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

``Rhizogenesis in vitro'' - as a model to study microgravity biological effects

NASA Astrophysics Data System (ADS)

Bulavin, Iliya

Functioning organisms is based on the physiological and biochemical processes in different tissues and cells. Numerous spaceflight biological experiments have shown the essential changes in cell behavior of multicellular and unicellular organisms in comparison with that on Earth. In our investigations, we used the model “Rhizogenesis in vitro” to study cell differentiation in the root cap and growth zones under clinorotation. Advantage of this model is the possibility to study the influence of clinorotation at the beginning of root initiation de novo and next morphogenetic processes unlike experiments in vivo with embryonal seedling roots formed in seeds. Arabidopsis thaliana plants of wild type and scr mutant (3999 by NASC database) were used. For rhizogenesis induction, rosette leaves with petioles were cut and transferred in Petri dishes on MS medium contained 1/10 of MS mineral salt, without vitamins and hormones. One half of Petri dishes were placed vertically (control), the other - on a slow horizontal clinostat (2 rpm). Anatomical investigation of A. thaliana wild type and scr mutant roots formed de novo showed that formation of root cap and growth zones (meristem, distal elongation zone (DEZ), central elongation zone (CEZ) and mature zone) under clinorotation was similar to that in control. A root cap consists of columella and peripheral cells. In the columella there are meristematic cells, statocytes (graviperceptive cells), and secretory cells. Epidermis, parenchyma, endodermis and central cylinder are distinguished in wild type roots. Unlike a wild type, a cortex of scr mutant was represented by one cell layer which had the parenchyma and endodermis characteristics. A root cap length and width were similar in control and under clinorotation. A cell number in the meristem and DEZ and a length of these growth zones did not differ in control and the experimental conditions. The ultrasructure of cap meristematic cells was typical for cells of this type. Statocytes preserved their polarity in control but it was disturbed under clinorotation due to amyloplast distribution in the cytoplasm whole volume and/or their localization in the cell center. Structural rearrangements occurred similarly in statocytes under their transformation in secretory cells in control and under clinorotation. A characteristic features of the root proper meristematic cells in the control and in the experiment are central nucleus location, the great diversity of a size and a shape of mitochondria and plastids, poorly ER development, the presence of some small ER-bodies. As cells passed in the DEZ, their size enlarged but a nucleus can preserve the central location. A quantity of ER-cistern, vacuoles, and ER-bodies increased also. Dictyosomes acquired polarity and produced many Golgi vesicles. In CEZ cells, a large vacuole occupied the cell center, and the cytoplasm with organelles was on the cell periphery. So, we can conclude that under clinorotation: 1) the structure of a cap and growth zones of A. thaliana wild type and scr mutant roots formed de novo in vitro as similar to that in control; 2) a gaviperceptive apparatus formed in both objects but did not function. The obtained data allow to propose the model “Rhizogenesis in vitro” for using in spaceflight experiments to study the influence of real microgravity on the cellular differentiation and basic processes.

A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability

PubMed Central

Davies, Benjamin; Brown, Laurence A; Cais, Ondrej; Clayton, Amber J; Chang, Veronica T; Biggs, Daniel; Preece, Christopher; Hernandez-Pliego, Polinka; Krohn, Jon; Bhomra, Amarjit; Twigg, Stephen R F; Rimmer, Andrew; Kanapin, Alexander; Sen, Arjune; Zaiwalla, Zenobia; McVean, Gil; Foster, Russell; Donnelly, Peter; Taylor, Jenny C; Blair, Edward; Nutt, David; Aricescu, A Radu; Greger, Ingo H; Peirson, Stuart N; Flint, Jonathan

2017-01-01

Abstract The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans. PMID:29016847

Establishment of a cell model of X-linked sideroblastic anemia using genome editing.

PubMed

Kaneko, Kiriko; Kubota, Yoshiko; Nomura, Kazumi; Hayashimoto, Haruka; Chida, Taisei; Yoshino, Naoto; Wayama, Marina; Ogasawara, Katsutoshi; Nakamura, Yukio; Tooyama, Ikuo; Furuyama, Kazumichi

2018-06-13

ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patient's bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, β-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared to wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition, and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, while the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency, in combination with increased iron import into mitochondria during erythroid differentiation, results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro. Copyright © 2018. Published by Elsevier Inc.

Evaluation of biological safety in vitro and immunogenicity in vivo of recombinant Escherichia coli Shiga toxoids as candidate vaccines in cattle.

PubMed

Kerner, Katharina; Bridger, Philip S; Köpf, Gabriele; Fröhlich, Julia; Barth, Stefanie; Willems, Hermann; Bauerfeind, Rolf; Baljer, Georg; Menge, Christian

2015-04-10

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa).

PubMed

Du, Yiwei; He, Wei; Deng, Changwang; Chen, Xi; Gou, Lanming; Zhu, Fugui; Guo, Wei; Zhang, Jianfu; Wang, Tao

2016-01-01

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23-26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

PubMed Central

Deng, Changwang; Chen, Xi; Gou, Lanming; Zhu, Fugui; Guo, Wei; Zhang, Jianfu; Wang, Tao

2016-01-01

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes. PMID:26934377

alphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein.

PubMed

Griffin, K J; Dwyer, T M; Manning, M C; Meyer, J D; Carpenter, J F; Frerman, F E

1997-04-08

Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.

Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane.

PubMed

McDowell, Stephen C; López-Marqués, Rosa L; Cohen, Taylor; Brown, Elizabeth; Rosenberg, Alexa; Palmgren, Michael G; Harper, Jeffrey F

2015-01-01

Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA) family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7) results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance of lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions.

Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane

PubMed Central

McDowell, Stephen C.; López-Marqués, Rosa L.; Cohen, Taylor; Brown, Elizabeth; Rosenberg, Alexa; Palmgren, Michael G.; Harper, Jeffrey F.

2015-01-01

Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA) family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7) results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance of lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions. PMID:25954280

Identification of Galectin-1 as a Critical Factor in Function of Mouse Mesenchymal Stromal Cell-Mediated Tumor Promotion

PubMed Central

Blazsó, Péter; Katona, Róbert László; Novák, Julianna; Szabó, Enikő; Czibula, Ágnes; Fajka-Boja, Roberta; Hegyi, Beáta; Uher, Ferenc; Krenács, László; Joó, Gabriella; Monostori, Éva

2012-01-01

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development. These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression. PMID:22844466

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

PubMed

Shi, Mei; Bennett, Teresa A; Cimino, Daniel F; Maestas, Diane C; Foutz, Terry D; Gurevich, Vsevolod V; Sklar, Larry A; Prossnitz, Eric R

2003-06-24

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

Shiver me titin! Elucidating titin's role in shivering thermogenesis.

PubMed

Taylor-Burt, Kari R; Monroy, Jenna; Pace, Cinnamon; Lindstedt, Stan; Nishikawa, Kiisa C

2015-03-01

Shivering frequency scales predictably with body mass and is 10 times higher in a mouse than a moose. The link between shivering frequency and body mass may lie in the tuning of muscle elastic properties. Titin functions as a muscle 'spring', so shivering frequency may be linked to titin's structure. The muscular dystrophy with myositis (mdm) mouse is characterized by a deletion in titin's N2A region. Mice that are homozygous for the mdm mutation have a lower body mass, stiffer gait and reduced lifespan compared with their wild-type and heterozygous siblings. We characterized thermoregulation in these mice by measuring metabolic rate and tremor frequency during shivering. Mutants were heterothermic at ambient temperatures of 20-37°C while wild-type and heterozygous mice were homeothermic. Metabolic rate increased at smaller temperature differentials (i.e. the difference between body and ambient temperatures) in mutants than in non-mutants. The difference between observed tremor frequencies and shivering frequencies predicted by body mass was significantly larger for mutant mice than for wild-type or heterozygous mice, even after accounting for differences in body temperature. Together, the heterothermy in mutants, the increase in metabolic rate at low temperature differentials and the decreased tremor frequency demonstrate the thermoregulatory challenges faced by mice with the mdm mutation. Oscillatory frequency is proportional to the square root of stiffness, and we observed that mutants had lower active muscle stiffness in vitro. The lower tremor frequencies in mutants are consistent with reduced active muscle stiffness and suggest that titin affects the tuning of shivering frequency. © 2015. Published by The Company of Biologists Ltd.

Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

PubMed

Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

2016-05-03

β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.

Comparative transcriptomic analysis of key genes involved in flavonoid biosynthetic pathway and identification of a flavonol synthase from Artemisia annua L.

PubMed

Liu, S; Liu, L; Tang, Y; Xiong, S; Long, J; Liu, Z; Tian, N

2017-07-01

The regulatory mechanism of flavonoids, which synergise anti-malarial and anti-cancer compounds in Artemisia annua, is still unclear. In this study, an anthocyanidin-accumulating mutant callus was induced from A. annua and comparative transcriptomic analysis of wild-type and mutant calli performed, based on the next-generation Illumina/Solexa sequencing platform and de novo assembly. A total of 82,393 unigenes were obtained and 34,764 unigenes were annotated in the public database. Among these, 87 unigenes were assigned to 14 structural genes involved in the flavonoid biosynthetic pathway and 37 unigenes were assigned to 17 structural genes related to metabolism of flavonoids. More than 30 unigenes were assigned to regulatory genes, including R2R3-MYB, bHLH and WD40, which might regulate flavonoid biosynthesis. A further 29 unigenes encoding flavonoid biosynthetic enzymes or transcription factors were up-regulated in the mutant, while 19 unigenes were down-regulated, compared with the wild type. Expression levels of nine genes involved in the flavonoid pathway were compared using semi-quantitative RT-PCR, and results were consistent with comparative transcriptomic analysis. Finally, a putative flavonol synthase gene (AaFLS1) was identified from enzyme assay in vitro and in vivo through heterogeneous expression, and confirmed comparative transcriptomic analysis of wild-type and mutant callus. The present work has provided important target genes for the regulation of flavonoid biosynthesis in A. annua. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis.

PubMed

Zimbler, Daniel L; Eddy, Justin L; Schroeder, Jay A; Lathem, Wyndham W

2016-01-01

Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production.

PubMed

Chutkow, William A; Patwari, Parth; Yoshioka, Jun; Lee, Richard T

2008-01-25

Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.

Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus-Mesorhizobium loti symbiosis.

PubMed

Fukudome, Mitsutaka; Calvo-Begueria, Laura; Kado, Tomohiro; Osuki, Ken-Ichi; Rubio, Maria Carmen; Murakami, Ei-Ichi; Nagata, Maki; Kucho, Ken-Ichi; Sandal, Niels; Stougaard, Jens; Becana, Manuel; Uchiumi, Toshiki

2016-09-01

Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia-legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5'-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

[6]-Gingerol Induces Cell Cycle Arrest and Cell Death of Mutant p53-expressing Pancreatic Cancer Cells

PubMed Central

Park, Yon Jung; Wen, Jing; Bang, Seungmin; Park, Seung Woo

2006-01-01

[6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild-type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21cip1, was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53-expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53-expressing cells by inducing temporal growth arrest. PMID:17066513

Genetic Phagocyte NADPH Oxidase Deficiency Enhances Nonviable Candida albicans-Induced Inflammation in Mouse Lungs.

PubMed

Endo, Daiki; Fujimoto, Kenta; Hirose, Rika; Yamanaka, Hiroko; Homme, Mizuki; Ishibashi, Ken-Ichi; Miura, Noriko; Ohno, Naohito; Aratani, Yasuaki

2017-02-01

Patients with chronic granulomatous disease (CGD) have mutated phagocyte NADPH oxidase, resulting in reduced production of reactive oxygen species (ROS). While the mechanism underlying hyperinfection in CGD is well understood, the basis for inflammatory disorders that arise in the absence of evident infection has not been fully explained. This study aimed to evaluate the effect of phagocyte NADPH oxidase deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice deficient in this enzyme (CGD mice) showed more severe neutrophilic pneumonia than nCA-treated wild-type mice, which exhibited significantly higher lung concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and keratinocyte-derived chemokine (KC). Neutralization of these proinflammatory mediators significantly reduced neutrophil infiltration. In vitro, production of IL-1β and TNF-α from neutrophils and that of KC from macrophages was enhanced in nCA-stimulated neutrophils from CGD mice. Expression of IL-1β mRNA was higher in the stimulated CGD neutrophils than in the stimulated wild-type cells, concomitant with upregulation of nuclear factor (NF)-κB and its upstream regulator extracellular-signal regulated kinase (ERK) 1/2. Pretreatment with an NADPH oxidase inhibitor significantly enhanced IL-1β production in the wild-type neutrophils stimulated with nCA. These results suggest that lack of ROS production because of NADPH oxidase deficiency results in the production of higher levels of proinflammatory mediators from neutrophils and macrophages, which may at least partly contribute to the exacerbation of nCA-induced lung inflammation in CGD mice.

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity

PubMed Central

Chittaranjan, Suganthi; Chan, Susanna; Yang, Cindy; Yang, Kevin C.; Chen, Vincent; Moradian, Annie; Firme, Marlo; Song, Jungeun; Go, Nancy E.; Blough, Michael D.; Chan, Jennifer A.; Cairncross, J. Gregory; Gorski, Sharon M.; Morin, Gregg B.; Yip, Stephen; Marra, Marco A.

2014-01-01

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism of reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples. PMID:25277207

Protective role of p53 in skin cancer: Carcinogenesis studies in mice lacking epidermal p53.

PubMed

Page, Angustias; Navarro, Manuel; Suarez-Cabrera, Cristian; Alameda, Josefa P; Casanova, M Llanos; Paramio, Jesús M; Bravo, Ana; Ramirez, Angel

2016-04-12

p53 is a protein that causes cell cycle arrest, apoptosis or senescence, being crucial in the process of tumor suppression in several cell types. Different in vitro and animal models have been designed for the study of p53 role in skin cancer. These models have revealed opposing results, as in some experimental settings it appears that p53 protects against skin cancer, but in others, the opposite conclusion emerges. We have generated cohorts of mice with efficient p53 deletion restricted to stratified epithelia and control littermates expressing wild type p53 and studied their sensitivity to both chemically-induced and spontaneous tumoral transformation, as well as the tumor types originated in each experimental group. Our results indicate that the absence of p53 in stratified epithelia leads to the appearance, in two-stage skin carcinogenesis experiments, of a higher number of tumors that grow faster and become malignant more frequently than tumors arisen in mice with wild type p53 genotype. In addition, the histological diversity of the tumor type is greater in mice with epidermal p53 loss, indicating the tumor suppressive role of p53 in different epidermal cell types. Aging mice with p53 inactivation in stratified epithelia developed spontaneous carcinomas in skin and other epithelia. Overall, these results highlight the truly protective nature of p53 functions in the development of cancer in skin and in other stratified epithelia.

Synthesis and evaluation of osimertinib derivatives as potent EGFR inhibitors.

PubMed

Gao, Hongying; Yang, Zimo; Yang, Xinglin; Rao, Yu

2017-09-01

Osimertinib has been identified as a promising therapeutic drug targeting for EGFR T790M mutant non-small cell lung cancer (NSCLC). A new series of N-oxidized and fluorinated osimertinib derivatives were designed and synthesized. The cellular anti-proliferative activity, kinase inhibitory activity and the activation of EGFR signaling pathways of 1-6 in vitro were determined against L858R/T790M and wild-type EGFR, the antitumor efficacy in NCI-H1975 xenografts in vivo were further studied. Compound 2, the newly synthesized N-oxide metabolite in N,N,N'-trimethylethylenediamine side chain of osimertinib, showed a comparable kinase selectivity in vitro and a slightly better antitumor efficacy in vivo to osimertinib, making it valuable and suitable for the potential lung cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

In Vitro Antifungal Susceptibility Testing of Candida Isolates with the EUCAST Methodology, a New Method for ECOFF Determination.

PubMed

Meletiadis, J; Curfs-Breuker, I; Meis, J F; Mouton, J W

2017-04-01

The in vitro susceptibilities of 1,099 molecularly identified clinical Candida isolates against 8 antifungal drugs were determined using the EUCAST microdilution method. A new simple, objective, and mathematically solid method for determining epidemiological cutoff values (ECOFFs) was developed by derivatizing the MIC distribution and determining the derivatized ECOFF (dECOFF) as the highest MIC with the maximum second derivative. The dECOFFs were similar (95% agreement within 1 dilution) to the EUCAST ECOFFs. Overall, low non-wild-type/resistance rates were found. The highest rates were found for azoles with C. parapsilosis (2.7 to 9.8%), C. albicans (7%), and C. glabrata (1.7 to 2.3%) and for echinocandins with C. krusei (3.3%), C. albicans (1%), and C. tropicalis (1.7%). Copyright © 2017 American Society for Microbiology.

Identification of phenolic compounds by liquid chromatography-mass spectrometry in seventeen species of wild mushrooms in Central Mexico and determination of their antioxidant activity and bioactive compounds.

