Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening

NASA Astrophysics Data System (ADS)

Kalid, Ori; Mense, Martin; Fischman, Sharon; Shitrit, Alina; Bihler, Hermann; Ben-Zeev, Efrat; Schutz, Nili; Pedemonte, Nicoletta; Thomas, Philip J.; Bridges, Robert J.; Wetmore, Diana R.; Marantz, Yael; Senderowitz, Hanoch

2010-12-01

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

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

PubMed

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

2004-03-01

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

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

PubMed Central

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

2004-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is defective during cystic fibrosis (CF). Activators of the CFTR Cl− channel may be useful for therapy of CF. Here, we demonstrate that a range of general anesthetics like normal-alkanols (n-alkanols) and related compounds can stimulate the Cl− channel activity of wild-type CFTR and delF508-CFTR mutant. The effects of n-alkanols like octanol on CFTR activity were measured by iodide (125I) efflux and patch-clamp techniques on three distinct cellular models: (1) CFTR-expressing Chinese hamster ovary cells, (2) human airway Calu-3 epithelial cells and (3) human airway JME/CF15 epithelial cells which express the delF508-CFTR mutant. Our data show for the first time that n-alkanols activate both wild-type CFTR and delF508-CFTR mutant. Octanol stimulated 125I efflux in a dose-dependent manner in CFTR-expressing cells (wild-type and delF508) but not in cell lines lacking CFTR. 125I efflux and Cl− currents induced by octanol were blocked by glibenclamide but insensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, as expected for a CFTR Cl− current. CFTR activation by octanol was neither due to cell-to-cell uncoupling properties of octanol nor to an intracellular cAMP increase. CFTR activation by octanol requires phosphorylation by protein kinase-A (PKA) since it was prevented by H-89, a PKA inhibitor. n-Alkanols chain length was an important determinant for channel activation, with rank order of potencies: 1-heptanol<1-octanol<2-octanol<1-decanol. Our findings may be of valuable interest for developing novel therapeutic strategies for CF. PMID:14967738

Strain-dependent airway hyperresponsiveness and a chromosome 7 locus of elevated lymphocyte numbers in cystic fibrosis transmembrane conductance regulator-deficient mice.

PubMed

Bazett, Mark; Stefanov, Anguel N; Paun, Alexandra; Paradis, Josee; Haston, Christina K

2012-03-01

We previously observed the lungs of naive BALB/cJ Cftr(tm1UNC) mice to have greater numbers of lymphocytes, by immunohistochemical staining, than did BALB wild type littermates or C57BL/6J Cftr(tm1UNC) mice. In the present study, we initially investigated whether this mutation in Cftr alters the adaptive immunity phenotype by measuring the lymphocyte populations in the lungs and spleens by FACS and by evaluating CD3-stimulated cytokine secretion, proliferation, and apoptosis responses. Next, we assessed a potential influence of this lymphocyte phenotype on lung function through airway resistance measures. Finally, we mapped the phenotype of pulmonary lymphocyte counts in BALB × C57BL/6J F2 Cftr(tm1UNC) mice and reviewed positional candidate genes. By FACS analysis, both the lungs and spleens of BALB Cftr(tm1UNC) mice had more CD3(+) (both CD4(+) and CD8(+)) cells than did littermates or C57BL/6J Cftr(tm1UNC) mice. Cftr(tm1UNC) and littermate mice of either strain did not differ in anti-CD3-stimulated apoptosis or proliferation levels. Lymphocytes from BALB Cftr(tm1UNC) mice produced more IL-4 and IL-5 and reduced levels of IFN-γ than did littermates, whereas lymphocytes from C57BL/6J Cftr(tm1UNC) mice demonstrated increased Il-17 secretion. BALB Cftr(tm1UNC) mice presented an enhanced airway hyperresponsiveness to methacholine challenge compared with littermates and C57BL/6J Cftr(tm1UNC) mice. A chromosome 7 locus was identified to be linked to lymphocyte numbers, and genetic evaluation of the interval suggests Itgal and Il4ra as candidate genes for this trait. We conclude that the pulmonary phenotype of BALB Cftr(tm1UNC) mice includes airway hyperresponsiveness and increased lymphocyte numbers, with the latter trait being influenced by a chromosome 7 locus.

A mouse model for the cystic fibrosis delta F508 mutation.

PubMed Central

van Doorninck, J H; French, P J; Verbeek, E; Peters, R H; Morreau, H; Bijman, J; Scholte, B J

1995-01-01

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block. Images PMID:7556083

Probing Conformational Rescue Induced by a Chemical Corrector of F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutant*

PubMed Central

Yu, Wilson; Chiaw, Patrick Kim; Bear, Christine E.

2011-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause loss of function of the CFTR channel on the apical surface of epithelial cells. The major CF-causing mutation, F508del-CFTR, is misfolded, retained in the endoplasmic reticulum, and degraded. Small molecule corrector compounds have been identified using high throughput screens, which partially rescue the trafficking defect of F508del-CFTR, allowing a fraction of the mutant protein to escape endoplasmic reticulum retention and traffic to the plasma membrane, where it exhibits partial function as a cAMP-regulated chloride channel. A subset of such corrector compounds binds directly to the mutant protein, prompting the hypothesis that they rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis directly by evaluating the consequences of a corrector compound on the conformation of each nucleotide binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. Interestingly, we found that VRT-325 was capable of partially restoring compactness in NBD1. However, VRT-325 had no detectable effect on the conformation of the second half of the molecule. In comparison, ablation of the di-arginine sequence, R553XR555 (F508del-KXK-CFTR), modified protease susceptibility of NBD1, NBD2, and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together, these interventions restored processing of F508del-CFTR to near wild type. Importantly, however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. Importantly, this defect in channel activation can be fully corrected by the addition of the potentiator, VX-770. PMID:21602569

Matrine in association with FD-2 stimulates F508del-cystic fibrosis transmembrane conductance regulator activity in the presence of corrector VX809