PubMed

Yahia, Elhadi M; Gutiérrez-Orozco, Fabiola; Moreno-Pérez, Marco A

2017-07-01

Wild mushrooms are important for the diet of some communities in Mexico. However, limited information exists on their chemical composition, contribution to the diet, and health effects. We characterized seventeen wild mushroom species growing in the state of Queretaro in Central Mexico. Most species analyzed were edible, but also included nonedible, medicinal, poisonous and toxic specimens. Whole mushrooms (caps and stipes) were characterized for water content, color, and total content of phenolic compounds, flavonoids and anthocyanins. In vitro antioxidant capacity was measured by FRAP and DPPH assays. Phenolic compounds were identified and quantified by HPLC-mass spectrometry. All species analyzed were found to possess antioxidant activity in vitro and a wide range of phenolic and organic compounds were identified. Our results add to the limited information available on the composition and potential nutritional and health value of wild mushrooms. Further analyses of their bioactivities are warranted. Copyright © 2017 Elsevier Ltd. All rights reserved.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells.

PubMed

Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H; Veits, Jutta; Gischke, Marcel; Mettenleiter, Thomas C; Abdelwhab, Elsayed M

2018-02-14

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host.

Impact of Mutations in the Hemagglutinin of H10N7 Viruses Isolated from Seals on Virus Replication in Avian and Human Cells

PubMed Central

Dittrich, Anne; Scheibner, David; Salaheldin, Ahmed H.; Veits, Jutta; Gischke, Marcel

2018-01-01

Wild birds are the reservoir for low-pathogenic avian influenza viruses, which are frequently transmitted to domestic birds and occasionally to mammals. In 2014, an H10N7 virus caused severe mortality in harbor seals in northeastern Europe. Although the hemagglutinin (HA) of this virus was closely related to H10 of avian H10N4 virus, it possessed unique nonsynonymous mutations, particularly in the HA1 subunit in or adjacent to the receptor binding domain and proteolytic cleavage site. Here, the impact of these mutations on virus replication was studied in vitro. Using reverse genetics, an avian H10N4 virus was cloned, and nine recombinant viruses carrying one of eight unique mutations or the complete HA from the seal virus were rescued. Receptor binding affinity, replication in avian and mammalian cell cultures, cell-to-cell spread, and HA cleavability of these recombinant viruses were studied. Results show that wild-type recombinant H10N4 virus has high affinity to avian-type sialic acid receptors and no affinity to mammalian-type receptors. The H10N7 virus exhibits dual receptor binding affinity. Interestingly, Q220L (H10 numbering) in the rim of the receptor binding pocket increased the affinity of the H10N4 virus to mammal-type receptors and completely abolished the affinity to avian-type receptors. No remarkable differences in cell-to-cell spread or HA cleavability were observed. All viruses, including the wild-type H10N7 virus, replicated at higher levels in chicken cells than in human cells. These results indicate that H10N7 acquired adaptive mutations (e.g., Q220L) to enhance replication in mammals and retained replication efficiency in the original avian host. PMID:29443887

TRAF family member-associated NF-κB activator (TANK) is a negative regulator of osteoclastogenesis and bone formation.

PubMed

Maruyama, Kenta; Kawagoe, Tatsukata; Kondo, Takeshi; Akira, Shizuo; Takeuchi, Osamu

2012-08-17

The differentiation of bone-resorbing osteoclasts is induced by RANKL signaling, and leads to the activation of NF-κB via TRAF6 activation. TRAF family member-associated NF-κB activator (TANK) acts as a negative regulator of Toll-like receptors (TLRs) and B-cell receptor (BCR) signaling by inhibiting TRAF6 activation. Tank(-/-) mice spontaneously develop autoimmune glomerular nephritis in an IL-6-dependent manner. Despite its importance in the TCRs and BCR-activated TRAF6 inhibition, the involvement of TANK in RANKL signaling is poorly understood. Here, we report that TANK is a negative regulator of osteoclast differentiation. The expression levels of TANK mRNA and protein were up-regulated during RANKL-induced osteoclastogenesis, and overexpression of TANK in vitro led to a decrease in osteoclast formation. The in vitro osteoclastogenesis of Tank(-/-) cells was significantly increased, accompanied by increased ubiquitination of TRAF6 and enhanced canonical NF-κB activation in response to RANKL stimulation. Tank(-/-) mice showed severe trabecular bone loss, but increased cortical bone mineral density, because of enhanced bone erosion and formation. TANK mRNA expression was induced during osteoblast differentiation and Tank(-/-) osteoblasts exhibited enhaced NF-κB activation, IL-11 expression, and bone nodule formation than wild-type control cells. Finally, wild-type mice transplanted with bone marrow cells from Tank(-/-) mice showed trabecular bone loss analogous to that in Tank(-/-) mice. These findings demonstrate that TANK is critical for osteoclastogenesis by regulating NF-κB, and is also important for proper bone remodeling.

TRAF Family Member-associated NF-κB Activator (TANK) Is a Negative Regulator of Osteoclastogenesis and Bone Formation*

PubMed Central

Maruyama, Kenta; Kawagoe, Tatsukata; Kondo, Takeshi; Akira, Shizuo; Takeuchi, Osamu

2012-01-01

The differentiation of bone-resorbing osteoclasts is induced by RANKL signaling, and leads to the activation of NF-κB via TRAF6 activation. TRAF family member-associated NF-κB activator (TANK) acts as a negative regulator of Toll-like receptors (TLRs) and B-cell receptor (BCR) signaling by inhibiting TRAF6 activation. Tank−/− mice spontaneously develop autoimmune glomerular nephritis in an IL-6-dependent manner. Despite its importance in the TCRs and BCR-activated TRAF6 inhibition, the involvement of TANK in RANKL signaling is poorly understood. Here, we report that TANK is a negative regulator of osteoclast differentiation. The expression levels of TANK mRNA and protein were up-regulated during RANKL-induced osteoclastogenesis, and overexpression of TANK in vitro led to a decrease in osteoclast formation. The in vitro osteoclastogenesis of Tank−/− cells was significantly increased, accompanied by increased ubiquitination of TRAF6 and enhanced canonical NF-κB activation in response to RANKL stimulation. Tank−/− mice showed severe trabecular bone loss, but increased cortical bone mineral density, because of enhanced bone erosion and formation. TANK mRNA expression was induced during osteoblast differentiation and Tank−/− osteoblasts exhibited enhaced NF-κB activation, IL-11 expression, and bone nodule formation than wild-type control cells. Finally, wild-type mice transplanted with bone marrow cells from Tank−/− mice showed trabecular bone loss analogous to that in Tank−/− mice. These findings demonstrate that TANK is critical for osteoclastogenesis by regulating NF-κB, and is also important for proper bone remodeling. PMID:22773835

Induction of cytochrome P450 3A by paclitaxel in mice: pivotal role of the nuclear xenobiotic receptor, pregnane X receptor.

PubMed

Nallani, Srikanth C; Goodwin, Bryan; Maglich, Jodi M; Buckley, Donna J; Buckley, Arthur R; Desai, Pankaj B

2003-05-01

Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.

Increased metastatic potential of tumor cells in von Willebrand factor-deficient mice.

PubMed

Terraube, V; Pendu, R; Baruch, D; Gebbink, M F B G; Meyer, D; Lenting, P J; Denis, C V

2006-03-01

The key role played by von Willebrand factor (VWF) in platelet adhesion suggests a potential implication in various pathologies, where this process is involved. In cancer metastasis development, tumor cells interact with platelets and the vessel wall to extravasate from the circulation. As a potential mediator of platelet-tumor cell interactions, VWF could influence this early step of tumor spread and therefore play a role in cancer metastasis. To investigate whether VWF is involved in metastasis development. In a first step, we characterized the interaction between murine melanoma cells B16-BL6 and VWF in vitro. In a second step, an experimental metastasis model was used to compare the formation of pulmonary metastatic foci in C57BL/6 wild-type and VWF-null mice following the injection of B16-BL6 cells or Lewis lung carcinoma cells. In vitro adhesion assays revealed that VWF is able to promote a dose-dependent adhesion of B16-BL6 cells via its Arg-Gly-Asp (RGD) sequence. In the experimental metastasis model, we found a significant increase in the number of pulmonary metastatic foci in VWF-null mice compared with the wild-type mice, a phenotype that could be corrected by restoring VWF plasma levels. We also showed that increased survival of the tumor cells in the lungs during the first 24 h in the absence of VWF was the cause of this increased metastasis. These findings suggest that VWF plays a protective role against tumor cell dissemination in vivo. Underlying mechanisms remain to be investigated.

DNA Mutations Mediate Microevolution between Host-Adapted Forms of the Pathogenic Fungus Cryptococcus neoformans

PubMed Central

Magditch, Denise A.; Liu, Tong-Bao; Xue, Chaoyang; Idnurm, Alexander

2012-01-01

The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a “switch” from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease. PMID:23055925

The expression of heterologous MAM-7 in Lactobacillus rhamnosus reduces its intrinsic capacity to inhibit colonization of pathogen Vibrio parahaemolyticus in vitro.

PubMed

Beltran, Sebastian; Munoz-Bergmann, Cristian A; Elola-Lopez, Ana; Quintana, Javiera; Segovia, Cristopher; Trombert, Annette N

2016-01-07

Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative, halophilic bacterium recognized as one of the most important foodborne pathogen. When ingested, V. parahaemolyticus causes a self-limiting illness (Vibriosis), characterized mainly by watery diarrhoea. Treatment is usually oral rehydration and/or antibiotics in complicated cases. Since 1996, the pathogenic and pandemic V. parahaemolyticus O3:K6 serotype has spread worldwide, increasing the reported number of vibriosis cases. Thus, the design of new strategies for pathogen control and illness prevention is necessary. Lactobacillus sp. grouped Gram positive innocuous bacteria, part of normal intestinal microbiota and usually used as oral vaccines for several diarrheic diseases. Recombinants strains of Lactobacillus (RL) expressing pathogen antigens can be used as part of an anti-adhesion strategy where RL block the pathogen union sites in host cells. Thus, we aimed to express MAM-7 V. parahaemolyticus adhesion protein in Lactobacillus sp. to generate an RL that prevents pathogen colonization. We cloned the MAM-7 gene from V. parahaemolyticus RIMD 2210633 in Lactobacillus expression vectors. Recombinant strains (Lactobacillus rhamnosus pSEC-MAM7 and L. rhamnosus pCWA-MAM7) adhered to CaCo-2 cells and competed with the pathogen. However, the L. rhamnosus wild type strain showed the best capacity to inhibit pathogen colonization in vitro. In addition, LDH-assay showed that recombinant strains were cytotoxic compared with the wild type isogenic strain. MAM-7 expression in lactobacilli reduces the intrinsic inhibitory capacity of L. rhamnosus against V. parahaemolyticus.

Effective targeting of STAT5-mediated survival in myeloproliferative neoplasms using ABT-737 combined with rapamycin

PubMed Central

Li, Geqiang; Miskimen, Kristy L.; Wang, Zhengqi; Xie, Xiu Yan; Tse, William; Gouilleux, Fabrice; Moriggl, Richard; Bunting, Kevin D.

2010-01-01

Signal transducer and activator of transcription-5 (STAT5) is a critical transcription factor for normal hematopoiesis and its sustained activation is associated with hematologic malignancy. A persistently active mutant of STAT5 (STAT5aS711F) associates with Grb2 associated binding protein 2 (Gab2) in myeloid leukemias and promotes growth in vitro through AKT activation. Here we have retrovirally transduced wild-type or Gab2−/− mouse bone marrow cells expressing STAT5aS711F and transplanted into irradiated recipient mice to test an in vivo myeloproliferative disease (MPD) model. To target Gab2-independent AKT/mTOR activation, wild-type mice were treated separately with rapamycin. In either case, mice lacking Gab2 or treated with rapamycin displayed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible. To improve upon this approach, in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin was combined with inhibition of the STAT5 direct target genes bcl-2 and bcl-XL using ABT-737. Striking synergy with both drugs was observed in mouse BaF3 cells expressing STAT5aS711F, TEL-JAK2, or BCR-ABL and in the relatively single agent-resistant human BCR-ABL positive K562 cell line. Therefore, targeting distinct STAT5 mediated survival signals, e.g. bcl-2/bcl-XL and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms. PMID:20535152

Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.

PubMed

Chandrayan, Sanjeev K; McTernan, Patrick M; Hopkins, R Christopher; Sun, Junsong; Jenney, Francis E; Adams, Michael W W

2012-01-27

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase*

PubMed Central

Chandrayan, Sanjeev K.; McTernan, Patrick M.; Hopkins, R. Christopher; Sun, Junsong; Jenney, Francis E.; Adams, Michael W. W.

2012-01-01

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production. PMID:22157005

Systemic Adenosine Triphosphate Impairs Neutrophil Chemotaxis and Host Defense in Sepsis.

PubMed

Li, Xiaoou; Kondo, Yutaka; Bao, Yi; Staudenmaier, Laura; Lee, Albert; Zhang, Jingping; Ledderose, Carola; Junger, Wolfgang G

2017-01-01

Sepsis remains an unresolved clinical problem. Therapeutic strategies focusing on inhibition of neutrophils (polymorphonuclear neutrophils) have failed, which indicates that a more detailed understanding of the underlying pathophysiology of sepsis is required. Polymorphonuclear neutrophil activation and chemotaxis require cellular adenosine triphosphate release via pannexin-1 channels that fuel autocrine feedback via purinergic receptors. In the current study, we examined the roles of endogenous and systemic adenosine triphosphate on polymorphonuclear neutrophil activation and host defense in sepsis. Prospective randomized animal investigation and in vitro studies. Preclinical academic research laboratory. Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies. Wild-type and pannexin-1 knockout mice were treated with suramin or apyrase to block the endogenous or systemic effects of adenosine triphosphate. Mice were subjected to cecal ligation and puncture and polymorphonuclear neutrophil activation (CD11b integrin expression), organ (liver) injury (plasma aspartate aminotransferase), bacterial spread, and survival were monitored. Human polymorphonuclear neutrophils were used to study the effect of systemic adenosine triphosphate and apyrase on chemotaxis. Inhibiting endogenous adenosine triphosphate reduced polymorphonuclear neutrophil activation and organ injury, but increased the spread of bacteria and mortality in sepsis. By contrast, removal of systemic adenosine triphosphate improved bacterial clearance and survival in sepsis by improving polymorphonuclear neutrophil chemotaxis. Systemic adenosine triphosphate impairs polymorphonuclear neutrophil functions by disrupting the endogenous purinergic signaling mechanisms that regulate cell activation and chemotaxis. Removal of systemic adenosine triphosphate improves polymorphonuclear neutrophil function and host defenses, making this a promising new treatment strategy for sepsis.

Evidence for a functional genetic polymorphism of the human thiosulfate sulfurtransferase (Rhodanese), a cyanide and H2S detoxification enzyme.

PubMed

Billaut-Laden, Ingrid; Allorge, Delphine; Crunelle-Thibaut, Aurélie; Rat, Emmanuel; Cauffiez, Christelle; Chevalier, Dany; Houdret, Nicole; Lo-Guidice, Jean-Marc; Broly, Franck

2006-08-01

Rhodanese or thiosulfate sulfurtransferase (TST) is a mitochondrial matrix enzyme that plays roles in cyanide detoxification, the formation of iron-sulfur proteins and the modification of sulfur-containing enzymes. Transsulfuration reaction catalyzed by TST is also involved in H(2)S detoxification. To date, no polymorphism of the human TST gene had been reported. We developed a screening strategy based on a PCR-SSCP method to search for mutations in the 3 exons of TST and their proximal flanking regions. This strategy has been applied to DNA samples from 50 unrelated French individuals of Caucasian origin. Eleven polymorphisms consisting in seven nucleotide substitutions in non-coding regions, two silent mutations and two missense mutations were characterized. The functional consequences of the identified mutations were assessed in vivo by measurement of erythrocyte TST activity and/or in vitro using heterologous expression in Saccharomyces cerevisiae or transient transfection assay in HT29 and Caco-2 cell lines. The P(285)A variant appears to encode a protein with a 50% decrease of in vitro intrinsic clearance compared to the wild-type enzyme. Additionally, the six polymorphisms located upstream the ATG initiation codon are responsible for a significant decrease (ranging from 40% to 73%) in promoter activity of a reporter gene compared to the corresponding wild-type sequence. This work constitutes the first report of the existence of a functional genetic polymorphism affecting TST activity and should be of great help to investigate certain disorders for which impairment of CN(-) or H(2)S detoxification have been suggested to be involved.

Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the FcɛRI Pathway in Mast Cells▿ †

PubMed Central

McPherson, Victor A.; Everingham, Stephanie; Karisch, Robert; Smith, Julie A.; Udell, Christian M.; Zheng, Jimin; Jia, Zongchao; Craig, Andrew W. B.

2009-01-01

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcɛRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcɛRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcɛRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcɛRI signaling and potential regulation the actin reorganization in mast cells. PMID:19001085

Alanine scan of core positions in ubiquitin reveals links between dynamics, stability, and function

PubMed Central

Lee, Shirley Y.; Pullen, Lester; Virgil, Daniel J.; Castañeda, Carlos A.; Abeykoon, Dulith; Bolon, Daniel N. A.; Fushman, David

2014-01-01

Mutations at solvent inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. The two null mutants (I30A and L43A) were both less stable to temperature-induced unfolding in vitro than wild-type, but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to wild-type. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high molecular weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high molecular weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation. PMID:24361330

Contributions of F-BAR and SH2 domains of Fes protein tyrosine kinase for coupling to the FcepsilonRI pathway in mast cells.

PubMed

McPherson, Victor A; Everingham, Stephanie; Karisch, Robert; Smith, Julie A; Udell, Christian M; Zheng, Jimin; Jia, Zongchao; Craig, Andrew W B

2009-01-01

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.

Hypoxic ventilatory response in Tac1-/- neonatal mice following exposure to opioids.

PubMed

Berner, J; Shvarev, Y; Zimmer, A; Wickstrom, R

2012-12-01

Morphine is the dominating analgetic drug used in neonates, but opioid-induced respiratory depression limits its therapeutic use. In this study, we examined acute morphine effects on respiration during intermittent hypoxia in newborn Tac1 gene knockout mice (Tac1-/-) lacking substance P and neurokinin A. In vivo, plethysmography revealed a blunted hypoxic ventilatory response (HVR) in Tac1-/- mice. Morphine (10 mg/kg) depressed the HVR in wild-type animals through an effect on respiratory frequency, whereas it increased tidal volumes in Tac1-/- during hypoxia, resulting in increased minute ventilation. Apneas were reduced during the first hypoxic episode in both morphine-exposed groups, but were restored subsequently in Tac1-/- mice. Morphine did not affect ventilation or apnea prevalence during baseline conditions. In vitro, morphine (50 nM) had no impact on anoxic response of brain stem preparations of either strain. In contrast, it suppressed the inspiratory rhythm during normoxia and potentiated development of posthypoxic neuronal arrest, especially in Tac1-/-. Thus this phenotype has a higher sensitivity to the depressive effects of morphine on inspiratory rhythm generation, but morphine does not modify the reactivity to oxygen deprivation. In conclusion, although Tac1-/- mice are similar to wild-type animals during normoxia, they differed by displaying a reversed pattern with an improved HVR during intermittent hypoxia both in vivo and in vitro. These data suggest that opioids and the substance P-ergic system interact in the HVR, and that reducing the activity in the tachykinin system may alter the respiratory effects of opioid treatment in newborns.

Lipid A’s Structure Mediates Neisseria gonorrhoeae Fitness during Experimental Infection of Mice and Men

PubMed Central

Hobbs, Marcia M.; Anderson, James E.; Balthazar, Jacqueline T.; Kandler, Justin L.; Carlson, Russell W.; Ganguly, Jhuma; Begum, Afrin A.; Duncan, Joseph A.; Lin, Jessica T.; Sparling, P. Frederick; Jerse, Ann E.; Shafer, William M.

2013-01-01

ABSTRACT Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments. IMPORTANCE Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A’s structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea. PMID:24255126

In vitro transport characteristics of EFdA, a novel nucleoside reverse transcriptase inhibitor using Caco-2 and MDCKII cell monolayers.

PubMed

Zhang, Wei; Parniak, Michael A; Sarafianos, Stefan G; Empey, Philip E; Rohan, Lisa C

2014-06-05

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a novel nucleoside reverse transcriptase inhibitor with a unique mechanism of action and highly potent activity against both wild-type and clinically relevant drug resistant HIV-1 variants. Furthermore, in vivo efficacy and safety evaluations have shown EFdA to be a promising therapeutic candidate for use in the treatment of HIV infection. However, little is known about the pharmacokinetic and biopharmaceutical properties of EFdA. In this study, we evaluated cellular EFdA transport using Caco-2 and Madin-Darby Canine Kidney II (MDCKII) in vitro cell models. Studies using Caco-2 cell monolayers showed that EFdA efflux ratios were >2.0, suggesting that active drug transport mechanisms may play a role in EFdA flux. ABCB1 transporter (PGP1) inhibition was assessed using the acetomethoxy derivate of calcein (calcein-AM) as a fluorescent probe in both wild-type MDCKII and PGP1 overexpressing MDCKII cells. Nonetheless, our data showed that EFdA is not a substrate of PGP1. Additionally, comparative bidirectional flux of EFdA and Lucifer yellow (LY, a well-known paracellular marker) was studied over a range of EFdA concentrations. In MDCKII monolayers, EFdA had an apparent permeability coefficient (Papp) (a-b) of <1×10(-6)cm/s. The Papp values significantly increased in the presence of the paracellular permeability enhancer, indicating that EFdA primarily permeates via the paracellular route. Copyright © 2014 Elsevier B.V. All rights reserved.

Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission.

PubMed

Zhang, Wei; Hu, Minlu; Shi, Yuan; Gong, Tiantian; Dezzutti, Charlene S; Moncla, Bernard; Sarafianos, Stefan G; Parniak, Michael A; Rohan, Lisa C

2015-09-01

EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission.

A pyruvate formate lyase-deficient Chlamydomonas reinhardtii strain provides evidence for a link between fermentation and hydrogen production in green algae.

PubMed

Philipps, Gabriele; Krawietz, Danuta; Hemschemeier, Anja; Happe, Thomas

2011-04-01

The green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism characterized by a plastidic hydrogenase (HYD1) coupled to photosynthesis and a bacterial-type fermentation system in which pyruvate formate lyase (PFL1) is the central fermentative enzyme. To identify mutant strains with altered hydrogen metabolism, a C. reinhardtii nuclear transformant library was screened. Mutant strain 48F5 showed lower light-dependent hydrogen (H₂) evolution rates and reduced in vitro hydrogenase activity, and fermentative H₂ production in the dark was enhanced. The transformant has a single integration of the paromomycin resistance cassette within the PFL1 gene, and is unable to synthesize PFL1 protein. 48F5 secretes no formate, but produces more ethanol, D-lactate and CO₂ than the wild type. Moreover, HYD1 transcript and HYD1 protein levels were lower in the pfl1 mutant strain. Complementation of strain 48F5 with an intact copy of the PFL1 gene restored formate excretion and hydrogenase activity to the wild type level. This analysis shows that the PFL1 pathway has a significant impact on hydrogen metabolism in C. reinhardtii. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Determination of the antioxidant activity and polyphenol contents of wild Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt. Fr.) P. Karst. (higher Basidiomycetes) from central Himalayan hills of India.

PubMed

Mohsin, Mohammed; Negi, P; Ahmed, Z

2011-01-01

The antioxidant potential of wild strain of Lingzhi or Reishi medicinal mushroom Ganoderma lucidum from Central Himalayan Hills (2000 m MSL) was evaluated, and compared with its in vitro cultured mycelia grown on malt extract broth in the laboratory. Antioxidant activities of both wild and cultivated G. lucidum in terms of IC₅₀ (mg/ mL) were determined against different in vitro radical systems such as DPPH (1, 1-diphenyl-2-picrylhydrazyl), ABTS [2,2'-azinobis (3-ethylenebenzothiazoline-6-sulphonic acid)] and hydroxyl radicals, in addition to ferric reducing antioxidant power assay. Polyphenol contents were also determined, in order to assess their effects on the antioxidant activity of extracts. All the extracts showed significant antioxidant activity, and maximum scavenging was observed in the case of methanolic extracts of wild G. lucidum with minimum IC50 values 0.953 ± 0.040, 0.690 ± 0.014 and 3.295 ± 0.027 mg/mL, respectively, for DPPH, ABTS, and hydroxyl radicals. The efficacy of wild G. lucidum as a rich source of natural antioxidant was established for nutraceutical development.

Impaired response of mature adipocytes of diabetic mice to hypoxia

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

Hong, Seok Jong, E-mail: seok-hong@northwestern.edu; Jin, Da P.; Buck, Donald W.

2011-10-01

Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role inmore » injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.« less

Somatic embryogenesis from corolla tubes of interspecific amphiploids between cultivated sunflower (Helianthus annuus L.) and its wild species

USDA-ARS?s Scientific Manuscript database

Somatic embryogenesis in vitro provides an efficient means of plant multiplication, facilitating sunflower improvement and germplasm innovation. In the present study, using interspecific amphiploids (2n=4x=68) between cultivated sunflower and wild perennial Helianthus species as explant donors, soma...

Development of in vitro and in vivo neutralization assays based on the pseudotyped H7N9 virus.

PubMed

Tian, Yabin; Zhao, Hui; Liu, Qiang; Zhang, Chuntao; Nie, Jianhui; Huang, Weijing; Li, Changgui; Li, Xuguang; Wang, Youchun

2018-05-31

H7N9 viral infections pose a great threat to both animal and human health. This avian virus cannot be handled in level 2 biocontainment laboratories, substantially hindering evaluation of prophylactic vaccines and therapeutic agents. Here, we report a high-titer pseudoviral system with a bioluminescent reporter gene, enabling us to visually and quantitatively conduct analyses of virus replications in both tissue cultures and animals. For evaluation of immunogenicity of H7N9 vaccines, we developed an in vitro assay for neutralizing antibody measurement based on the pseudoviral system; results generated by the in vitro assay were found to be strongly correlated with those by either hemagglutination inhibition (HI) or micro-neutralization (MN) assay. Furthermore, we injected the viruses into Balb/c mice and observed dynamic distributions of the viruses in the animals, which provides an ideal imaging model for quantitative analyses of prophylactic and therapeutic monoclonal antibodies. Taken together, the pseudoviral systems reported here could be of great value for both in vitro and in vivo evaluations of vaccines and antiviral agents without the need of wild type H7N9 virus.

Cell type of origin as well as genetic alterations contribute to breast cancer phenotypes

PubMed Central

West, William W.; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

Breast cancer is classified into different subtypes that are associated with different patient survival outcomes, underscoring the importance of understanding the role of precursor cell and genetic alterations in determining tumor subtypes. In this study, we evaluated the oncogenic phenotype of two distinct mammary stem/progenitor cell types designated as K5+/K19− or K5+/K19+ upon introduction of identical combinations of oncogenes-mutant H-Ras (mRas) and mutant p53 (mp53), together with either wild-type ErbB2(wtErbB2) or wild-type EGFR (wtEGFR). We examined their tumor forming and metastasis potential, using both in-vitro and in-vivo assays. Both the combinations efficiently transformed K5+/K19− or K5+/K19+ cells. Xenograft tumors formed by these cells were histologically heterogeneous, with variable proportions of luminal, basal-like and claudin-low type components depending on the cell types and oncogene combinations. Notably, K5+/K19− cells transformed with mRas/mp53/wtEGFR combination had a significantly longer latency for primary tumor development than other cell lines but more lung metastasis incidence than same cells expressing mRas/mp53/wtErbB2. K5+/K19+ cells exhibit shorter overall tumor latency, and high metastatic potential than K5+/K19− cells, suggesting that these K19+ progenitors are more susceptible to oncogenesis and metastasis. Our results suggest that both genetic alterations and cell type of origin contribute to oncogenic phenotype of breast tumors. PMID:25940703

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

PubMed

Holmes, Tarquin

2017-06-01

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

Type I interferon regulates pDC maturation and Ly49Q expression.

PubMed

Toma-Hirano, Makiko; Namiki, Sahori; Miyatake, Shoichiro; Arai, Ken-Ichi; Kamogawa-Schifter, Yumiko

2007-10-01

Ly49Q is expressed on peripheral mouse plasmacytoid dendritic cells (pDC). Immature Ly49Q-negative pDC precursors acquire Ly49Q in the bone marrow and then migrate into the periphery. While searching for molecules that regulate pDC maturation, we found that type I interferon (IFN) inhibited Ly49Q acquisition in vitro. Infections that induce type I IFN production by cells other than pDC (a condition mimicked by poly(I:C) injection in vivo) increase the prevalence of Ly49Q(-) pDC in the bone marrow and peripheral lymphoid organs in wild-type but not IFN-alpha/beta receptor knockout BALB/c mice. Moreover, in vivo exposure to type I IFN causes some Ly49Q(-), but not Ly49Q(+), pDC to convert to conventional DC, defined as B220(-) CD11c(+) CD11b(+) cells. These data suggest that type I IFN regulates pDC development and affects their distribution in the body.

A Dentin Sialophosphoprotein Mutation That Partially Disrupts a Splice Acceptor Site Causes Type II Dentin Dysplasia

PubMed Central

Lee, Sook-Kyung; Hu, Jan C.-C.; Lee, Kyung-Eun; Simmer, James P.; Kim, Jung-Wook

2009-01-01

The dentin sialophosphoprotein (DSPP) gene on chromosome 4q21.3 encodes the major noncollagenous protein in tooth dentin. DSPP mutations are the principal cause of dentin dysplasia type II, dentinogenesis imperfecta type II, and dentinogenesis imperfecta type III. We have identified a DSPP splice junction mutation (IVS2-6T>G) in a family with dentin dysplasia type II. The primary dentition is discolored brown with severe attrition. The mildly discolored permanent dentition has thistle-shaped pulp chambers, pulp stones, and eventual pulp obliteration. The mutation is in the sixth nucleotide from the end of intron 2, perfectly segregates with the disease phenotype, and is absent in 200 normal control chromosomes. An in vitro splicing assay shows that pre-mRNA splicing of the mutant allele generates wild-type mRNA and mRNA lacking exon 3 in approximately equal amounts. Skipping exon 3 might interfere with signal peptide cleavage, causing endoplasmic reticulum stress, and also reduce DSPP secretion, leading to haploinsufficiency. PMID:19026876

Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease.

PubMed

Laprairie, Robert B; Bagher, Amina M; Kelly, Melanie E M; Denovan-Wright, Eileen M

2016-03-01

Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

A Herpes Simplex Virus Type 1 Mutant Expressing a Baculovirus Inhibitor of Apoptosis Gene in Place of Latency-Associated Transcript Has a Wild-Type Reactivation Phenotype in the Mouse

PubMed Central

Jin, Ling; Perng, Guey-Chuen; Mott, Kevin R.; Osorio, Nelson; Naito, Julia; Brick, David J.; Carpenter, Dale; Jones, Clinton; Wechsler, Steven L.

2005-01-01

The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT− mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT− mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT− mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse. PMID:16160155

Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse

PubMed Central

Hossain-Ibrahim, Mohammed K; Rezajooi, Kia; Stallcup, William B; Lieberman, Alexander R; Anderson, Patrick N

2007-01-01

Background The chondroitin sulphate proteoglycan NG2 blocks neurite outgrowth in vitro and has been proposed as a major inhibitor of axonal regeneration in the CNS. Although a substantial body of evidence underpins this hypothesis, it is challenged by recent findings including strong expression of NG2 in regenerating peripheral nerve. Results We studied axonal regeneration in the PNS and CNS of genetically engineered mice that do not express NG2, and in sex and age matched wild-type controls. In the CNS, we used anterograde tracing with BDA to study corticospinal tract (CST) axons after spinal cord injury and transganglionic labelling with CT-HRP to trace ascending sensory dorsal column (DC) axons after DC lesions and a conditioning lesion of the sciatic nerve. Injury to these fibre tracts resulted in no difference between knockout and wild-type mice in the ability of CST axons or DC axons to enter or cross the lesion site. Similarly, after dorsal root injury (with conditioning lesion), most regenerating dorsal root axons failed to grow across the dorsal root entry zone in both transgenic and wild-type mice. Following sciatic nerve injuries, functional recovery was assessed by analysis of the toe-spreading reflex and cutaneous sensitivity to Von Frey hairs. Anatomical correlates of regeneration were assessed by: retrograde labelling of regenerating dorsal root ganglion (DRG) cells with DiAsp; immunostaining with PGP 9.5 to visualise sensory reinnervation of plantar hindpaws; electron microscopic analysis of regenerating axons in tibial and digital nerves; and by silver-cholinesterase histochemical study of motor end plate reinnervation. We also examined functional and anatomical correlates of regeneration after injury of the facial nerve by assessing the time taken for whisker movements and corneal reflexes to recover and by retrograde labelling of regenerated axons with Fluorogold and DiAsp. None of the anatomical or functional analyses revealed significant differences between wild-type and knockout mice. Conclusion These findings show that NG2 is unlikely to be a major inhibitor of axonal regeneration after injury to the CNS, and, further, that NG2 is unlikely to be necessary for regeneration or functional recovery following peripheral nerve injury. PMID:17900358

Cable Pili and the Associated 22 Kda Adhesin Contribute to Burkholderia Cenocepacia Persistence In Vivo

PubMed Central

Goldberg, Joanna B.; Ganesan, Shyamala; Comstock, Adam T.; Zhao, Ying; Sajjan, Uma S.