PubMed Central

Marengo, Barbara; Speciale, Andrea; Senatore, Lisa; Garibaldi, Silvano; Musumeci, Francesca; Nieddu, Erika; Pollarolo, Benedetta; Pronzato, Maria Adelaide; Schenone, Silvia; Mazzei, Mauro; Domenicotti, Cinzia

2017-01-01

Cystic fibrosis is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the predominant mutation is termed Phe508del (F508del). Therapy for F508del-CFTR patients is based on the use of Orkambi®, a combination of VX809 and VX770. However, though Orkambi leads to an improvement in the lung function of patients, a progressive reduction in its efficacy has been observed. In order to overcome this effect, the aim of the present study was to investigate the role of matrine and the in-house compound FD-2 in increasing the action of VX809 and VX770. Fischer rat thyroid cells overexpressing F508del-CFTR were treated with matrine, VX809 (corrector) and/or with a number of potentiators (VX770, FD-1 and FD-2). The results demonstrated that matrine was able to stimulate CFTR activity and, in association with FD-2, increased the functionality of the channel in the presence of VX809. Based on these results, it may be hypothesized that FD-2 may be a novel and more effective potentiator compared with VX770. PMID:29039559

Defects in Gallbladder Emptying and Bile Acid Homeostasis in Mice With Cystic Fibrosis Transmembrane Conductance Regulator Deficiencies

PubMed Central

Debray, Dominique; Rainteau, Dominique; Barbu, Véronique; Rouahi, Myriam; Mourabit, Haquima El; Lerondel, Stéphanie; Rey, Colette; Humbert, Lydie; Wendum, Dominique; Cottart, Charles-Henry; Dawson, Paul; Chignard, Nicolas; Housset, Chantal

2013-01-01

Background & Aims Patients with cystic fibrosis (CF) have poorly defined defects in biliary function. We evaluated the effects of cystic fibrosis transmembrane conductance regulator (CFTR) deficiency on the enterohepatic disposition of bile acids (BAs). Methods Bile secretion and BA homeostasis were investigated in Cftrtm1Unc (Cftr−/−) and CftrΔF508 (ΔF508) mice. Results Cftr−/− and ΔF508 mice did not grow to normal size, but did not have liver abnormalities. The gallbladders of Cftr−/− mice were enlarged and had defects in emptying, based on99mtechnetiummebrofenin scintigraphy or post-prandial variationsn gallbladder volume; gallbladder contraction in response to cholecystokinin-8 was normal. Cftr−/− mice had abnormal gallbladder bile and duodenal acidity, and overexpressed the vasoactive intestinal peptide—a myorelaxant factor for the gallbladder. The BA pool was larger in Cftr−/− than wild-type mice, although there were no differences in fecal loss of BAs. Amounts of secondary BAs in portal blood, liver, and bile of Cftr−/− mice were much lower than normal. Expression of genes that are induced by BAs, including fibroblast growth factor-15 and BA transporters, was lower in the ileum but higher in the gallbladders of Cftr−/− mice, compared with wild-type mice, whereas enzymes that synthesize BA were down-regulated in livers of Cftr−/− mice. This indicates that BAs underwent a cholecystohepatic shunt, which was confirmed using cholyl-(Ne-NBD)-lysine as a tracer. In Cftr−/− mice, cholecystectomy reversed most changes in gene expression and partially restored circulating levels of secondary BAs. The ΔF508 mice overexpressed vasoactive intestinal peptide and had defects in gallbladder emptying and in levels of secondary BAs, but these features were less severe than in Cftr−/− mice. Conclusions Cftr−/− and CftrΔF508 mice have defects in gallbladder emptying that disrupt enterohepatic circulation of BAs. These defects

Impact of the F508del mutation on ovine CFTR, a Cl− channel with enhanced conductance and ATP-dependent gating

PubMed Central

Cai, Zhiwei; Palmai-Pallag, Timea; Khuituan, Pissared; Mutolo, Michael J; Boinot, Clément; Liu, Beihui; Scott-Ward, Toby S; Callebaut, Isabelle; Harris, Ann; Sheppard, David N

2015-01-01

Cross-species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that

Compounds that correct F508del-CFTR trafficking can also correct other protein trafficking diseases: an in vitro study using cell lines

PubMed Central

2013-01-01

Background Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER), and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR. Most of these correctors do not act directly as ligands of CFTR, but indirectly on other pathways to promote folding and correction. We hypothesize that these proteostasis regulators may also correct other protein trafficking diseases. Methods To test our hypothesis, we used stable cell lines or transient transfection to express 2 well-studied trafficking disease mutations in each of 3 different proteins: the arginine-vasopressin receptor 2 (AVPR2, also known as V2R), the human ether-a-go-go-related gene (KCNH2, also known as hERG), and finally the sulfonylurea receptor 1 (ABCC8, also known as SUR1). We treated cells expressing these mutant proteins with 9 structurally diverse F508del-CFTR correctors that function through different cellular mechanisms and assessed whether correction occurred via immunoblotting and functional assays. Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). Results Here we show that F508del-CFTR correctors RDR1, KM60 and KM57 also correct some mutant alleles of other protein trafficking diseases. We also show that one corrector, the cardiac glycoside ouabain, was found to alter the glycosylation of all mutant alleles tested. Conclusions Correctors of F508del-CFTR trafficking might have broader applications to other protein trafficking diseases. PMID:23316740

Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR.

PubMed

Farinha, Carlos M; Sousa, Marisa; Canato, Sara; Schmidt, André; Uliyakina, Inna; Amaral, Margarida D

2015-08-01

Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue.

Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR

PubMed Central

Farinha, Carlos M; Sousa, Marisa; Canato, Sara; Schmidt, André; Uliyakina, Inna; Amaral, Margarida D

2015-01-01

Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX-809, a corrector of F508del-CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX-809 is additive to two other correctors (VRT-325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell-specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro-Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse-chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX-809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX-809 significantly stabilizes F508del-CFTR immature form, an effect that is not observed for C3 nor C4. VX-809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX-809 increases the stability of F508del-CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue. PMID:26171232

The ΔF508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Is Associated With Progressive Insulin Resistance and Decreased Functional β-Cell Mass in Mice.