2011-01-01

Background Infection by Burkholderia cenocepacia in cystic fibrosis (CF) patients is associated with poor clinical prognosis. Previously, we demonstrated that one of the highly transmissible strains, BC7, expresses cable pili and the associated 22 kDa adhesin, both of which contribute to BC7 binding to airway epithelial cells. However, the contribution of these factors to induce inflammation and bacterial persistence in vivo is not known. Methodology/Principal Findings Wild-type BC7 stimulated higher IL-8 responses than the BC7 cbl and BC7 adhA mutants in both CF and normal bronchial epithelial cells. To determine the role of cable pili and the associated adhesin, we characterized a mouse model of B. cenocepacia, where BC7 are suspended in Pseudomonas aeruginosa alginate. C57BL/6 mice were infected intratracheally with wild-type BC7 suspended in either alginate or PBS and were monitored for lung bacterial load and inflammation. Mice infected with BC7 suspended in PBS completely cleared the bacteria by 3 days and resolved the inflammation. In contrast, mice infected with BC7 suspended in alginate showed persistence of bacteria and moderate lung inflammation up to 5 days post-infection. Using this model, mice infected with the BC7 cbl and BC7 adhA mutants showed lower bacterial loads and mild inflammation compared to mice infected with wild-type BC7. Complementation of the BC7 cblS mutation in trans restored the capacity of this strain to persist in vivo. Immunolocalization of bacteria revealed wild-type BC7 in both airway lumen and alveoli, while the BC7 cbl and BC7 adhA mutants were found mainly in airway lumen and peribronchiolar region. Conclusions and Significance B. cenocepacia suspended in alginate can be used to determine the capacity of bacteria to persist and cause lung inflammation in normal mice. Both cable pili and adhesin contribute to BC7-stimulated IL-8 response in vitro, and BC7 persistence and resultant inflammation in vivo. PMID:21811611

Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

PubMed

Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

2016-09-01

Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Can we induce spermatogenesis in the domestic cat using an in vitro tissue culture approach?

PubMed Central

Amaral, Sandra; Tavares, Renata S.; Schlatt, Stefan; Ramalho-Santos, João

2018-01-01

The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia regardless of the initial stage of tubule development in the donor tissue. Moreover, the number of spermatogonia decreased with time in culture in all media tested, especially in the medium supplemented with KSR, while AlbuMax had a slight protective effect. The combination of AlbuMax and beta-estradiol led to an increase in the area occupied by seminiferous tubules, and thus to an increase in total number of spermatogonial cells. Considering all the media combinations tested the stimulus for felid germ cell differentiation in this type of system seems to be different from the mouse. Studies using other triggers of differentiation and tissue survival factors should be performed to pursue this technology for the genetic diversity preservation in wild felids. PMID:29414992

Products of Dark CO2 Fixation in Pea Root Nodules Support Bacteroid Metabolism 1

PubMed Central

Rosendahl, Lis; Vance, Carroll P.; Pedersen, Walther B.

1990-01-01

Products of the nodule cytosol in vivo dark [14C]CO2 fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO2 fixation products was compared in effective (fix+) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix−) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The 14C incorporation from [14C]CO2 was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the 14C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining 14C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the 14C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive 14C labeling of malate and aspartate from nodule dark [14C]CO2 fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO2 fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more 14C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated 14C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained 14C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid 14C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of 14C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO2 fixation in the supply of the bacteroids with dicarboxylic acids. PMID:16667422

ABO3, a WRKY transcription factor, mediates plant responses to abscisic acid and drought tolerance in Arabidopsis.

PubMed

Ren, Xiaozhi; Chen, Zhizhong; Liu, Yue; Zhang, Hairong; Zhang, Min; Liu, Qian; Hong, Xuhui; Zhu, Jian-Kang; Gong, Zhizhong

2010-08-01

The biological functions of WRKY transcription factors in plants have been widely studied, but their roles in abiotic stress are still not well understood. We isolated an ABA overly sensitive mutant, abo3, which is disrupted by a T-DNA insertion in At1g66600 encoding a WRKY transcription factor AtWRKY63. The mutant was hypersensitive to ABA in both seedling establishment and seedling growth. However, stomatal closure was less sensitive to ABA, and the abo3 mutant was less drought tolerant than the wild type. Northern blot analysis indicated that the expression of the ABA-responsive transcription factor ABF2/AREB1 was markedly lower in the abo3 mutant than in the wild type. The abo3 mutation also reduced the expression of stress-inducible genes RD29A and COR47, especially early during ABA treatment. ABO3 is able to bind the W-box in the promoter of ABF2in vitro. These results uncover an important role for a WRKY transcription factor in plant responses to ABA and drought stress. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

Interferon-alpha/beta deficiency greatly exacerbates arthritogenic disease in mice infected with wild-type chikungunya virus but not with the cell culture-adapted live-attenuated 181/25 vaccine candidate

PubMed Central

Gardner, Christina L.; Burke, Crystal W.; Higgs, Stephen T.; Klimstra, William B.; Ryman, Kate D.

2012-01-01

In humans, chikungunya virus (CHIKV) infection causes fever, rash, and acute and persisting polyarthalgia/arthritis associated with joint swelling. We report a new CHIKV disease model in adult mice that distinguishes the wild-type CHIKV-LR strain from the live-attenuated vaccine strain (CHIKV-181/25). Although eight-week old normal mice inoculated in the hind footpad developed no hind limb swelling with either virus, CHIKV-LR replicated in musculoskeletal tissues and caused detectable inflammation. In mice deficient in STAT1-dependent interferon (IFN) responses, CHIKV-LR caused significant swelling of the inoculated and contralateral limbs and dramatic inflammatory lesions, while CHIKV-181/25 vaccine and another arthritogenic alphavirus, Sindbis, failed to induce swelling. IFN responses suppressed CHIKV-LR and CHIKV-181/25 replication equally in dendritic cells in vitro whereas macrophages were refractory to infection independently of STAT1-mediated IFN responses. Glycosaminoglycan (GAG) binding may be a CHIKV vaccine attenuation mechanism as CHIKV-LR infectivity was not dependent upon GAG, while CHIKV-181/25 was highly dependent. PMID:22305131

Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution.

PubMed

Schlinkmann, Karola M; Hillenbrand, Matthias; Rittner, Alexander; Künz, Madeleine; Strohner, Ralf; Plückthun, Andreas

2012-09-21

To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution. Copyright © 2012 Elsevier Ltd. All rights reserved.

Diminished but Not Abolished Effect of Two His351 Mutants of Anthrax Edema Factor in a Murine Model

PubMed Central

Zhao, Taoran; Zhao, Xinghui; Liu, Ju; Meng, Yingying; Feng, Yingying; Fang, Ting; Zhang, Jinlong; Yang, Xiuxu; Li, Jianmin; Xu, Junjie; Chen, Wei

2016-01-01

Edema toxin (ET), which is composed of a potent adenylate cyclase (AC), edema factor (EF), and protective antigen (PA), is one of the major toxicity factors of Bacillus anthracis. In this study, we introduced mutations in full-length EF to generate alanine EF(H351A) and arginine EF(H351R) variants. In vitro activity analysis displayed that the adenylyl cyclase activity of both the mutants was significantly diminished compared with the wild-type EF. When the native and mutant toxins were administered subcutaneously in a mouse footpad edema model, severe acute swelling was evoked by wild-type ET, while the symptoms induced by mutant toxins were very minor. Systemic administration of these EF variants caused non-lethal hepatotoxicity. In addition, EF(H351R) exhibited slightly higher activity in causing more severe edema than EF(H351A). Our findings demonstrate that the toxicity of ET is not abolished by substitution of EF residue His351 by alanine or arginine. These results also indicate the potential of the mouse footpad edema model as a sensitive method for evaluating both ET toxicity and the efficacy of candidate therapeutic agents. PMID:26848687

Cardiomyocyte mitochondrial respiration is reduced by receptor for advanced glycation end-product signaling in a ceramide-dependent manner.

PubMed

Nelson, Michael B; Swensen, Adam C; Winden, Duane R; Bodine, Jared S; Bikman, Benjamin T; Reynolds, Paul R

2015-07-01

Cigarette smoke exposure is associated with an increased risk of cardiovascular complications. The role of advanced glycation end products (AGEs) is already well established in numerous comorbidities, including cardiomyopathy. Given the role of AGEs and their receptor, RAGE, in activating inflammatory pathways, we sought to determine whether ceramides could be a mediator of RAGE-induced altered heart mitochondrial function. Using an in vitro model, we treated H9C2 cardiomyocytes with the AGE carboxy-methyllysine before mitochondrial respiration assessment. We discovered that mitochondrial respiration was significantly impaired in AGE-treated cells, but not when cotreated with myriocin, an inhibitor of de novo ceramide biosynthesis. Moreover, we exposed wild-type and RAGE knockout mice to secondhand cigarette smoke and found reduced mitochondrial respiration in the left ventricular myocardium from wild-type mice, but RAGE knockout mice were protected from this effect. Finally, conditional overexpression of RAGE in the lungs of transgenic mice elicited a robust increase in left ventricular ceramides in the absence of smoke exposure. Taken together, these findings suggest a RAGE-ceramide axis as an important contributor to AGE-mediated disrupted cardiomyocyte mitochondrial function. Copyright © 2015 the American Physiological Society.

Arabidopsis shaker pollen inward K+ channel SPIK functions in SnRK1 complex-regulated pollen hydration on the stigma.

PubMed

Li, Dan-Dan; Guan, Huan; Li, Fei; Liu, Chang-Zhen; Dong, Yu-Xiu; Zhang, Xian-Sheng; Gao, Xin-Qi

2017-09-01

Pollen hydration is a critical step that determines pollen germination on the stigma. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex (SnRK1 complex). In pollen of the Arabidopsis kinβγ mutant, the levels of reactive oxygen species were decreased which lead to compromised hydration of the mutant pollen on the stigma. In this study, we analyzed gene expression in kinβγ mutant pollen by RNA-seq and found the expression of inward shaker K + channel SPIK was down-regulated in the kinβγ pollen. Furthermore, we showed that the pollen hydration of the Arabidopsis spik mutant was defective on the wild-type stigma, although the mutant pollen demonstrated normal hydration in vitro. Additionally, the defective hydration of spik mutant pollen could not be rescued by the wild-type pollen on the stigma, indicating that the spik mutation deprived the capability of pollen absorption on the stigma. Our results suggest that the Arabidopsis SnRK1 complex regulates SPIK expression, which functions in determining pollen hydration on the stigma. © 2017 Institute of Botany, Chinese Academy of Sciences.

Activation of Intestinal Human Pregnane X Receptor Protects against Azoxymethane/Dextran Sulfate Sodium–Induced Colon Cancer

PubMed Central

Cheng, Jie; Fang, Zhong-Ze; Nagaoka, Kenjiro; Okamoto, Minoru; Qu, Aijuan; Tanaka, Naoki; Kimura, Shioko

2014-01-01

The role of intestinal human pregnane X receptor (PXR) in colon cancer was determined through investigation of the chemopreventive role of rifaximin, a specific agonist of intestinal human PXR, toward azoxymethane (AOM)/dextran sulfate sodium (DSS)–induced colon cancer. Rifaximin treatment significantly decreased the number of colon tumors induced by AOM/DSS treatment in PXR-humanized mice, but not wild-type or Pxr-null mice. Additionally, rifaximin treatment markedly increased the survival rate of PXR-humanized mice, but not wild-type or Pxr-null mice. These data indicated a human PXR–dependent therapeutic chemoprevention of rifaximin toward AOM/DSS-induced colon cancer. Nuclear factor κ-light-chain-enhancer of activated B cells–mediated inflammatory signaling was upregulated in AOM/DSS-treated mice, and inhibited by rifaximin in PXR-humanized mice. Cell proliferation and apoptosis were also modulated by rifaximin treatment in the AOM/DSS model. In vitro cell-based assays further revealed that rifaximin regulated cell apoptosis and cell cycle in a human PXR-dependent manner. These results suggested that specific activation of intestinal human PXR exhibited a chemopreventive role toward AOM/DSS-induced colon cancer by mediating anti-inflammation, antiproliferation, and proapoptotic events. PMID:25277138

The non-essential UL50 gene of avian infectious laryngotracheitis virus encodes a functional dUTPase which is not a virulence factor.

PubMed

Fuchs, W; Ziemann, K; Teifke, J P; Werner, O; Mettenleiter, T C

2000-03-01

The DNA sequence of the infectious laryngotracheitis virus (ILTV) UL50, UL51 and UL52 gene homologues was determined. Although the deduced UL50 protein lacks the first of five conserved domains of the corresponding proteins of mammalian alphaherpesviruses, the ILTV gene product was also shown to possess dUTPase activity. The generation of UL50-negative ILTV mutants was facilitated by recombination plasmids encoding green fluorescent protein (GFP), and expression constructs of predicted transactivator proteins of ILTV (alphaTIF, ICP4) were successfully used to increase the infectivity of viral genomic DNA. A GFP-expressing UL50-deletion mutant of ILTV showed reduced cell-to-cell spread in vitro, and was attenuated in vivo. A similar deletion mutant without the foreign gene, however, propagated like wild-type ILTV in cell culture and was pathogenic in chickens. We conclude that the viral dUTPase is not required for efficient replication of ILTV in the respiratory tract of infected animals. The replication defect of the GFP-expressing ILTV recombinant is most likely caused by toxic effects of the reporter gene product, since spontaneously occurring inactivation mutants exhibited wild-type-like growth.

Exploiting Drug-Resistant Enzymes as Tools to Identify Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

PubMed Central

Masaoka, Takashi; Chung, Suhman; Caboni, Pierluigi; Rausch, Jason W.; Wilson, Jennifer A.; Taskent-Sezgin, Humeyra; Beutler, John A.; Tocco, Graziella; Le Grice, Stuart F. J.

2013-01-01

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally-designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3′,4′-dihydroxyphenyl (catechol)-substituted thienopyrimidinones with sub-micromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize the enzyme, in some instances reducing the Tm by 5°C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthens the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy. PMID:23631411

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model

PubMed Central

Szentpáli-Gavallér, Katalin; Lim, Stephanie M.; Dencső, László; Bányai, Krisztián; Koraka, Penelope; Osterhaus, Albert D.M.E.; Martina, Byron E.E.; Bakonyi, Tamás; Bálint, Ádám

2016-01-01

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA. PMID:26907325

T-Lymphocytes Enable Osteoblast Maturation via IL-17F during the Early Phase of Fracture Repair

PubMed Central

Nam, Diane; Mau, Elaine; Wang, Yufa; Wright, David; Silkstone, David; Whetstone, Heather; Whyne, Cari; Alman, Benjamin

2012-01-01

While it is well known that the presence of lymphocytes and cytokines are important for fracture healing, the exact role of the various cytokines expressed by cells of the immune system on osteoblast biology remains unclear. To study the role of inflammatory cytokines in fracture repair, we studied tibial bone healing in wild-type and Rag1−/− mice. Histological analysis, µCT stereology, biomechanical testing, calcein staining and quantitative RNA gene expression studies were performed on healing tibial fractures. These data provide support for Rag1−/− mice as a model of impaired fracture healing compared to wild-type. Moreover, the pro-inflammatory cytokine, IL-17F, was found to be a key mediator in the cellular response of the immune system in osteogenesis. In vitro studies showed that IL-17F alone stimulated osteoblast maturation. We propose a model in which the Th17 subset of T-lymphocytes produces IL-17F to stimulate bone healing. This is a pivotal link in advancing our current understanding of the molecular and cellular basis of fracture healing, which in turn may aid in optimizing fracture management and in the treatment of impaired bone healing. PMID:22768215

Co-precipitation of phosphate and iron limits mitochondrial phosphate availability in Saccharomyces cerevisiae lacking the yeast frataxin homologue (YFH1).

PubMed

Seguin, Alexandra; Santos, Renata; Pain, Debkumar; Dancis, Andrew; Camadro, Jean-Michel; Lesuisse, Emmanuel

2011-02-25

Saccharomyces cerevisiae cells lacking the yeast frataxin homologue (Δyfh1) accumulate iron in the mitochondria in the form of nanoparticles of ferric phosphate. The phosphate content of Δyfh1 mitochondria was higher than that of wild-type mitochondria, but the proportion of mitochondrial phosphate that was soluble was much lower in Δyfh1 cells. The rates of phosphate and iron uptake in vitro by isolated mitochondria were higher for Δyfh1 than wild-type mitochondria, and a significant proportion of the phosphate and iron rapidly became insoluble in the mitochondrial matrix, suggesting co-precipitation of these species after oxidation of iron by oxygen. Increasing the amount of phosphate in the medium decreased the amount of iron accumulated by Δyfh1 cells and improved their growth in an iron-dependent manner, and this effect was mostly transcriptional. Overexpressing the major mitochondrial phosphate carrier, MIR1, slightly increased the concentration of soluble mitochondrial phosphate and significantly improved various mitochondrial functions (cytochromes, [Fe-S] clusters, and respiration) in Δyfh1 cells. We conclude that in Δyfh1 cells, soluble phosphate is limiting, due to its co-precipitation with iron.

Novel role of transient receptor potential vanilloid 2 in the regulation of cardiac performance

PubMed Central

Lasko, Valerie M.; Koch, Sheryl E.; Singh, Vivek P.; Carreira, Vinicius; Robbins, Nathan; Patel, Amit R.; Jiang, Min; Bidwell, Philip; Kranias, Evangelia G.; Jones, W. Keith; Lorenz, John N.

2013-01-01

Transient receptor potential cation channels have been implicated in the regulation of cardiovascular function, but only recently has our laboratory described the vanilloid-2 subtype (TRPV2) in the cardiomyocyte, though its exact mechanism of action has not yet been established. This study tests the hypothesis that TRPV2 plays an important role in regulating myocyte contractility under physiological conditions. Therefore, we measured cardiac and vascular function in wild-type and TRPV2−/− mice in vitro and in vivo and found that TRPV2 deletion resulted in a decrease in basal systolic and diastolic function without affecting loading conditions or vascular tone. TRPV2 stimulation with probenecid, a relatively selective TRPV2 agonist, caused an increase in both inotropy and lusitropy in wild-type mice that was blunted in TRPV2−/− mice. We examined the mechanism of TRPV2 inotropy/lusitropy in isolated myocytes and found that it modulates Ca2+ transients and sarcoplasmic reticulum Ca2+ loading. We show that the activity of this channel is necessary for normal cardiac function and that there is increased contractility in response to agonism of TRPV2 with probenecid. PMID:24322617

Superior anti-tumor activity of the MDM2 antagonist idasanutlin and the Bcl-2 inhibitor venetoclax in p53 wild-type acute myeloid leukemia models.

PubMed

Lehmann, Christian; Friess, Thomas; Birzele, Fabian; Kiialainen, Anna; Dangl, Markus

2016-06-28

Venetoclax, a small molecule BH3 mimetic which inhibits the anti-apoptotic protein Bcl-2, and idasanutlin, a selective MDM2 antagonist, have both shown activity as single-agent treatments in pre-clinical and clinical studies in acute myeloid leukemia (AML). In this study, we deliver the rationale and molecular basis for the combination of idasanutlin and venetoclax for treatment of p53 wild-type AML. The effect of idasanutlin and venetoclax combination on cell viability, apoptosis, and cell cycle progression was investigated in vitro using established AML cell lines. In vivo efficacy was demonstrated in subcutaneous and orthotopic xenograft models generated in female nude or non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Mode-of-action analyses were performed by means of cell cycle kinetic studies, RNA sequencing as well as western blotting experiments. Combination treatment with venetoclax and idasanutlin results in synergistic anti-tumor activity compared with the respective single-agent treatments in vitro, in p53 wild-type AML cell lines, and leads to strongly superior efficacy in vivo, in subcutaneous and orthotopic AML models. The inhibitory effects of idasanutlin were cell-cycle dependent, with cells arresting in G1 in consecutive cycles and the induction of apoptosis only evident after cells had gone through at least two cell cycles. Combination treatment with venetoclax removed this dependency, resulting in an acceleration of cell death kinetics. As expected, gene expression studies using RNA sequencing showed significant alterations to pathways associated with p53 signaling and cell cycle arrest (CCND1 pathway) in response to idasanutlin treatment. Only few gene expression changes were observed for venetoclax treatment and combination treatment, indicating that their effects are mediated mainly at the post-transcriptional level. Protein expression studies demonstrated that inhibition of the anti-apoptotic protein Mcl-1 contributed to the activity of venetoclax and idasanutlin, with earlier inhibition of Mcl-1 in response to combination treatment contributing to the superior combined activity. The role of Mcl-1 was confirmed by small hairpin RNA gene knockdown studies. Our findings provide functional and molecular insight on the superior anti-tumor activity of combined idasanutlin and venetoclax treatment in AML and support its further exploration in clinical studies.

The role and relevance of phospholipase D1 during growth and dimorphism of Candida albicans.

PubMed

Hube, B; Hess, D; Baker, C A; Schaller, M; Schäfer, W; Dolan, J W

2001-04-01

The phosphatidylcholine-specific phospholipase D1 (PLD1) in Saccharomyces cerevisiae is involved in vesicle transport and is essential for sporulation. The gene encoding the homologous phospholipase D1 from Candida albicans (PLD1) was used to study the role of PLD1 in this pathogenic fungus. In vitro and in vivo expression studies using Northern blots and reverse transcriptase-PCR showed low PLD1 mRNA levels in defined media supporting yeast growth and during experimental infection, while enhanced levels of PLD1 transcripts were detected during the yeast to hyphal transition. To study the relevance of PLD1 during yeast and hyphal growth, an essential part of the gene was deleted in both alleles of two isogenic strains. In vitro PLD1 activity assays showed that pld1 mutants produced no detectable levels of phosphatidic acid, the hydrolytic product of PLD1 activity, and strongly reduced levels of diacylglycerol, the product of lipid phosphate phosphohydrolase, suggesting no or a negligible background PLD1 activity in the pld1 mutants. The pld1 mutants showed no growth differences compared to the parental wild-type in liquid complex and minimal media, independent of the growth temperature. In addition, growth rates of pld1 mutants in media with protein as the sole source of nitrogen were similar to growth rates of the wild-type, indicating that secretion of proteinases was not reduced. Chlamydospore formation was normal in pld1 mutants. When germ tube formation was induced in liquid media, pld1 mutants showed similar rates of yeast to hyphal transition compared to the wild-type. However, no hyphae formation was observed on solid Spider medium, and cell growth on cornmeal/Tween 80 medium indicated aberrant morphogenesis. In addition, pld1 mutants growing on solid media had an attenuated ability to invade the agar. In a model of oral candidosis, pld1 mutants showed no attenuation of virulence. In contrast, the mutant was less virulent in two different mouse models. These data suggest that PLD1 is not essential for growth and oral infections. However, they also suggest that a prominent part of the phosphatidic acid and diacylglycerol pools is produced by PLD1 and that the level of these components is important for morphological transitions under certain conditions in C. albicans.

Mutant Huntingtin Impairs Axonal Trafficking in Mammalian Neurons In Vivo and In Vitro

PubMed Central

Trushina, Eugenia; Dyer, Roy B.; Badger, John D.; Ure, Daren; Eide, Lars; Tran, David D.; Vrieze, Brent T.; Legendre-Guillemin, Valerie; McPherson, Peter S.; Mandavilli, Bhaskar S.; Van Houten, Bennett; Zeitlin, Scott; McNiven, Mark; Aebersold, Ruedi; Hayden, Michael; Parisi, Joseph E.; Seeberg, Erling; Dragatsis, Ioannis; Doyle, Kelly; Bender, Anna; Chacko, Celin; McMurray, Cynthia T.

2004-01-01

Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntington's disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction. PMID:15340079

Cryopreservation for preservation of potato genetic resources

PubMed Central

Niino, Takao; Arizaga, Miriam Valle

2015-01-01

Cryopreservation is becoming a very important tool for the long-term storage of plant genetic resources and efficient cryopreservation protocols have been developed for a large number of plant species. Practical procedures, developed using in vitro tissue culture, can be a simple and reliable preservation option of potato genetic resources rather than maintaining by vegetative propagation in genebanks due their allogamous nature. Cryopreserved materials insure a long-term backup of field collections against loss of plant germplasm. Occurrence of genetic variation, in tissue culture cells during prolonged subcultures, can be avoided with suitable cryopreservation protocols that provide high regrowth, leading and facilitating a systematic and strategic cryo-banking of plant genetic resources. Cryopreservation protocols for potato reviewed here, can efficiently complement field and in vitro conservation, providing for preservation of genotypes difficult to preserve by other methods, wild types and other species decided as priority collections. PMID:25931979

The fish pathogen Yersinia ruckeri produces holomycin and uses an RNA methyltransferase for self-resistance.

PubMed

Qin, Zhiwei; Baker, Alexander Thomas; Raab, Andrea; Huang, Sheng; Wang, Tiehui; Yu, Yi; Jaspars, Marcel; Secombes, Christopher J; Deng, Hai

2013-05-24

Holomycin and its derivatives belong to a class of broad-spectrum antibacterial natural products containing a rare dithiolopyrrolone heterobicyclic scaffold. The antibacterial mechanism of dithiolopyrrolone compounds has been attributed to the inhibition of bacterial RNA polymerase activities, although the exact mode of action has not been established in vitro. Some dithiopyrrolone derivatives display potent anticancer activities. Recently the biosynthetic gene cluster of holomycin has been identified and characterized in Streptomyces clavuligerus. Here we report that the fish pathogen Yersinia ruckeri is a holomycin producer, as evidenced through genome mining, chemical isolation, and structural elucidation as well as genetic manipulation. We also identified a unique regulatory gene hom15 at one end of the gene cluster encoding a cold-shock-like protein that likely regulates the production of holomycin in low cultivation temperatures. Inactivation of hom15 resulted in a significant loss of holomycin production. Finally, gene disruption of an RNA methyltransferase gene hom12 resulted in the sensitivity of the mutant toward holomycin. A complementation experiment of hom12 restored the resistance against holomycin. Although the wild-type Escherichia coli BL21(DE3) Gold is susceptible to holomycin, the mutant harboring hom12 showed tolerance toward holomycin. High resolution liquid chromatography (LC)-ESI/MS analysis of digested RNA fragments demonstrated that the wild-type Y. ruckeri and E. coli harboring hom12 contain a methylated RNA fragment, whereas the mutated Y. ruckeri and the wild-type E. coli only contain normal non-methylated RNA fragments. Taken together, our results strongly suggest that this putative RNA methyltransferase Hom12 is the self-resistance protein that methylates the RNA of Y. ruckeri to reduce the cytotoxic effect of holomycin during holomycin production.

Lon protease affects the RdxA nitroreductase activity and metronidazole susceptibility in Helicobacter pylori.

PubMed

Tu, I-Fan; Liao, Jiahn-Haur; Yang, Feng-Ling; Lin, Nien-Tsung; Chan, Hong-Lin; Wu, Shih-Hsiung

2014-10-01

The lon gene of Helicobacter pylori strains is constitutively expressed during growth. However, virtually nothing is understood concerning the role of Lon in H. pylori. This study examined the function and physiological role of Lon in H. pylori (HpLon) using a trapping approach to identify putative Lon binding partners in the bacterium. Protease-deficient Lon was expressed and served as the bait in trapping approach to capture the interacting partners in H. pylori. The antibiotic susceptibility of wild-type and lon derivative mutants was determined by the E test trips and the disc diffusion assay. The effect of HpLon on RdxA activity was detected the change in NADPH oxidation and metronidazole reduction by spectrophotometer. Lon in Helicobacter pylori (HpLon) interacting partners are mostly associated with metronidazole activation. lon mutant presents more susceptible to metronidazole than that of the wild type, and this phenotype is recovered by complementation of the wild-type Lon. We found that the ATPases associated with a variety of cellular activities (AAA(+) ) module of HpLon causes a decrease in both NADPH oxidase and Mtz reductase activity in RdxA, a major Mtz-activating enzyme in H. pylori. Metronidazole resistance of H. pylori causes the serious medical problem worldwide. In this study, HpLon is involved in metronidazole susceptibility among H. pylori strains. We provide the evidence that HpLon alters RdxA activity in vitro. The decrease in metronidazole activation caused by HpLon is possibly prior to accumulate mutation in rdxA gene before the metronidazole-resistant strains to be occurred. © 2014 John Wiley & Sons Ltd.

mlh3 mutations in baker’s yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide

PubMed Central

Al-Sweel, Najla; Raghavan, Vandana; Khondakar, Nabila; Manhart, Carol M.; Surtees, Jennifer A.

2017-01-01

Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated. PMID:28827832

Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana.

PubMed

Stührwohldt, Nils; Dahlke, Renate I; Kutschmar, Anke; Peng, Xiongbo; Sun, Meng-Xiang; Sauter, Margret

2015-04-01

Phytosulfokine (PSK) is a peptide growth factor that requires tyrosine sulfation carried out by tyrosylprotein sulfotransferase (TPST) for its activity. PSK is processed from precursor proteins encoded by five genes in Arabidopsis thaliana and perceived by receptor kinases encoded by two genes in Arabidopsis. pskr1-3 pskr2-1 and tpst-1 knockout mutants displayed reduced seed production, indicative of a requirement for PSK peptide signaling in sexual plant reproduction. Expression analysis revealed PSK precursor and PSK receptor gene activity in reproductive organs with strong expression of PSK2 in pollen. In support of a role for PSK signaling in pollen, in vitro pollen tube (PT) growth was enhanced by exogenously added PSK while PTs of pskr1-3 pskr2-1 and of tpst-1 were shorter. In planta, growth of wild-type pollen in pskr1-3 pskr2-1 and tpst-1 flowers appeared slower than growth in wild-type flowers. But PTs did eventually reach the base of the style, suggesting that PT elongation rate may not be responsible for the reduced fertility. Detailed analysis of anthers, style and ovules did not reveal obvious developmental defects. By contrast, a high percentage of unfertilized ovules in pskr1-3 pskr2-1 and in tpst-1 siliques displayed loss of funicular PT guidance, suggesting that PSK signaling is required to guide the PT from the transmitting tract to the embryo sac. Cross-pollination experiments with wild-type, pskr1-3 pskr2-1 and tpst-1 male and female parents revealed that both the PT and the female sporophytic tissue and/or female gametophyte contribute to successful PT guidance via PSK signaling and to fertilization success. © 2014 Scandinavian Plant Physiology Society.

Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization

PubMed Central

Haq, Imran; Saleh, Aarash D.; Dron, Louis; Regan-Mochrie, Gemma L.; Motamedi-Shad, Neda; Hurst, John R.; Gooptu, Bibek

2016-01-01

Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate—and therefore polymerize—more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the “breach” region and “shutter” region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care. PMID:26091018

Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization.

PubMed

Haq, Imran; Irving, James A; Saleh, Aarash D; Dron, Louis; Regan-Mochrie, Gemma L; Motamedi-Shad, Neda; Hurst, John R; Gooptu, Bibek; Lomas, David A

2016-01-01

Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.

Absence of fibroblast growth factor 2 promotes oligodendroglial repopulation of demyelinated white matter.

PubMed

Armstrong, Regina C; Le, Tuan Q; Frost, Emma E; Borke, Rosemary C; Vana, Adam C

2002-10-01

This study takes advantage of fibroblast growth factor 2 (FGF2) knock-out mice to determine the contribution of FGF2 to the regeneration of oligodendrocytes in the adult CNS. The role of FGF2 during spontaneous remyelination was examined using two complementary mouse models of experimental demyelination. The murine hepatitis virus strain A59 (MHV-A59) model produces focal areas of spinal cord demyelination with inflammation. The cuprizone neurotoxicant model causes extensive corpus callosum demyelination without a lymphocytic cell response. In both models, FGF2 expression is upregulated in areas of demyelination in wild-type mice. Surprisingly, in both models, oligodendrocyte repopulation of demyelinated white matter was significantly increased in FGF2 -/- mice compared with wild-type mice and even surpassed the oligodendrocyte density of nonlesioned mice. This dramatic result indicated that the absence of FGF2 promoted oligodendrocyte regeneration, possibly by enhancing oligodendrocyte progenitor proliferation and/or differentiation. FGF2 -/- and +/+ mice had similar oligodendrocyte progenitor densities in normal adult CNS, as well as similar progenitor proliferation and accumulation during demyelination. To directly analyze progenitor differentiation, glial cultures from spinal cords of wild-type mice undergoing remyelination after MHV-A59 demyelination were treated for 3 d with either exogenous FGF2 or an FGF2 neutralizing antibody. Elevating FGF2 favored progenitor proliferation, whereas attenuating endogenous FGF2 activity promoted the differentiation of progenitors into oligodendrocytes. These in vitro results are consistent with enhanced progenitor differentiation in FGF2 -/- mice. These studies demonstrate that the FGF2 genotype regulates oligodendrocyte regeneration and that FGF2 appears to inhibit oligodendrocyte lineage differentiation during remyelination.

Developing rods transplanted into the degenerating retina of Crx-knockout mice exhibit neural activity similar to native photoreceptors

PubMed Central

Homma, Kohei; Okamoto, Satoshi; Mandai, Michiko; Gotoh, Norimoto; Rajasimha, Harsha K.; Chang, Yi-Sheng; Chen, Shan; Li, Wei; Cogliati, Tiziana; Swaroop, Anand; Takahashi, Masayo

2013-01-01

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age-related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with GFP driven by the promoter of rod differentiation factor, Nrl. GFP-tagged developing rods show Ca2+ responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization-activated current (Ih) induced by the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. When transplanted into the subretinal space of wild type or retinal degeneration mice, GFP-tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild-type mouse retina, and exhibit Ca2+ responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin-positive outer segment-like structures within two weeks after transplantation into the retina of Crx-knockout mice, and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP-positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod-specific phototransduction genes, and HCN-1 channels. We conclude that Nrl-promoter driven GFP-tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell-derived rods in vitro may provide a renewable source for cell replacement therapy. PMID:23495178

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease

PubMed Central

Manocha, Gunjan D.; Floden, Angela M.; Rausch, Keiko; Kulas, Joshua A.; McGregor, Brett A.; Rojanathammanee, Lalida; Puig, Kelley R.; Puig, Kendra L.; Karki, Sanjib; Nichols, Michael R.; Darland, Diane C.; Porter, James E.

2016-01-01

Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP−/−) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP−/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP−/− mice compared with wild-type mice. The mAPP−/− mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains. PMID:27511018

Molecular characterization, fitness and mycotoxin production of Fusarium graminearum laboratory strains resistant to benzimidazoles.