PubMed

Fontés, Ghislaine; Ghislain, Julien; Benterki, Isma; Zarrouki, Bader; Trudel, Dominique; Berthiaume, Yves; Poitout, Vincent

2015-12-01

Cystic fibrosis (CF) is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF-related diabetes affects 50% of adult CF patients. How CFTR deficiency predisposes to diabetes is unknown. Herein, we examined the impact of the most frequent cftr mutation in humans, deletion of phenylalanine at position 508 (ΔF508), on glucose homeostasis in mice. We compared ΔF508 mutant mice with wild-type (WT) littermates. Twelve-week-old male ΔF508 mutants had lower body weight, improved oral glucose tolerance, and a trend toward higher insulin tolerance. Glucose-induced insulin secretion was slightly diminished in ΔF508 mutant islets, due to reduced insulin content, but ΔF508 mutant islets were not more sensitive to proinflammatory cytokines than WT islets. Hyperglycemic clamps confirmed an increase in insulin sensitivity with normal β-cell function in 12- and 18-week-old ΔF508 mutants. In contrast, 24-week-old ΔF508 mutants exhibited insulin resistance and reduced β-cell function. β-Cell mass was unaffected at 11 weeks of age but was significantly lower in ΔF508 mutants versus controls at 24 weeks. This was not associated with gross pancreatic pathology. We conclude that the ΔF508 CFTR mutation does not lead to an intrinsic β-cell secretory defect but is associated with insulin resistance and a β-cell mass deficit in aging mutants. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR.

PubMed

Trzcińska-Daneluti, Agata M; Chen, Anthony; Nguyen, Leo; Murchie, Ryan; Jiang, Chong; Moffat, Jason; Pelletier, Lawrence; Rotin, Daniela

2015-06-01

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR). ΔF508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ΔF508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ∼750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ΔF508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ΔF508-CFTR and measuring the amount of surface ΔF508-CFTR by ELISA. The role of FGFR signaling on ΔF508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLCγ-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ΔF508-CFTR homozygous mice resulted in a robust ΔF508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ΔF508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ΔF508/ΔF508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ΔF508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of CF. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased NF-κB Activity and Decreased Wnt/β-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice.

PubMed

Le Henaff, Carole; Mansouri, Rafik; Modrowski, Dominique; Zarka, Mylène; Geoffroy, Valérie; Marty, Caroline; Tarantino, Nadine; Laplantine, Emmanuel; Marie, Pierre J

2015-07-17

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased β-catenin phosphorylation, reduced osteoblast β-catenin expression, and altered expression of Wnt/β-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/β-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/β-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased NF-κB Activity and Decreased Wnt/β-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice*

PubMed Central

Le Henaff, Carole; Mansouri, Rafik; Modrowski, Dominique; Zarka, Mylène; Geoffroy, Valérie; Marty, Caroline; Tarantino, Nadine; Laplantine, Emmanuel; Marie, Pierre J.

2015-01-01

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased β-catenin phosphorylation, reduced osteoblast β-catenin expression, and altered expression of Wnt/β-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/β-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/β-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis. PMID:26060255

High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue.

PubMed

Perkins, Lydia A; Fisher, Gregory W; Naganbabu, Matharishwan; Schmidt, Brigitte F; Mun, Frederick; Bruchez, Marcel P

2018-03-05

The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon's ON-TARGET plus SMARTpool siRNA Kinase library (715 target kinases) with and without 10 μM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment.

Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor).

PubMed

Matthes, Elizabeth; Goepp, Julie; Carlile, Graeme W; Luo, Yishan; Dejgaard, Kurt; Billet, Arnaud; Robert, Renaud; Thomas, David Y; Hanrahan, John W

2016-02-01

The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a 'potentiator' to increase channel open probability and a 'corrector' that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood. CF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin-stimulated short-circuit current (Isc ) across airway epithelial monolayers expressing F508del CFTR. The potentiated Isc response during forskolin stimulation was increased sixfold after pretreatment with VX-809 alone and reached ~11% that measured across non-CF monolayers. VX-770 (100 nM) and genistein (50 μM) caused similar levels of potentiation, which were not additive and were abolished by the CFTR inhibitor CFTRinh -172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which together with previous measurements in patients given 250 mg p.o. twice daily, suggests a peak free plasma concentration of 1.5-8.5 nM. Chronic exposure to high VX-770 concentrations (>1 μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20-40 mg·mL(-1) ). Chronic exposure to a low concentration of VX-770 (100 nM) together with VX-809 (1 μM) also did not reduce the forskolin-stimulated Isc , relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator. Chronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770. © 2015

Fatty Acid Cysteamine Conjugates as Novel and Potent Autophagy Activators That Enhance the Correction of Misfolded F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

PubMed

Vu, Chi B; Bridges, Robert J; Pena-Rasgado, Cecilia; Lacerda, Antonio E; Bordwell, Curtis; Sewell, Abby; Nichols, Andrew J; Chandran, Sachin; Lonkar, Pallavi; Picarella, Dominic; Ting, Amal; Wensley, Allison; Yeager, Maisy; Liu, Feng

2017-01-12

A depressed autophagy has previously been reported in cystic fibrosis patients with the common F508del-CFTR mutation. This report describes the synthesis and preliminary biological characterization of a novel series of autophagy activators involving fatty acid cysteamine conjugates. These molecular entities were synthesized by first covalently linking cysteamine to docosahexaenoic acid. The resulting conjugate 1 synergistically activated autophagy in primary homozygous F508del-CFTR human bronchial epithelial (hBE) cells at submicromolar concentrations. When conjugate 1 was used in combination with the corrector lumacaftor and the potentiator ivacaftor, it showed an additive effect, as measured by the increase in the chloride current in a functional assay. In order to obtain a more stable form for oral dosing, the sulfhydryl group in conjugate 1 was converted into a functionalized disulfide moiety. The resulting conjugate 5 is orally bioavailable in the mouse, rat, and dog and allows a sustained delivery of the biologically active conjugate 1.

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator.

PubMed

Luciani, Alessandro; Villella, Valeria Rachela; Esposito, Speranza; Gavina, Manuela; Russo, Ilaria; Silano, Marco; Guido, Stefano; Pettoello-Mantovani, Massimo; Carnuccio, Rosa; Scholte, Bob; De Matteis, Antonella; Maiuri, Maria Chiara; Raia, Valeria; Luini, Alberto; Kroemer, Guido; Maiuri, Luigi

2012-11-01

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.

Aspergillus fumigatus generates an enhanced Th2-biased immune response in mice with defective cystic fibrosis transmembrane conductance regulator.

PubMed

Allard, Jenna B; Poynter, Matthew E; Marr, Kieren A; Cohn, Lauren; Rincon, Mercedes; Whittaker, Laurie A

2006-10-15

Cystic fibrosis (CF) lung disease is characterized by persistent airway inflammation and airway infection that ultimately leads to respiratory failure. Aspergillus sp. are present in the airways of 20-40% of CF patients and are of unclear clinical significance. In this study, we demonstrate that CF transmembrane conductance regulator (CFTR)-deficient (CFTR knockout, Cftr(tm1Unc-)TgN(fatty acid-binding protein)CFTR) and mutant (DeltaF508) mice develop profound lung inflammation in response to Aspergillus fumigatus hyphal Ag exposure. CFTR-deficient mice also develop an enhanced Th2 inflammatory response to A. fumigatus, characterized by elevated IL-4 in the lung and IgE and IgG1 in serum. In contrast, CFTR deficiency does not promote a Th1 immune response. Furthermore, we demonstrate that CD4+ T cells from naive CFTR-deficient mice produce higher levels of IL-4 in response to TCR ligation than wild-type CD4+ T cells. The Th2 bias of CD4+ T cells in the absence of functional CFTR correlates with elevated nuclear levels of NFAT. Thus, CFTR is important to maintain the Th1/Th2 balance in CD4+ T cells.

Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation.

PubMed

Clancy, J P; Rowe, Steven M; Accurso, Frank J; Aitken, Moira L; Amin, Raouf S; Ashlock, Melissa A; Ballmann, Manfred; Boyle, Michael P; Bronsveld, Inez; Campbell, Preston W; De Boeck, Kris; Donaldson, Scott H; Dorkin, Henry L; Dunitz, Jordan M; Durie, Peter R; Jain, Manu; Leonard, Anissa; McCoy, Karen S; Moss, Richard B; Pilewski, Joseph M; Rosenbluth, Daniel B; Rubenstein, Ronald C; Schechter, Michael S; Botfield, Martyn; Ordoñez, Claudia L; Spencer-Green, George T; Vernillet, Laurent; Wisseh, Steve; Yen, Karl; Konstan, Michael W

2012-01-01

VX-809, a cystic fibrosis transmembrane conductance regulator (CFTR) modulator, has been shown to increase the cell surface density of functional F508del-CFTR in vitro. A randomised, double-blind, placebo-controlled study evaluated the safety, tolerability and pharmacodynamics of VX-809 in adult patients with cystic fibrosis (n=89) who were homozygous for the F508del-CFTR mutation. Subjects were randomised to one of four VX-809 28 day dose groups (25, 50, 100 and 200 mg) or matching placebo. The type and incidence of adverse events were similar among VX-809- and placebo-treated subjects. Respiratory events were the most commonly reported and led to discontinuation by one subject in each active treatment arm. Pharmacokinetic data supported a once-daily oral dosing regimen. Pharmacodynamic data suggested that VX-809 improved CFTR function in at least one organ (sweat gland). VX-809 reduced elevated sweat chloride values in a dose-dependent manner (p=0.0013) that was statistically significant in the 100 and 200 mg dose groups. There was no statistically significant improvement in CFTR function in the nasal epithelium as measured by nasal potential difference, nor were there statistically significant changes in lung function or patient-reported outcomes. No maturation of immature F508del-CFTR was detected in the subgroup that provided rectal biopsy specimens. In this study, VX-809 had a similar adverse event profile to placebo for 28 days in F508del-CFTR homozygous patients, and demonstrated biological activity with positive impact on CFTR function in the sweat gland. Additional data are needed to determine how improvements detected in CFTR function secondary to VX-809 in the sweat gland relate to those measurable in the respiratory tract and to long-term measures of clinical benefit. NCT00865904.

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

PubMed Central

Luciani, Alessandro; Villella, Valeria Rachela; Esposito, Speranza; Gavina, Manuela; Russo, Ilaria; Silano, Marco; Guido, Stefano; Pettoello-Mantovani, Massimo; Carnuccio, Rosa; Scholte, Bob; De Matteis, Antonella; Maiuri, Maria Chiara; Raia, Valeria; Luini, Alberto; Kroemer, Guido; Maiuri, Luigi

2012-01-01

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators. PMID:22874563

Mechanism-based corrector combination restores ΔF508-CFTR folding and function

PubMed Central

Okiyoneda, Tsukasa; Veit, Guido; Dekkers, Johanna F.; Bagdany, Miklos; Soya, Naoto; Xu, Haijin; Roldan, Ariel; Verkman, Alan S.; Kurth, Mark; Simon, Agnes; Hegedus, Tamas; Beekman, Jeffrey M.; Lukacs, Gergely L.

2013-01-01

The most common cystic fibrosis (CF) mutation, ΔF508 in the nucleotide binding domain-1 (NBD1), impairs CFTR coupled-domain folding, plasma membrane (PM) expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and incompletely understood mechanism, hampering drug development. Based on the effect of second site suppressor mutations, robust ΔF508-CFTR correction likely requires stabilization of NBD1 and the membrane spanning domains (MSDs)-NBD1 interface, both established primary conformational defects. Here, we elucidated the molecular targets of available correctors; class-I stabilizes the NBD1-MSD1/2 interface, class-II targets NBD2, and only chemical chaperones, surrogates of class-III correctors, stabilize the human ΔF508-NBD1. While VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional PM expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in CF bronchial epithelial cells and intestinal organoids. Thus, structure-guided corrector combination represents an effective approach for CF therapy. PMID:23666117

Lumacaftor/Ivacaftor in Patients Aged 6-11 Years with Cystic Fibrosis and Homozygous for F508del-CFTR.