PubMed

Sevastos, A; Markoglou, A; Labrou, N E; Flouri, F; Malandrakis, A

2016-03-01

Six benzimidazole (BMZ)-resistant Fusarium graminearum strains were obtained after UV mutagenesis and selection on carbendazim (MBC)-amended medium. In vitro bioassays resulted in the identification of two resistant phenotypes that were highly HR (Rf: 40-170, based on EC50) and moderately MR (Rf: 10-20) resistant to carbendazim. Cross resistance studies with other fungicides showed that all mutant strains tested were also resistant to other BMZs, such as benomyl and thiabendazole, but retained their parental sensitivity to fungicides belonging to other chemical groups. A point mutation at codon 6 (His6Asn) was found in the β2-tubulin gene of MR isolates while another mutation at codon 200 (Phe200Tyr) was present in one MR and one HR isolates. Interestingly, low temperatures suppressed MBC-resistance in all isolates bearing the H6N mutation. The three-dimensional homology model of the wild-type and mutants of β-tubulins were constructed, and the possible carbendazim binding site was analyzed. Studies on fitness parameters showed that the mutation(s) for resistance to BMZs did not affect the mycelial growth rate whereas adverse effects were found in sporulation and conidial germination in most of the resistant mutants. Pathogenicity tests on corn cobs revealed that mutants were less or equally aggressive to the wild-type strain but expressed their BMZ-resistance after inoculation on maize cobs treated with MBC. Analysis of mycotoxin production by high performance liquid chromatography revealed that only two HR strains produced zearalenone (ZEA) at concentrations similar to that of the wild-type strain, while no ZEA levels were detected in the rest of the mutants. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro and in vivo host range of Anticarsia gemmatalis multiple nuclear polyhedrosis virus.

PubMed

Grasela, J J; McIntosh, A H

1998-01-01

A clone of the wild type (wt) Anticarsia gemmatalis multiple nuclear polyhedrosis virus AgMNPV, derived from a geographical isolate (Hondrina, Brazil) and designated AgMNPV-CL4-3A1, was used to determine the host range of this virus in six established lepidopteran cell lines: Anticarsia gemmatalis (BCIRL-AG-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Heliothis virescens (BCIRL-HV-AM1), Helicoverpa armigera (BCIRL-HA-AM1), Trichoplusia ni (TN-CL1), Bombyx mori (BMN), and a coleopteran cell line Anthonomus grandis (BRL-AG-1). In addition, the in vivo host range of this clone was also assayed in larvae of Helicoverpa zea, Heliothis virescens, Trichoplusia ni, and the homologous species Anticarsia gemmatalis by probit analysis. On the basis of temporal studies of TCID50 values, BCIRL-HV-AM1 cells gave the highest extracellular virus (ECV) titer (9.7 x 10(6) TCID50/ml) followed by BCIRL-HA-AM1 cells (8.3 x 10(5) TCID50/ml) and BCIRL-AG-AM1 cells (3.2 x 10(5) TCID50/ml). In addition, a low ECV titer of 1.37 x 10(3) TCID50/ml was detected from TN-CL1 cells 96 h postinoculation, while BRL-AG-1, BMN, and BCIRL-HZ-AM1 cells were nonpermissive to AgMNPV-CL4-3A1 on the basis of TCID50 results. AgMNPV-CL4-3A1 and the wild type AgMNPV had similar restriction profiles that were different from wild type AcMNPV. The LC50 values were 96.9, 564.6, 733.3, and 1.1 x 10(4) occlusion bodies/cm2 of diet for A. gemmatalis, Helicoverpa zea, Heliothis virescens, and T. ni, respectively.

Copper/zinc superoxide dismutase insufficiency impairs progesterone secretion and fertility in female mice.

PubMed

Noda, Yoshihiro; Ota, Kuniaki; Shirasawa, Takuji; Shimizu, Takahiko

2012-01-01

Copper/zinc superoxide dismutase (CuZn-SOD, SOD1) is one of the major antioxidant enzymes, and is localized in the cytoplasm to scavenge superoxide. To investigate the physiological role of SOD1 in the ovaries, we analyzed the fertility of Sod1-deficient female mice. To evaluate their hormonal metabolism, we measured pituitary and ovarian hormone levels in the plasma of the mutant mice. Plasma follicle-stimulating hormone, luteinizing hormone, and estradiol were not altered in the mutant compared to the wild-type females, while the plasma progesterone level was significantly reduced in the mutant females. Furthermore, the mutant mice showed decreased progesterone secretion under the condition of superovulation. In a histochemical analysis, we observed a remarkable reduction in the corpus luteum area in the mutant ovaries without atrophic changes. The mutant mice also displayed enhanced superoxide generation in the region surrounding the corpora lutea, which was associated with increased apoptotic cells and suppressed vasculature. These results suggested that SOD1 deficiency dysregulated luteal formation because of increased superoxide generation in the ovary. In vitro fertilization experiments showed no abnormal fertilization of Sod1-deficient oocytes. In addition, when Sod1-deficient embryos were transferred into the oviducts of wild-type females, mutant embryos developed at a normal rate, indicating that SOD1 deficiency in embryos did not cause miscarriage in the uterus of wild-type females. These results indicated that increased intracellular ROS impaired luteal formation and progesterone production in the mutant females, thus suggesting that SOD1 plays a crucial role in both the luteal function and the maintenance of fertility in female mice.

Antioxidant and neuroprotective effects of Dictyophora indusiata polysaccharide in Caenorhabditis elegans.

PubMed

Zhang, Ju; Shi, Ruona; Li, Haifeng; Xiang, Yanxia; Xiao, Lingyun; Hu, Minghua; Ma, Fangli; Ma, Chung Wah; Huang, Zebo

2016-11-04

Dictyophora indusiata is a medicinal mushroom traditionally used in China for a variety of conditions, including inflammatory and neural diseases. D. indusiata polysaccharides (DiPS) are shown to have in vitro antioxidant activity but in vivo evidence is lacking. This study aimes to explore the antioxidant capacity and related neuroptotective activities of DiPS using wild-type and neurodegenerative Caenorhabditis elegans models. The antioxidant capacities of DiPS were first determined using paraquat survival and Pgst-4::GFP expression assays in wild-type and transgenic C. elegans models, respectively, and then further investigated by determining reactive oxygen species (ROS) level, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity as well as functional parameters of mitochondria. The activation of stress response transcription factors and neuroptotective activities were examined using nuclear localization and chemosensory behavioral assays in transgenic nematodes, respectively. DiPS was shown not only to increase survival rate and reduce stress level under paraquat-induced oxidative conditions but also to decrease ROS and MDA levels and increase SOD activity in C. elegans models. Moreover, DiPS was also able to restore the functional parameters of mitochondria, including membrane potential and ATP content, in paraquat-stressed nematodes. In addition, nuclear translocation assays demonstrate that the stress response transcription factor DAF-16/FOXO was involved in the antioxidant activity of the polysaccharide. Further experiments reveal that DiPS was capable of reducing ROS levels and alleviating chemosensory behavior dysfunction in transgenic nematode models of neurodegenerative diseases mediated by polyglutamine and amyloid-β protein. These findings demonstrate the antioxidant and neuroprotective activities of the D. indusiata polysaccharide DiPS in wild-type and neurodegenerative C. elegans models, and thus provide an important pharmacological basis for the therapeutic potential of D. indusiata in neurodegeneration. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Interleukin 6 Influences Germinal Center Development and Antibody Production via a Contribution of C3 Complement Component

PubMed Central

Kopf, Manfred; Herren, Suzanne; Wiles, Michael V.; Pepys, Mark B.; Kosco-Vilbois, Marie H.

1998-01-01

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell–dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6–deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti–mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3–deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6–deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses. PMID:9815267

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

NASA Technical Reports Server (NTRS)

Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

1999-01-01

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

Probing the Role of Nascent Helicity in p27 Function as a Cell Cycle Regulator

PubMed Central

Otieno, Steve; Kriwacki, Richard

2012-01-01

p27 regulates the activity of Cdk complexes which are the principal governors of phase transitions during cell division. Members of the p27 family of proteins, which also includes p21 and p57, are called the Cip/Kip cyclin-dependent kinase regulators (CKRs). Interestingly, the Cip/Kip CKRs play critical roles in cell cycle regulation by being intrinsically unstructured, a characteristic contrary to the classical structure-function paradigm. They exhibit nascent helicity which has been localized to a segment referred to as sub-domain LH. The nascent helicity of this sub-domain is conserved and we hypothesize that it is an important determinant of their functional properties. To test this hypothesis, we successfully designed and prepared p27 variants in which domain LH was either more or less helical with respect to the wild-type protein. Thermal denaturation experiments showed that the ternary complexes of the p27 variants bound to Cdk2/Cyclin A were less stable compared to the wild-type complex. Isothermal titration calorimetry experiments showed a decrease in the enthalpy of binding for all the mutants with respect to p27. The free energies of binding varied within a much narrower range. In vitro Cdk2 inhibition assays showed that the p27 variants exhibited disparate inhibitory potencies. Furthermore, when over-expressed in NIH 3T3 mouse fibroblast cells, the less helical p27 variants were less effective in causing cell cycle arrest relative to the wild-type p27. Our results indicate that the nascent helicity of sub-domain LH plays a key role mediating the biological function of p27. PMID:23071750

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1.

PubMed

De Luca, Gabriele; Ventura, Ilenia; Sanghez, Valentina; Russo, Maria Teresa; Ajmone-Cat, Maria Antonietta; Cacci, Emanuele; Martire, Alberto; Popoli, Patrizia; Falcone, Germana; Michelini, Flavia; Crescenzi, Marco; Degan, Paolo; Minghetti, Luisa; Bignami, Margherita; Calamandrei, Gemma

2013-08-01

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1-Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1-Tg mice express high levels of the hMTH1 hydrolase that degrades 8-oxodGTP and 8-oxoGTP and excludes 8-oxoguanine from both DNA and RNA. Compared to wild-type animals, hMTH1-overexpressing mice have significantly lower steady-state levels of 8-oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age-dependent accumulation of DNA 8-oxoguanine that occurs in wild-type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1-Tg animals live significantly longer than their wild-type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1-Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1-Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1-Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids. © 2013 John Wiley & Sons Ltd and the Anatomical Society.

Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.

PubMed

Timm, Kerstin N; Hartl, Johannes; Keller, Markus A; Hu, De-En; Kettunen, Mikko I; Rodrigues, Tiago B; Ralser, Markus; Brindle, Kevin M

2015-12-01

A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells. © 2014 Wiley Periodicals, Inc.

A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

PubMed Central

Ohkawa, T; Majima, K; Maeda, S

1994-01-01

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi.

PubMed

Costes, B; Fournier, G; Michel, B; Delforge, C; Raj, V Stalin; Dewals, B; Gillet, L; Drion, P; Body, A; Schynts, F; Lieffrig, F; Vanderplasschen, A

2008-05-01

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

Evidence for organ-specific stem cell microenvironments.

PubMed

Ghinassi, Barbara; Martelli, Fabrizio; Verrucci, Maria; D'Amore, Emanuela; Migliaccio, Giovanni; Vannucchi, Alessandro Maria; Hoffman, Ronald; Migliaccio, Anna Rita

2010-05-01

The X-linked Gata1(low) mutation in mice induces strain-restricted myeloproliferative disorders characterized by extramedullary hematopoiesis in spleen (CD1 and DBA/2) and liver (CD1 only). To assess the role of the microenvironment in establishing this myeloproliferative trait, progenitor cell compartments of spleen and marrow from wild-type and Gata1(low) mice were compared. Phenotype and clonal assay of non-fractionated cells indicated that Gata1(low) mice contain progenitor cell numbers 4-fold lower and 10-fold higher than normal in marrow and spleen, respectively. However, progenitor cells prospectively isolated from spleen, but not from marrow, of Gata1(low) mice expressed colony-forming function in vitro. Therefore, calculation of cloning activity of purified cells demonstrated that the total number of Gata1(low) progenitor cells was 10- to 100-fold lower than normal in marrow and >1,000 times higher than normal in spleen. This observation indicates that Gata1(low) hematopoiesis is favored by the spleen and is in agreement with our previous report that removal of this organ induces wild-type hematopoiesis in heterozygous Gata1(low/+) females (Migliaccio et al., 2009, Blood 114:2107). To clarify if rescue of wild-type hematopoiesis by splenectomy prevented extramedullary hematopoiesis in liver, marrow cytokine expression profile and liver histopathology of splenectomized Gata1(low/+) females were investigated. After splenectomy, the marrow expression levels of TGF-beta, VEGF, osteocalcin, PDGF-alpha, and SDF-1 remained abnormally high while Gata1(low) hematopoiesis was detectable in liver of both CD1 and DBA/2 mutants. Therefore, in the absence of the spleen, Gata1(low) hematopoiesis is supported by the liver suggesting that treatment of myelofibrosis in these animals requires the rescue of both stem cell and microenvironmental functions.

IDENTIFICATION OF NOVEL TOXICITY-ASSOCIATED METABOLITES BY METABOLOMICS AND MASS ISOTOPOMER ANALYSIS OF ACETAMINOPHEN METABOLISM IN WILD-TYPE AND CYP2E1-NULL MICE

PubMed Central

Chen, Chi; Krausz, Kristopher W.; Idle, Jeffrey R.; Gonzalez, Frank J.

2008-01-01

CYP2E1 is recognized as the most important enzyme for initiation of acetaminophen (APAP)-induced toxicity. In this study, the resistance of Cyp2e1-null mice to APAP treatment was confirmed by comparing serum aminotransferase activities and blood urea nitrogen levels in wild-type and Cyp2e1-null mice. However, unexpectedly, profiling of major known APAP metabolites in urine and serum revealed that the contribution of CYP2E1 to APAP metabolism decreased with increasing APAP doses administered. Measurement of hepatic glutathione and hydrogen peroxide levels exposed the importance of oxidative stress in determining the consequence of APAP overdose. Subsequent metabolomic analysis was capable of constructing a principal components analysis (PCA) model that delineated a relationship between urinary metabolomes and the responses to APAP treatment. Urinary ions high in wild-type mice treated with 400 mg/kg APAP were elucidated as 3-methoxy-APAP glucuronide (VII) and three novel APAP metabolites, including S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid (VI, formed by a Cys-APAP transamination reaction in kidney), 3,3′-biacetaminophen (VIII, an APAP dimer) and a benzothiazine compound (IX, originated from deacetylated APAP), through mass isotopomer analysis, accurate mass measurement, tandem MS fragmentation, in vitro reactions and chemical treatments. Dose-, time- and genotype-dependent appearance of these minor APAP metabolites implied their association with the APAP-induced toxicity and potential biomarker application. Overall, the oxidative stress elicited by CYP2E1-mediated APAP metabolism might significantly contribute to APAP-induced toxicity. The combination of genetically-modified animal models, mass isotopomer analysis and metabolomics provides a powerful and efficient technical platform to characterize APAP-induced toxicity through identifying novel biomarkers and unravelling novel mechanisms. PMID:18093979

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines.

PubMed

Peng, Jiaojiao; Zhu, Shenghe; Hu, Lili; Ye, Pingping; Wang, Yifei; Tian, Qin; Mei, Mingzhu; Chen, Hao; Guo, Xiaofeng

2016-10-02

Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.

Amphipathic helical peptides hamper protein-protein interactions of the intrinsically disordered chromatin nuclear protein 1 (NUPR1).

PubMed

Santofimia-Castaño, Patricia; Rizzuti, Bruno; Abián, Olga; Velázquez-Campoy, Adrián; Iovanna, Juan L; Neira, José L

2018-06-01

NUPR1 is a multifunctional intrinsically disordered protein (IDP) involved, among other functions, in chromatin remodelling, and development of pancreatic ductal adenocarcinoma (PDAC). It interacts with several biomolecules through hydrophobic patches around residues Ala33 and Thr68. The drug trifluoperazine (TFP), which hampers PDAC development in xenografted mice, also binds to those regions. Because of the large size of the hot-spot interface of NUPR1, small molecules could not be adequate to modulate its functions. We explored how amphipathic helical-designed peptides were capable of interacting with wild-type NUPR1 and the Thr68Gln mutant, inhibiting the interaction with NUPR1 protein partners. We used in vitro biophysical techniques (fluorescence, circular dichroism (CD), nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC)), in silico studies (docking and molecular dynamics (MD)), and in cellulo protein ligation assays (PLAs) to study the interaction. Peptide dissociation constants towards wild-type NUPR1 were ~ 3 μM, whereas no interaction was observed with the Thr68Gln mutant. Peptides interacted with wild-type NUPR1 residues around Ala33 and residues at the C terminus, as shown by NMR. The computational results clarified the main determinants of the interactions, providing a mechanism for the ligand-capture that explains why peptide binding was not observed for Thr68Gln mutant. Finally, the in cellulo assays indicated that two out of four peptides inhibited the interaction of NUPR1 with the C-terminal region of the Polycomb RING protein 1 (C-RING1B). Designed peptides can be used as lead compounds to inhibit NUPR1 interactions. Peptides may be exploited as drugs to target IDPs. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes.