PubMed

Milla, Carlos E; Ratjen, Felix; Marigowda, Gautham; Liu, Fang; Waltz, David; Rosenfeld, Margaret

2017-04-01

Combination lumacaftor/ivacaftor has been shown to improve lung function and other endpoints in patients aged 12 years and older with cystic fibrosis and homozygous for F508del-CFTR, but it has not been assessed in younger patients. In this open-label phase III trial, we evaluated the safety, tolerability, pharmacodynamics, and efficacy of lumacaftor/ivacaftor combination therapy in patients aged 6-11 years with cystic fibrosis who were homozygous for F508del-CFTR. Patients (N = 58) received 200 mg lumacaftor/250 mg ivacaftor orally every 12 hours for 24 weeks in addition to their existing cystic fibrosis medications. Lumacaftor/ivacaftor was well tolerated; the safety profile was generally similar to that observed in larger lumacaftor/ivacaftor trials with older patients. Four patients discontinued (two because of drug-related adverse events: elevated liver transaminases, n = 1; rash, n = 1). No safety concerns were associated with spirometry. No significant changes in percent predicted FEV 1 were observed (change from baseline at Week 24, +2.5 percentage points; 95% confidence interval [CI], -0.2 to 5.2; P = 0.0671). At Week 24, significant improvements from baseline were observed in sweat chloride (-24.8 mmol/L; 95% CI, -29.1 to -20.5; P < 0.0001), body mass index z score (+0.15; 95% CI, 0.08 to 0.22; P < 0.0001), Cystic Fibrosis Questionnaire-Revised respiratory domain score (+5.4; 95% CI, 1.4 to 9.4; P = 0.0085), and lung clearance index based on lung volume turnover required to reach 2.5% of starting N 2 concentration (-0.88; 95% CI, -1.40 to -0.37; P = 0.0018). Lumacaftor/ivacaftor was well tolerated in this young population; no new safety concerns were identified. Improvements in lung clearance index, sweat chloride, nutritional status, and health-related quality of life were observed after 24 weeks of treatment. Clinical trial registered with www.clinicaltrials.gov (NCT01897233).

SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation.

PubMed

Ramachandran, Shyam; Osterhaus, Samantha R; Parekh, Kalpaj R; Jacobi, Ashley M; Behlke, Mark A; McCray, Paul B

2016-12-02

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl - conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl - transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl - conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Induction of HSP70 promotes DeltaF508 CFTR trafficking.

PubMed

Choo-Kang, L R; Zeitlin, P L

2001-07-01

The DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is a temperature-sensitive trafficking mutant that is detected as an immature 160-kDa form (band B) in gel electrophoresis. The goal of this study was to test the hypothesis that HSP70, a member of the 70-kDa heat shock protein family, promotes DeltaF508 CFTR processing to the mature 180-kDa form (band C). Both pharmacological and genetic techniques were used to induce HSP70. IB3-1 cells were treated with sodium 4-phenylbutyrate (4PBA) to promote maturation of DeltaF508 CFTR to band C. A dose-dependent increase in band C and total cellular HSP70 was observed. Under these conditions, HSP70-CFTR complexes were increased and 70-kDa heat shock cognate protein-CFTR complexes were decreased. Increased DeltaF508 CFTR maturation was also seen after transfection with an HSP70 expression plasmid and exposure to glutamine, an inducer of HSP70. With immunofluorescence techniques, the increased appearance of CFTR band C correlated with CFTR distribution beyond the perinuclear regions. These data suggest that induction of HSP70 promotes DeltaF508 CFTR maturation and trafficking.

Epithelial IgG and its relationship to the loss of F508 in the common mutant form of the cystic fibrosis transmembrane conductance regulator.

PubMed

Treharne, Kate J; Cassidy, Diane; Goddard, Catharine; Colledge, William H; Cassidy, Andrew; Mehta, Anil

2009-08-06

The most debilitating feature of cystic fibrosis (CF) disease is uncontrolled inflammation of respiratory epithelium. The relationship between the commonest mutated form of CFTR (F508del or DeltaF508) and inflammation has not yet been elucidated. Here, we present a new paradigm suggesting that CFTR can interact with intra-epithelial IgG, establishing a direct link between normal CFTR and the immune system. Further, our data show that the amino-acid sequence local to F508 can bind IgG with high affinity, dependent on F508, such that loss of F508 abolishes this link both in vitro and in the intact cell.

Defective Fluid Secretion from Submucosal Glands of Nasal Turbinates from CFTR-/- and CFTRΔF508/ΔF508 Pigs

PubMed Central

Cho, Hyung-Ju; Joo, Nam Soo; Wine, Jeffrey J.

2011-01-01

Background Cystic fibrosis (CF), caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion. Methodology/Principal Findings Unexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area), more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands). Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls) in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland) were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CFΔF508/ΔF508 with CFTR-/- piglets and found significantly greater forskolin-stimulated secretion rates in the ΔF508 vs. the null piglets (1.4±0.8, n = 4 vs. 0.2±0.1, n = 7). An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ∼4 times greater in adult than in neonatal nasal glands. Conclusions/Significance These findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ΔF508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections. PMID:21935358

∆F508 CFTR interactome remodelling promotes rescue of cystic fibrosis.

PubMed

Pankow, Sandra; Bamberger, Casimir; Calzolari, Diego; Martínez-Bartolomé, Salvador; Lavallée-Adam, Mathieu; Balch, William E; Yates, John R

2015-12-24

Deletion of phenylalanine 508 of the cystic fibrosis transmembrane conductance regulator (∆F508 CFTR) is the major cause of cystic fibrosis, one of the most common inherited childhood diseases. The mutated CFTR anion channel is not fully glycosylated and shows minimal activity in bronchial epithelial cells of patients with cystic fibrosis. Low temperature or inhibition of histone deacetylases can partly rescue ∆F508 CFTR cellular processing defects and function. A favourable change of ∆F508 CFTR protein-protein interactions was proposed as a mechanism of rescue; however, CFTR interactome dynamics during temperature shift and inhibition of histone deacetylases are unknown. Here we report the first comprehensive analysis of the CFTR and ∆F508 CFTR interactome and its dynamics during temperature shift and inhibition of histone deacetylases. By using a novel deep proteomic analysis method, we identify 638 individual high-confidence CFTR interactors and discover a ∆F508 deletion-specific interactome, which is extensively remodelled upon rescue. Detailed analysis of the interactome remodelling identifies key novel interactors, whose loss promote ∆F508 CFTR channel function in primary cystic fibrosis epithelia or which are critical for CFTR biogenesis. Our results demonstrate that global remodelling of ∆F508 CFTR interactions is crucial for rescue, and provide comprehensive insight into the molecular disease mechanisms of cystic fibrosis caused by deletion of F508.