PubMed

De Filippis, Lidia; Lamorte, Giuseppe; Snyder, Evan Y; Malgaroli, Antonio; Vescovi, Angelo L

2007-09-01

The discovery and study of neural stem cells have revolutionized our understanding of the neurogenetic process, and their inherent ability to adopt expansive growth behavior in vitro is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high-throughput assays for drug development and gene discovery dictate the need for rapid, reproducible, long-term expansion of human neural stem cells (hNSCs). In this view, the complement of wild-type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (immortalized human NSCs [IhNSCs]) by v-myc-mediated immortalization of previously derived wild-type hNSCs. These cells demonstrate three- to fourfold faster proliferation than wild-type cells in response to growth factors but retain rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy, and electrophysiology, we show that upon growth factor removal, IhNSCs completely downregulate v-myc expression, cease proliferation, and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes, and neurons. The latter are functional, mature cells displaying clear-cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the GABAergic, glutamatergic, and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to 20% of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high-throughput gene and drug screening that are essential in both diagnostics and cell therapy studies.

TraJ-dependent Escherichia coli K1 interactions with professional phagocytes are important for early systemic dissemination of infection in the neonatal rat.

PubMed

Hill, Val T; Townsend, Stacy M; Arias, Robyn S; Jenabi, Jasmine M; Gomez-Gonzalez, Ignacio; Shimada, Hiroyuki; Badger, Julie L

2004-01-01

Escherichia coli is a major cause of neonatal bacterial sepsis and meningitis. We recently identified a gene, traJ, which contributes to the ability of E. coli K1 to penetrate the blood-brain barrier in the neonatal rat. Because very little is known regarding the most critical step in disease progression, translocation to the gut and dissemination to the lymphoid tissues after a natural route of infection, we assessed the ability of a traJ mutant to cause systemic disease in the neonatal rat. Our studies determined that the traJ mutant is significantly less virulent than the wild type in the neonatal rat due to a decreased ability to disseminate from the mesenteric lymph nodes to the deeper tissues of the liver and spleen and to the blood during the early stages of systemic disease. Histopathologic studies determined that although significantly less or no mutant bacteria were recovered from the spleen and livers of infected neonatal rats, the inflammatory response was considerably greater than that in wild-type-colonized tissues. In vitro studies revealed that macrophages internalize the traJ mutant less frequently than they do the wild type and by a morphologically distinct process. Furthermore, we determined that tissue macrophages and dendritic cells within the liver and spleen are the major cellular targets of E. coli K1 and that TraJ significantly contributes to the predominantly intracellular nature of E. coli K1 within these professional phagocytes exclusively during the early stages of systemic disease. These data indicate that, contrary to earlier indications, E. coli K1 resides within professional phagocytes, and this is essential for the efficient progression of systemic disease.

Impaired Sperm Maturation in Rnase9 Knockout Mice1

PubMed Central

Westmuckett, Andrew D.; Nguyen, Edward B.; Herlea-Pana, Oana M.; Alvau, Antonio; Salicioni, Ana M.; Moore, Kevin L.

2014-01-01

ABSTRACT Ribonuclease, RNase A family, 9 (RNASE9) is a ribonuclease A superfamily member that is expressed only in the epididymis. It is a small, secreted polypeptide, it lacks ribonuclease activity, and its function(s) is unknown. However, epididymis-specific expression suggests a role in sperm maturation. We generated Rnase9−/− mice to study RNASE9 function in vivo. We confirm that RNASE9 expression is restricted to the epididymis. Within the epididymis, RNASE9 is first detected in midcaput, persists through the distal caput and corpus, and wanes in the cauda. Rnase9−/− mice are born at the expected Mendelian ratio, have normal postnatal growth and development, and have no outwardly apparent phenotype. Spermatogenesis is normal, and Rnase9-null sperm are morphologically normal. Rnase9−/− males have normal fertility in unrestricted mating trials, and fertilization rates in in vitro fertilization assays are indistinguishable from wild-type mice. Visual observations coupled with analyses of sperm velocities shortly after swim out from the corpus shows that motility of Rnase9-null sperm is significantly impaired. However, no differences between wild-type and Rnase9-null sperm are detected by computer-assisted sperm analysis 10–90 min after sperm isolation from the corpus or cauda. Assessment of capacitation-dependent signaling pathways in Rnase9-null sperm showed that, while levels of tyrosine phosphorylation of sperm proteins were normal, there was decreased phosphorylation of protein kinase A substrates upon capacitation compared to wild-type mice. In conclusion, RNASE9 is dispensable for fertility, but the absence of RNASE9 during epididymal transit results in impaired sperm maturation. PMID:24719258

Eosinophils contribute to early clearance of Pneumocystis murina infection

PubMed Central

Eddens, Taylor; Elsegeiny, Waleed; Nelson, Michael P.; Horne, William; Campfield, Brian T.; Steele, Chad; Kolls, Jay K.

2015-01-01

Pneumocystis pneumonia remains a common opportunistic infection in the diverse immunosuppressed population. One clear risk factor for susceptibility to Pneumocystis is a declining CD4+ T-cell counts in the setting of HIV/AIDS or primary immunodeficiency. Non-HIV infected individuals taking immunosuppressive drug regimens targeting T-cell activation are also susceptible. Given the crucial role of CD4+ T-cells in host defense against Pneumocystis, we used RNA-sequencing of whole lung early in infection in wild type and CD4-depleted animals as an unbiased approach to examine mechanisms of fungal clearance. In wild type mice, a strong eosinophil signature was observed at day 14 post-Pneumocystis challenge and eosinophils were increased in the bronchoalveolar lavage fluid of wild type mice. Furthermore, eosinophilopoiesis-deficient Gata1tm6Sho/J mice were more susceptible to Pneumocystis infection when compared to BALB/c controls and bone marrow derived eosinophils had in vitro Pneumocystis killing activity. To drive eosinophilia in vivo, Rag1−/− mice were treated with a plasmid expressing IL-5 (pIL5) or an empty plasmid control via hydrodynamic injection. pIL5 treated mice had increased serum IL-5 and eosinophilia in the lung, as well as reduced Pneumocystis burden compared to mice treated with control plasmid. Additionally, pIL5 treatment could induce eosinophilia and reduce Pneumocystis burden in CD4-depleted C57Bl/6 and BALB/c mice, but not eosinophilopoiesis-deficient Gata1tm6Sho/J mice. Taken together, these results demonstrate that an early role of CD4+ T-cells is to recruit eosinophils to the lung and that eosinophils are a novel candidate for future therapeutic development for Pneumocystis pneumonia in the immunosuppressed population. PMID:25994969

Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics.

PubMed

Walker, Aisha L; Lancaster, Cynthia S; Finkelstein, David; Ware, Russell E; Sparreboom, Alex

2013-12-15

Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assessing the role of OATP1B transporters in modulating hydroxyurea PK. Using wild-type and Oatp1b knockout (Oatp1b(-/-)) mice, hydroxyurea PK was analyzed in vivo by measuring [(14)C]hydroxyurea distribution in plasma, kidney, liver, urine, or the exhaled (14)CO2 metabolite. Plasma levels were significantly reduced by 20% in Oatp1b(-/-) mice compared with wild-type (area under the curve of 38.64 or 48.45 μg·h(-1)·ml(-1), respectively) after oral administration, whereas no difference was observed between groups following intravenous administration. Accumulation in the kidney was significantly decreased by twofold in Oatp1b(-/-) mice (356.9 vs. 748.1 pmol/g), which correlated with a significant decrease in urinary excretion. Hydroxyurea accumulation in the liver was also decreased (136.6 vs. 107.3 pmol/g in wild-type or Oatp1b(-/-) mice, respectively) correlating with a decrease in exhaled (14)CO2. These findings illustrate that deficiency of Oatp1b transporters alters the absorption, distribution, and elimination of hydroxyurea thus providing the first in vivo evidence that cell membrane transporters may play a significant role in modulating hydroxyurea PK. Future studies to investigate other transporters and their role in hydroxyurea disposition are warranted for understanding the sources of variation in hydroxyurea's PK.

Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics

PubMed Central

Lancaster, Cynthia S.; Finkelstein, David; Ware, Russell E.; Sparreboom, Alex

2013-01-01

Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assessing the role of OATP1B transporters in modulating hydroxyurea PK. Using wild-type and Oatp1b knockout (Oatp1b−/−) mice, hydroxyurea PK was analyzed in vivo by measuring [14C]hydroxyurea distribution in plasma, kidney, liver, urine, or the exhaled 14CO2 metabolite. Plasma levels were significantly reduced by 20% in Oatp1b−/− mice compared with wild-type (area under the curve of 38.64 or 48.45 μg·h−1·ml−1, respectively) after oral administration, whereas no difference was observed between groups following intravenous administration. Accumulation in the kidney was significantly decreased by twofold in Oatp1b−/− mice (356.9 vs. 748.1 pmol/g), which correlated with a significant decrease in urinary excretion. Hydroxyurea accumulation in the liver was also decreased (136.6 vs. 107.3 pmol/g in wild-type or Oatp1b−/− mice, respectively) correlating with a decrease in exhaled 14CO2. These findings illustrate that deficiency of Oatp1b transporters alters the absorption, distribution, and elimination of hydroxyurea thus providing the first in vivo evidence that cell membrane transporters may play a significant role in modulating hydroxyurea PK. Future studies to investigate other transporters and their role in hydroxyurea disposition are warranted for understanding the sources of variation in hydroxyurea's PK. PMID:23986199

Analysis and Manipulation of Aspartate Pathway Genes for l-Lysine Overproduction from Methanol by Bacillus methanolicus▿

PubMed Central

Nærdal, Ingemar; Netzer, Roman; Ellingsen, Trond E.; Brautaset, Trygve

2011-01-01

We investigated the regulation and roles of six aspartate pathway genes in l-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by l-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the l-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has—in addition to a hom-1 mutation—chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for l-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased l-lysine production levels. PMID:21724876

Analysis and manipulation of aspartate pathway genes for L-lysine overproduction from methanol by Bacillus methanolicus.

PubMed

Nærdal, Ingemar; Netzer, Roman; Ellingsen, Trond E; Brautaset, Trygve

2011-09-01

We investigated the regulation and roles of six aspartate pathway genes in L-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by L-lysine and induced 2-fold by dl-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by dl-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the L-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has-in addition to a hom-1 mutation-chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by meso-diaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for L-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased L-lysine production levels.

Losartan increases bone mass and accelerates chondrocyte hypertrophy in developing skeleton

PubMed Central

Rianon, Nahid; Rajagopal, Abbhirami; Munivez, Elda; Bertin, Terry; Dawson, Brian; Chen, Yuqing; Jiang, Ming-Ming; Lee, Brendan; Yang, Tao; Bae, Yangjin

2015-01-01

Angiotensin receptor blockers (ARBs) are a group of anti-hypertensive drugs that are widely used to treat pediatric hypertension. Recent application of ARBs to treat diseases such as Marfan syndrome or Alport syndrome has shown positive outcomes in animal and human studies, suggesting a broader therapeutic potential for this class of drugs. Multiple studies have reported a benefit of ARBs on adult bone homeostasis; however, its effect on the growing skeleton in children is unknown. We investigated the effect of Losartan, an ARB, in regulating bone mass and cartilage during development in mice. Wild type mice were treated with Losartan from birth until 6 weeks of age, after which bones were collected for microCT and histomorphometric analyses. Losartan increased trabecular bone volume vs. tissue volume (a 98% increase) and cortical thickness (a 9% increase) in 6-weeks old wild type mice. The bone changes were attributed to decreased osteoclastogenesis as demonstrated by reduced osteoclast number per bone surface in vivo and suppressed osteoclast differentiation in vitro. At the molecular level, Angiotensin II-induced ERK1/2 phosphorylation in RAW cells was attenuated by Losartan. Similarly, RANKL-induced ERK1/2 phosphorylation was suppressed by Losartan, suggesting a convergence of RANKL and angiotensin signaling at the level of ERK1/2 regulation. To assess the effect of Losartan on cartilage development, we examined the cartilage phenotype of wild type mice treated with Losartan in utero from conception to 1 day of age. Growth plates of these mice showed an elongated hypertrophic chondrocyte zone and increased Col10a1 expression level, with minimal changes in chondrocyte proliferation. Altogether, inhibition of the angiotensin pathway by Losartan increases bone mass and accelerates chondrocyte hypertrophy in growth plate during skeletal development. PMID:25779879

Losartan increases bone mass and accelerates chondrocyte hypertrophy in developing skeleton.

PubMed

Chen, Shan; Grover, Monica; Sibai, Tarek; Black, Jennifer; Rianon, Nahid; Rajagopal, Abbhirami; Munivez, Elda; Bertin, Terry; Dawson, Brian; Chen, Yuqing; Jiang, Ming-Ming; Lee, Brendan; Yang, Tao; Bae, Yangjin

2015-05-01

Angiotensin receptor blockers (ARBs) are a group of anti-hypertensive drugs that are widely used to treat pediatric hypertension. Recent application of ARBs to treat diseases such as Marfan syndrome or Alport syndrome has shown positive outcomes in animal and human studies, suggesting a broader therapeutic potential for this class of drugs. Multiple studies have reported a benefit of ARBs on adult bone homeostasis; however, its effect on the growing skeleton in children is unknown. We investigated the effect of Losartan, an ARB, in regulating bone mass and cartilage during development in mice. Wild type mice were treated with Losartan from birth until 6 weeks of age, after which bones were collected for microCT and histomorphometric analyses. Losartan increased trabecular bone volume vs. tissue volume (a 98% increase) and cortical thickness (a 9% increase) in 6-weeks old wild type mice. The bone changes were attributed to decreased osteoclastogenesis as demonstrated by reduced osteoclast number per bone surface in vivo and suppressed osteoclast differentiation in vitro. At the molecular level, Angiotensin II-induced ERK1/2 phosphorylation in RAW cells was attenuated by Losartan. Similarly, RANKL-induced ERK1/2 phosphorylation was suppressed by Losartan, suggesting a convergence of RANKL and angiotensin signaling at the level of ERK1/2 regulation. To assess the effect of Losartan on cartilage development, we examined the cartilage phenotype of wild type mice treated with Losartan in utero from conception to 1 day of age. Growth plates of these mice showed an elongated hypertrophic chondrocyte zone and increased Col10a1 expression level, with minimal changes in chondrocyte proliferation. Altogether, inhibition of the angiotensin pathway by Losartan increases bone mass and accelerates chondrocyte hypertrophy in growth plate during skeletal development. Copyright © 2015 Elsevier Inc. All rights reserved.

A Recurrent Missense Mutation in ZP3 Causes Empty Follicle Syndrome and Female Infertility.

PubMed

Chen, Tailai; Bian, Yuehong; Liu, Xiaoman; Zhao, Shigang; Wu, Keliang; Yan, Lei; Li, Mei; Yang, Zhenglin; Liu, Hongbin; Zhao, Han; Chen, Zi-Jiang

2017-09-07

Empty follicle syndrome (EFS) is defined as the failure to aspirate oocytes from mature ovarian follicles during in vitro fertilization. Except for some cases caused by pharmacological or iatrogenic problems, the etiology of EFS remains enigmatic. In the present study, we describe a large family with a dominant inheritance pattern of female infertility characterized by recurrent EFS. Genome-wide linkage analyses and whole-exome sequencing revealed a paternally transmitted heterozygous missense mutation of c.400 G>A (p.Ala134Thr) in zona pellucida glycoprotein 3 (ZP3). The same mutation was identified in an unrelated EFS pedigree. Haplotype analysis revealed that the disease allele of these two families came from different origins. Furthermore, in a cohort of 21 cases of EFS, two were also found to have the ZP3 c.400 G>A mutation. Immunofluorescence and histological analysis indicated that the oocytes of the EFS female had degenerated and lacked the zona pellucida (ZP). ZP3 is a major component of the ZP filament. When mutant ZP3 was co-expressed with wild-type ZP3, the interaction between wild-type ZP3 and ZP2 was markedly decreased as a result of the binding of wild-type ZP3 and mutant ZP3, via dominant negative inhibition. As a result, the assembly of ZP was impeded and the communication between cumulus cells and the oocyte was prevented, resulting in oocyte degeneration. These results identified a genetic basis for EFS and oocyte degeneration and, moreover, might pave the way for genetic diagnosis of infertile females with this phenotype. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis

PubMed Central

Hsu, Ron-Bin; Shun, Chia-Tung; Hsu, Chih-Chieh

2017-01-01

ABSTRACT Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis. PMID:28674029

Secreted Fungal Effector Lipase Releases Free Fatty Acids to Inhibit Innate Immunity-Related Callose Formation during Wheat Head Infection[W][OPEN

PubMed Central

Blümke, Antje; Falter, Christian; Herrfurth, Cornelia; Sode, Björn; Bode, Rainer; Schäfer, Wilhelm; Feussner, Ivo; Voigt, Christian A.