Association between F508 deletion in CFTR and chronic pancreatitis risk.

PubMed

Zhao, Dong; Xu, Yanzhen; Li, Jiatong; Fu, Shien; Xiao, Feifan; Song, Xiaowei; Xie, Zhibin; Jiang, Min; He, Yan; Liu, Chengwu; Wen, Qiongxian; Yang, Xiaoli

2017-09-01

The cystic fibrosis transmembrane conductance regulator (CFTR) has been reported to influence individual susceptibility to chronic pancreatitis (CP), but the results of previous studies are controversial. We performed a study to demonstrate the relationship between CFTR and CP. We searched PubMed, Scopus, and Embase for studies of patients with CP. Seven studies from 1995 to 2016 were identified, and included 64,832 patients. Pooled prevalence and 95% confidence intervals (CIs) were calculated. F508 deletion in CFTR was significantly positively associated with CP risk in the overall analysis (odds ratio [OR]=3.20, 95% CI: 2.30-4.44, I 2 =31.7%). In subgroup analysis stratified by ethnicity, F508 deletion was significantly associated with CP risk in Indian populations, using a fixed effects model (ORs=5.45, 95% CI: 2.52-11.79, I 2 =0.0%), and in non-Indian populations, using a random effects model (ORs=3.59, 95% CI: 1.73-7.48, I 2 =60.9%). At the same time, we found that Indians with F508 deletion had much higher CP prevalence than non-Indians. Interestingly, F508 deletion was also associated with CP and idiopathic CP risk in subgroup analysis stratified by aeitiology, using the fixed effects model. Based on current evidence, F508 deletion is a risk factor for CP, and Indians with F508 deletion have much higher CP morbidity. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Missense variations in the cystic fibrosis gene: Heteroduplex formation in the F508C mutation

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

Macek, M. Jr.; Ladanyi, L.; Buerger, J.

1992-11-01

Kobayashi et al. (1990) have described missense variations in the conserved region of exon 10 of the cystic fibrosis (CF) transmembrane conductance regulator gene. In their paper, two [Delta]F508/F508C compound heterozygous individuals were reported. Clinical and epithelial physiological studies in both cases were normal, suggesting that the substitution of cysteine for phenylalanine at position 508, the F508C mutation, is benign. However, Kerem et al. reported a patient with this substitution who had typical symptoms of CF. In routine [Delta]F508 mutation screening by visualization of the 3-bp deletion on a 12% polyacrylamide gel the authors detected an abnormal heteroduplex in themore » father of a CF patient of German origin. Subsequent direct sequencing of the PCR product confirmed that this clinically normal father is a compound heterozygote for the [Delta]F508/F508C mutations. This heteroduplex is slightly different from the usual heteroduplex in [Delta]F508/F508C heteroduplex was not published, it is likely that similar cases can be overseen during the widely performed [Delta]F508 mutation screening by PAGE. Detection of more cases, such as the one presented here, together with careful, standardized clinical examination of the proband, would be valuable to verify the nature of this mutation. 4 refs., 1 fig.« less

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation.

PubMed

Ferrari, Eleonora; Monzani, Romina; Villella, Valeria R; Esposito, Speranza; Saluzzo, Francesca; Rossin, Federica; D'Eletto, Manuela; Tosco, Antonella; De Gregorio, Fabiola; Izzo, Valentina; Maiuri, Maria C; Kroemer, Guido; Raia, Valeria; Maiuri, Luigi

2017-01-12

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation

PubMed Central

Ferrari, Eleonora; Monzani, Romina; Villella, Valeria R; Esposito, Speranza; Saluzzo, Francesca; Rossin, Federica; D'Eletto, Manuela; Tosco, Antonella; De Gregorio, Fabiola; Izzo, Valentina; Maiuri, Maria C; Kroemer, Guido; Raia, Valeria; Maiuri, Luigi

2017-01-01

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF. PMID:28079883

Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing.

PubMed

Chung, Woo Young; Song, Myungjae; Park, Jinhong; Namkung, Wan; Lee, Jinu; Kim, Hyongbum; Lee, Min Goo; Kim, Joo Young

2016-12-01

To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.

Nebulized hyaluronan ameliorates lung inflammation in cystic fibrosis mice.

PubMed

Gavina, Manuela; Luciani, Alessandro; Villella, Valeria R; Esposito, Speranza; Ferrari, Eleonora; Bressani, Ilaria; Casale, Alida; Bruscia, Emanuela M; Maiuri, Luigi; Raia, Valeria