2014-01-01

The deposition of the (1,3)-β-glucan cell wall polymer callose at sites of attempted penetration is a common plant defense response to intruding pathogens and part of the plant’s innate immunity. Infection of the Fusarium graminearum disruption mutant Δfgl1, which lacks the effector lipase FGL1, is restricted to inoculated wheat (Triticum aestivum) spikelets, whereas the wild-type strain colonized the whole wheat spike. Our studies here were aimed at analyzing the role of FGL1 in establishing full F. graminearum virulence. Confocal laser-scanning microscopy revealed that the Δfgl1 mutant strongly induced the deposition of spot-like callose patches in vascular bundles of directly inoculated spikelets, while these callose deposits were not observed in infections by the wild type. Elevated concentrations of the polyunsaturated free fatty acids (FFAs) linoleic and α-linolenic acid, which we detected in F. graminearum wild type-infected wheat spike tissue compared with Δfgl1-infected tissue, provided clear evidence for a suggested function of FGL1 in suppressing callose biosynthesis. These FFAs not only inhibited plant callose biosynthesis in vitro and in planta but also partially restored virulence to the Δfgl1 mutant when applied during infection of wheat spikelets. Additional FFA analysis confirmed that the purified effector lipase FGL1 was sufficient to release linoleic and α-linolenic acids from wheat spike tissue. We concluded that these two FFAs have a major function in the suppression of the innate immunity-related callose biosynthesis and, hence, the progress of F. graminearum wheat infection. PMID:24686113

FOLFIRI plus panitumumab in the treatment of wild-type KRAS and wild-type NRAS metastatic colorectal cancer.

PubMed

Geredeli, Caglayan; Yasar, Nurgul

2018-03-27

The aim of this study was to investigate the efficacy and safety of first-line panitumumab plus folinic acid, 5-fluorouracil and irinotecan (FOLFIRI) in patients with wild-type KRAS and wild-type NRAS metastatic colorectal cancer (mCRC). Patients with wild-type KRAS and wild-type NRAS mCRC presenting to the medical oncology department of the Okmeydani Training and Research Hospital in Istanbul, Turkey, between April 2014 and January 2018 were enrolled in this study. A total of 64 patients (35 males and 29 females) with a median age of 59 (35-81) years old were enrolled. The median follow-up was 18.9 months, and the median progression-free survival was 13 months. The median overall survival (OS) was 26 months in the patients with wild-type KRAS and wild-type NRAS mCRC. It was 90.4% for the 6-month OS, 79.5% for the 1-year OS, 53.7% for the 2-year OS and 31.1% for the 3-year OS. The median OS of the patients who underwent metastasectomies was 40 [95% confidence interval (CI) = 19.9-60.1] months, and the median OS of the patients without metastasectomies was 22 (95% CI = 17.7-26.4) months. There was a statistically significant difference between these (P = 0.007). The first-line FOLFIRI plus panitumumab was associated with favourable efficacy in the patients with wild-type KRAS and wild-type NRAS mCRC, and it was well tolerated. The removal of the metastases that became resectable after chemotherapy further prolonged the patients' survival. Retrospectively registered: 33886.

MTBDRplus and MTBDRsl Assays: Absence of Wild-Type Probe Hybridization and Implications for Detection of Drug-Resistant Tuberculosis

PubMed Central

Georghiou, Sophia B.; Catanzaro, Donald; Rodrigues, Camilla; Crudu, Valeriu; Victor, Thomas C.; Garfein, Richard S.; Catanzaro, Antonino; Rodwell, Timothy C.

2016-01-01

Accurate identification of drug-resistant Mycobacterium tuberculosis is imperative for effective treatment and subsequent reduction in disease transmission. Line probe assays rapidly detect mutations associated with resistance and wild-type sequences associated with susceptibility. Examination of molecular-level performance is necessary for improved assay result interpretation and for continued diagnostic development. Using data collected from a large, multisite diagnostic study, probe hybridization results from line probe assays, MTBDRplus and MTBDRsl, were compared to those of sequencing, and the diagnostic performance of each individual mutation and wild-type probe was assessed. Line probe assay results classified as resistant due to the absence of wild-type probe hybridization were compared to those of sequencing to determine if novel mutations were inhibiting wild-type probe hybridization. The contribution of absent wild-type probe hybridization to the detection of drug resistance was assessed via comparison to a phenotypic reference standard. In our study, mutation probes demonstrated significantly higher specificities than wild-type probes and wild-type probes demonstrated marginally higher sensitivities than mutation probes, an ideal combination for detecting the presence of resistance conferring mutations while yielding the fewest number of false-positive results. The absence of wild-type probe hybridization without mutation probe hybridization was determined to be primarily the result of failure of mutation probe hybridization and not the result of novel or rare mutations. Compared to phenotypic culture-based drug susceptibility testing, the absence of wild-type probe hybridization without mutation probe hybridization significantly contributed to the detection of phenotypic rifampin and fluoroquinolone resistance with negligible increases in false-positive results. PMID:26763971

Dual HER2\\PIK3CA targeting overcomes single-agent acquired resistance in HER2 amplified uterine serous carcinoma cell lines in vitro and in vivo

PubMed Central

Lopez, Salvatore; Cocco, Emiliano; Black, Jonathan; Bellone, Stefania; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Schwab, Carlton L.; English, Diana P.; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E.; Terranova, Corrado; Angioli, Roberto; Santin, Alessandro D.

2015-01-01

HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC), and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA mutated and PIK3CA-wild type HER2/neu amplified USC cell lines. Cell viability and cell cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC-xenografts. We found both single agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long lasting growth inhibition in both USC xenografts when compared to single agent therapy. Combined targeting of HER2 and PIK3CA was associated with a significant and dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and a dose-dependent decline in the phosphorylation of S6. Importantly, dual inhibition therapy initiated after tumor progression in single agent-treated mice was still remarkably effective at inducing tumor regression in both large PIK3CA or pan-ErbB inhibitor-resistant USC xenografts. Dual HER2/PIK3CA blockade may represent a novel therapeutic option for USC patients harboring tumors with HER2/neu gene amplification and mutated or wild type PIK3CA resistant to chemotherapy. PMID:26333383

Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion

PubMed Central

Roeder, Sebastian S.; Barnes, Taylor J.; Lee, Jonathan S.; Kato, India; Eng, Diana G.; Kaverina, Natalya V.; Sunseri, Maria W.; Daniel, Christoph; Amann, Kerstin; Pippin, Jeffrey W.; Shankland, Stuart J.

2017-01-01

The glycoprotein CD44 is barely detected in normal mouse and human glomeruli, but is increased in glomerular parietal epithelial cells following podocyte injury in focal segmental glomerulosclerosis (FSGS). To determine the biological role and regulation of CD44 in these cells, we employed an in vivo and in vitro approach. Experimental FSGS was induced in CD44 knockout and wildtype mice with a cytotoxic podocyte antibody. Albuminuria, focal and global glomerulosclerosis (periodic acid-Schiff stain) and collagen IV staining were lower in CD44 knockout compared with wild type mice with FSGS. Parietal epithelial cells had lower migration from Bowman’s capsule to the glomerular tuft in CD44 knockout mice with disease compared with wild type mice. In cultured murine parietal epithelial cells, overexpressing CD44 with a retroviral vector encoding CD44 was accompanied by significantly increased collagen IV expression and parietal epithelial cells migration. Because our results showed de novo co-staining for activated ERK1/2 (pERK) in parietal epithelial cells in experimental FSGS, and also in biopsies from patients with FSGS, two in vitro strategies were employed to prove that pERK regulated CD44 levels. First, mouse parietal epithelial cells were infected with a retroviral vector for the upstream kinase MEK-DD to increase pERK, which was accompanied by increased CD44 levels. Second, in CD44 overexpressing parietal epithelial cells, decreasing pERK with U0126 was accompanied by reduced CD44. Finally, parietal epithelial cell migration was higher in cells with increased and reduced in cells with decreased pERK. Thus, pERK is a regulator of CD44 expression and increased CD44 expression leads to a pro-sclerotic and migratory parietal epithelial cells phenotype. PMID:27998643

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

PubMed Central

Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R.; Rommelaere, Jean; Lacroix, Jeannine

2017-01-01

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo. PMID:29039746

Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y1 and Y2 receptors

PubMed Central

Tough, IR; Forbes, S; Tolhurst, R; Ellis, M; Herzog, H; Bornstein, JC; Cox, HM

2011-01-01

BACKGROUND AND PURPOSE Peptide YY (PYY) and neuropeptide Y (NPY) activate Y receptors, targets under consideration as treatments for diarrhoea and other intestinal disorders. We investigated the gastrointestinal consequences of selective PYY or NPY ablation on mucosal ion transport, smooth muscle activity and transit using wild-type, single and double peptide knockout mice, comparing mucosal responses with those from human colon. EXPERIMENTAL APPROACH Mucosae were pretreated with a Y1 (BIBO3304) or Y2 (BIIE0246) receptor antagonist and changes in short-circuit current recorded. Colonic transit and colonic migrating motor complexes (CMMCs) were assessed in vitro and upper gastrointestinal and colonic transit measured in vivo. KEY RESULTS Y receptor antagonists revealed tonic Y1 and Y2 receptor-mediated antisecretory effects in human and wild-type mouse colon mucosae. In both, Y1 tone was epithelial while Y2 tone was neuronal. Y1 tone was reduced 90% in PYY−/− mucosa but unchanged in NPY−/− tissue. Y2 tone was partially reduced in NPY−/− or PYY−/− mucosae and abolished in tetrodotoxin-pretreated PYY−/− tissue. Y1 and Y2 tone were absent in NPYPYY−/− tissue. Colonic transit was inhibited by Y1 blockade and increased by Y2 antagonism indicating tonic Y1 excitation and Y2 inhibition respectively. Upper GI transit was increased in PYY−/− mice only. Y2 blockade reduced CMMC frequency in isolated mouse colon. CONCLUSIONS AND IMPLICATIONS Endogenous PYY and NPY induced significant mucosal antisecretory tone mediated by Y1 and Y2 receptors, via similar mechanisms in human and mouse colon mucosa. Both peptides contributed to tonic Y2-receptor-mediated inhibition of colonic transit in vitro but only PYY attenuated upper GI transit. PMID:21457230

A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model.

PubMed

Mohanty, Sujit K; Donnelly, Bryan; Dupree, Phylicia; Lobeck, Inna; Mowery, Sarah; Meller, Jaroslaw; McNeal, Monica; Tiao, Greg

2017-08-01

Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRV VP4-R446G ) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRV VP4-R446G ) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro , the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo , it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia. Copyright © 2017 American Society for Microbiology.

The chemokine CCL2 protects against methylmercury neurotoxicity.

PubMed

Godefroy, David; Gosselin, Romain-Daniel; Yasutake, Akira; Fujimura, Masatake; Combadière, Christophe; Maury-Brachet, Régine; Laclau, Muriel; Rakwal, Randeep; Melik-Parsadaniantz, Stéphane; Bourdineaud, Jean-Paul; Rostène, William

2012-01-01

Industrial pollution due to heavy metals such as mercury is a major concern for the environment and public health. Mercury, in particular methylmercury (MeHg), primarily affects brain development and neuronal activity, resulting in neurotoxic effects. Because chemokines can modulate brain functions and are involved in neuroinflammatory and neurodegenerative diseases, we tested the possibility that the neurotoxic effect of MeHg may interfere with the chemokine CCL2. We have used an original protocol in young mice using a MeHg-contaminated fish-based diet for 3 months relevant to human MeHg contamination. We observed that MeHg induced in the mice cortex a decrease in CCL2 concentrations, neuronal cell death, and microglial activation. Knock-out (KO) CCL2 mice fed with a vegetal control food already presented a decrease in cortical neuronal cell density in comparison with wild-type animals under similar diet conditions, suggesting that the presence of CCL2 is required for normal neuronal survival. Moreover, KO CCL2 mice showed a pronounced neuronal cell death in response to MeHg. Using in vitro experiments on pure rat cortical neurons in culture, we observed by blockade of the CCL2/CCR2 neurotransmission an increased neuronal cell death in response to MeHg neurotoxicity. Furthermore, we showed that sod genes are upregulated in brain of wild-type mice fed with MeHg in contrast to KO CCL2 mice and that CCL2 can blunt in vitro the decrease in glutathione levels induced by MeHg. These original findings demonstrate that CCL2 may act as a neuroprotective alarm system in brain deficits due to MeHg intoxication.

Immunoglobulin class switch recombination is impaired in Atm-deficient mice.

PubMed

Lumsden, Joanne M; McCarty, Thomas; Petiniot, Lisa K; Shen, Rhuna; Barlow, Carrolee; Wynn, Thomas A; Morse, Herbert C; Gearhart, Patricia J; Wynshaw-Boris, Anthony; Max, Edward E; Hodes, Richard J

2004-11-01

Immunoglobulin class switch recombination (Ig CSR) involves DNA double strand breaks (DSBs) at recombining switch regions and repair of these breaks by nonhomologous end-joining. Because the protein kinase ataxia telengiectasia (AT) mutated (ATM) plays a critical role in DSB repair and AT patients show abnormalities of Ig isotype expression, we assessed the role of ATM in CSR by examining ATM-deficient mice. In response to T cell-dependent antigen (Ag), Atm-/- mice secreted substantially less Ag-specific IgA, IgG1, IgG2b, and IgG3, and less total IgE than Atm+/+ controls. To determine whether Atm-/- B cells have an intrinsic defect in their ability to undergo CSR, we analyzed in vitro responses of purified B cells. Atm-/- cells secreted substantially less IgA, IgG1, IgG2a, IgG3, and IgE than wild-type (WT) controls in response to stimulation with lipopolysaccharide, CD40 ligand, or anti-IgD plus appropriate cytokines. Molecular analysis of in vitro responses indicated that WT and Atm-/- B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm-/- B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm-/- B cells occurs at the level of genomic DNA recombination as measured by digestion-circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the mu-gamma1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR.

Vaginal microbicide film combinations of two reverse transcriptase inhibitors, EFdA and CSIC, for the prevention of HIV-1 sexual transmission

PubMed Central

Zhang, Wei; Hu, Minlu; Shi, Yuan; Gong, Tiantian; Dezzutti, Charlene S.; Moncla, Bernard; Sarafianos, Stefan G.; Parniak, Michael A.; Rohan, Lisa C.

2015-01-01

Purpose EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. Methods Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. Results No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. Conclusions Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission. PMID:25794967

Glutamate dehydrogenase (RocG) in Bacillus licheniformis WX-02: Enzymatic properties and specific functions in glutamic acid synthesis for poly-γ-glutamic acid production.

PubMed

Tian, Guangming; Wang, Qin; Wei, Xuetuan; Ma, Xin; Chen, Shouwen

2017-04-01

Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway. However, little is known about the glutamate synthesis pathway in B. licheniformis. The aim of this work was to characterize the glutamate dehydrogenase (RocG) in glutamic acid synthesis from B. licheniformis with both in vivo and in vitro experiments. By re-directing the carbon flux distribution, the rocG gene deletion mutant WX-02ΔrocG produced intracellular glutamic acid with a concentration of 90ng/log(CFU), which was only 23.7% that of the wild-type WX-02 (380ng/log(CFU)). Furthermore, the γ-PGA yield of mutant WX-02ΔrocG was 5.37g/L, a decrease of 45.3% compared to the wild type (9.82g/L). In vitro enzymatic assays of RocG showed that RocG has higher affinity for 2-oxoglutarate than glutamate, and the glutamate synthesis rate was far above degradation. This is probably the first study to reveal the glutamic acid synthesis pathway and the specific functions of RocG in B. licheniformis. The results indicate that γ-PGA production can be enhanced through improving intracellular glutamic acid synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Vildagliptin Stimulates Endothelial Cell Network Formation and Ischemia-induced Revascularization via an Endothelial Nitric-oxide Synthase-dependent Mechanism*

PubMed Central

Ishii, Masakazu; Shibata, Rei; Kondo, Kazuhisa; Kambara, Takahiro; Shimizu, Yuuki; Tanigawa, Tohru; Bando, Yasuko K.; Nishimura, Masahiro; Ouchi, Noriyuki; Murohara, Toyoaki

2014-01-01

Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production. PMID:25100725

Vildagliptin stimulates endothelial cell network formation and ischemia-induced revascularization via an endothelial nitric-oxide synthase-dependent mechanism.

PubMed

Ishii, Masakazu; Shibata, Rei; Kondo, Kazuhisa; Kambara, Takahiro; Shimizu, Yuuki; Tanigawa, Tohru; Bando, Yasuko K; Nishimura, Masahiro; Ouchi, Noriyuki; Murohara, Toyoaki

2014-09-26

Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Absence of Activation-induced Cytidine Deaminase, a Regulator of Class Switch Recombination and Hypermutation in B Cells, Suppresses Aorta Allograft Vasculopathy in Mice.

PubMed

Nakanishi, Tomonori; Xu, Xiaoyan; Wynn, Carmen; Yamada, Toshiko; Pan, Fan; Erickson, Laurie; Teo, Haeman; Nakagawa, Terry; Masunaga, Taro; Abe, Jumpei; Akamatsu, Masahiko; Tamura, Kouichi; Jiang, Hongsi

2015-08-01

Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process. Splenocytes from C57BL/6 J (B6) AID(−/−) mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID(−/−) mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses. The AID(−/−) splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID(−/−) recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID(−/−) recipients with further reduction of intimal hyperplasia and fibrosis scores. The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.