2013-08-01

Chronic lung inflammation with increased susceptibility to bacterial infections cause much of the morbidity and mortality in patients with cystic fibrosis (CF), the most common severe, autosomal recessively inherited disease in the Caucasian population. Exogenous inhaled hyaluronan (HA) can exert a protective effect against injury and beneficial effects of HA have been shown in experimental models of chronic respiratory diseases. Our objective was to examine whether exogenous administration of nebulized HA might interfere with lung inflammation in CF. F508del homozygous mice (Cftr(F508del) ) and transgenic mice overexpressing the ENaC channel β-subunit (Scnn1b-Tg) were treated with nebulized HA (0.5 mg/mouse/day for 7 days). Tumor necrosis factor-alpha (TNFα), macrophage inflammatory protein-2 (MIP-2), myeloperoxidase (MPO) levels, and macrophage infiltration were assessed on lung tissues. IB3-1 and CFBE41o-epithelial cell lines were cultured with HA (24 hr, 100 µg/ml) and Reactive Oxygen Species (ROS), Tissue Transglutaminase (TG2) SUMOylation and Peroxisome Proliferator Activated Receptor gamma (PPARγ) and phospho-p42/p44 levels were measured by dichlorodihydrofluorescein assay, or fluorescence resonance energy transfer (FRET) microscopy or immunoblots. Nebulized HA reduced TNFα expression (P < 0.005); TNFα, MIP-2, and MPO protein levels (P < 0.05); MPO activity (P < 0.05); and CD68+ cells counts (P < 0.005) in lung tissues of Cftr(F508del) and Scnn1b-Tg mice, compared with saline-treated mice. HA reduced ROS, TG2 SUMOylation, TG2 activity, phospho-p42-44, and increased PPARγ protein in both IB3-1 and CFBE41o cells (P < 0.05). Nebulized HA is effective in controlling inflammation in vivo in mice CF airways and in vitro in human airway epithelial cells. We provide the proof of concept for the use of inhaled HA as a potential anti-inflammatory drug in CF therapy. Copyright © 2012 Wiley Periodicals, Inc.

Some gating potentiators, including VX-770, diminish ΔF508-CFTR functional expression

PubMed Central

Veit, Guido; Avramescu, Radu G.; Perdomo, Doranda; Phuan, Puay-Wah; Bagdany, Miklos; Apaja, Pirjo M.; Borot, Florence; Szollosi, Daniel; Wu, Yu-Sheng; Finkbeiner, Walter E.; Hegedus, Tamas; Verkman, Alan S.; Lukacs, Gergely L.

2015-01-01

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane regulator (CFTR) that result in reduced anion conductance at the apical membrane of secretory epithelia. Treatment of CF patients carrying the G551D gating mutation with the potentiator VX-770 (ivacaftor) largely restores channel activity and has shown substantial clinical benefit. However, most CF patients carry the ΔF508 mutation, which impairs CFTR folding, processing, function, and stability. Studies in homozygous ΔF508 CF patients indicated little clinical benefit of monotherapy with the investigational corrector VX-809 (lumacaftor) or VX-770, whereas combination clinical trials show limited but significant improvements in lung function. We show that VX-770, as well as most other potentiators, reduces the correction efficacy of VX-809 and another investigational corrector, VX-661. To mimic the administration of VX-770 alone or in combination with VX-809, we examined its long-term effect in immortalized and primary human respiratory epithelia. VX-770 diminished the folding efficiency and the metabolic stability of ΔF508-CFTR at the endoplasmic reticulum (ER) and post-ER compartments, respectively, causing reduced cell surface ΔF508-CFTR density and function. VX-770–induced destabilization of ΔF508-CFTR was influenced by second-site suppressor mutations of the folding defect and was prevented by stabilization of the nucleotide-binding domain 1 (NBD1)–NBD2 interface. The reduced correction efficiency of ΔF508-CFTR, as well as of two other processing mutations in the presence of VX-770, suggests the need for further optimization of potentiators to maximize the clinical benefit of corrector-potentiator combination therapy in CF. PMID:25101887

5'-adenosine monophosphate mediated cooling treatment enhances ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) stability in vivo.

PubMed

Zhang, Yueqiang; O'Brien, William G; Zhao, Zhaoyang; Lee, Cheng Chi

2015-09-04

Gene mutations that produce misprocessed proteins are linked to many human disorders. Interestingly, some misprocessed proteins retained their biological function when stabilized by low temperature treatment of cultured cells in vitro. Here we investigate whether low temperature treatment in vivo can rescue misfolded proteins by applying 5'-AMP mediated whole body cooling to a Cystic Fibrosis (CF) mouse model carrying a mutant cystic fibrosis transmembrane conductance regulator (CFTR) with a deletion of the phenylalanine residue in position 508 (ΔF508-CFTR). Low temperature treatment of cultured cells was previously shown to be able to alleviate the processing defect of ΔF508-CFTR, enhancing its plasma membrane localization and its function in mediating chloride ion transport. Here, we report that whole body cooling enhanced the retention of ΔF508-CFTR in intestinal epithelial cells. Functional analysis based on β-adrenergic dependent salivary secretion and post-natal mortality rate revealed a moderate but significant improvement in treated compared with untreated CF mice. Our findings demonstrate that temperature sensitive processing of mutant proteins can be responsive to low temperature treatment in vivo.

Some gating potentiators, including VX-770, diminish ΔF508-CFTR functional expression.

PubMed

Veit, Guido; Avramescu, Radu G; Perdomo, Doranda; Phuan, Puay-Wah; Bagdany, Miklos; Apaja, Pirjo M; Borot, Florence; Szollosi, Daniel; Wu, Yu-Sheng; Finkbeiner, Walter E; Hegedus, Tamas; Verkman, Alan S; Lukacs, Gergely L

2014-07-23

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane regulator (CFTR) that result in reduced anion conductance at the apical membrane of secretory epithelia. Treatment of CF patients carrying the G551D gating mutation with the potentiator VX-770 (ivacaftor) largely restores channel activity and has shown substantial clinical benefit. However, most CF patients carry the ΔF508 mutation, which impairs CFTR folding, processing, function, and stability. Studies in homozygous ΔF508 CF patients indicated little clinical benefit of monotherapy with the investigational corrector VX-809 (lumacaftor) or VX-770, whereas combination clinical trials show limited but significant improvements in lung function. We show that VX-770, as well as most other potentiators, reduces the correction efficacy of VX-809 and another investigational corrector, VX-661. To mimic the administration of VX-770 alone or in combination with VX-809, we examined its long-term effect in immortalized and primary human respiratory epithelia. VX-770 diminished the folding efficiency and the metabolic stability of ΔF508-CFTR at the endoplasmic reticulum (ER) and post-ER compartments, respectively, causing reduced cell surface ΔF508-CFTR density and function. VX-770-induced destabilization of ΔF508-CFTR was influenced by second-site suppressor mutations of the folding defect and was prevented by stabilization of the nucleotide-binding domain 1 (NBD1)-NBD2 interface. The reduced correction efficiency of ΔF508-CFTR, as well as of two other processing mutations in the presence of VX-770, suggests the need for further optimization of potentiators to maximize the clinical benefit of corrector-potentiator combination therapy in CF. Copyright © 2014, American Association for the Advancement of Science.

Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR

PubMed Central

Aleksandrov, Andrei A.; Kota, Pradeep; Cui, Liying; Jensen, Tim; Alekseev, Alexey E.; Reyes, Santiago; He, Lihua; Gentzsch, Martina; Aleksandrov, Luba A.; Dokholyan, Nikolay V.; Riordan, John R.

2013-01-01

Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remain incomplete. Here we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in the first nucleotide domain (NBD1), including the structurally diverse region (SDR), the gamma phosphate switch loop and the Regulatory Insertion (RI). Molecular Dynamic analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild-type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the SDR, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein’s inherent stability and channel activity returns a near-normal state. PMID:22406676

Activation of 3-Phosphoinositide-dependent Kinase 1 (PDK1) and Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) by Short-chain Sphingolipid C4-ceramide Rescues the Trafficking Defect of ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR)*

PubMed Central

Caohuy, Hung; Yang, Qingfeng; Eudy, Yvonne; Ha, Thien-An; Xu, Andrew E.; Glover, Matthew; Frizzell, Raymond A.; Jozwik, Catherine; Pollard, Harvey B.

2014-01-01

Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser422. SGK1[Ser(P)422] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser241. Then PDK1[Ser(P)241] phosphorylates SGK1[Ser(P)422] at Thr256 to generate fully activated SGK1[Ser422, Thr(P)256]. SGK1[Ser(P)422,Thr(P)256] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day. PMID:25384981

Genetic deletion of keratin 8 corrects the altered bone formation and osteopenia in a mouse model of cystic fibrosis.

PubMed

Le Henaff, Carole; Faria Da Cunha, Mélanie; Hatton, Aurélie; Tondelier, Danielle; Marty, Caroline; Collet, Corinne; Zarka, Mylène; Geoffroy, Valérie; Zatloukal, Kurt; Laplantine, Emmanuel; Edelman, Aleksander; Sermet-Gaudelus, Isabelle; Marie, Pierre J

2016-04-01

Patients with cystic fibrosis (CF) display low bone mass and alterations in bone formation. Mice carrying the F508del genetic mutation in the cystic fibrosis conductance regulator (Cftr) gene display reduced bone formation and decreased bone mass. However, the underlying molecular mechanisms leading to these skeletal defects are unknown, which precludes the development of an efficient anti-osteoporotic therapeutic strategy. Here we report a key role for the intermediate filament protein keratin 8 (Krt8), in the osteoblast dysfunctions in F508del-Cftr mice. We found that murine and human osteoblasts express Cftr and Krt8 at low levels. Genetic studies showed that Krt8 deletion (Krt8(-/-)) in F508del-Cftr mice increased the levels of circulating markers of bone formation, corrected the expression of osteoblast phenotypic genes, promoted trabecular bone formation and improved bone mass and microarchitecture. Mechanistically, Krt8 deletion in F508del-Cftr mice corrected overactive NF-κB signaling and decreased Wnt-β-catenin signaling induced by the F508del-Cftr mutation in osteoblasts. In vitro, treatment with compound 407, which specifically disrupts the Krt8-F508del-Cftr interaction in epithelial cells, corrected the abnormal NF-κB and Wnt-β-catenin signaling and the altered phenotypic gene expression in F508del-Cftr osteoblasts. In vivo, short-term treatment with 407 corrected the altered Wnt-β-catenin signaling and bone formation in F508del-Cftr mice. Collectively, the results show that genetic or pharmacologic targeting of Krt8 leads to correction of osteoblast dysfunctions, altered bone formation and osteopenia in F508del-Cftr mice, providing a therapeutic strategy targeting the Krt8-F508del-CFTR interaction to correct the abnormal bone formation and bone loss in cystic fibrosis. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Efficacy and safety of lumacaftor and ivacaftor in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR: a randomised, placebo-controlled phase 3 trial.

PubMed

Ratjen, Felix; Hug, Christopher; Marigowda, Gautham; Tian, Simon; Huang, Xiaohong; Stanojevic, Sanja; Milla, Carlos E; Robinson, Paul D; Waltz, David; Davies, Jane C

2017-07-01

Lumacaftor and ivacaftor combination treatment showed efficacy in patients aged 12 years or older with cystic fibrosis homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in placebo-controlled studies and patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR in an open-label study. We report efficacy and safety of lumacaftor and ivacaftor in patients with cystic fibrosis aged 6-11 years homozygous for F508del-CFTR. In this phase 3, randomised, double-blind, placebo-controlled, multicentre study, patients were enrolled at 54 hospitals and medical centres in nine countries (the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK). Eligible patients weighed at least 15 kg, with a confirmed diagnosis of cystic fibrosis, percent predicted forced expiratory volume in 1 s (FEV 1 ) of 70 or more, and lung clearance index 2·5 (LCI 2·5 ) of 7·5 or more at screening (values less than these thresholds were permitted at day 1). All patients were tested for CFTR genotype at screening; eligible patients had to have the F508del-CFTR mutation on both alleles. Exclusion criteria included any comorbidity or laboratory abnormality that might confound the study results or pose additional risk to the patient. Patients were stratified by weight (<25 kg vs ≥25 kg) and ppFEV 1 severity (<90 vs ≥90) determined at the screening visit, and randomly assigned 1:1 to treatment using an interactive web response system to receive 200 mg lumacaftor and 250 mg ivacaftor every 12 hours or placebo for 24 weeks. Patients, all site personnel including the investigator and the site monitor, and the study team were blinded, with the exception of site personnel needing this information in the event of medical emergency or pregnancy and patient safety and regulatory affairs personnel to meet serious adverse event reporting requirements. The primary endpoint was the mean absolute change in LCI 2·5 from all on-treatment study

A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator

PubMed Central

Bali, Vedrana; Lazrak, Ahmed; Guroji, Purushotham; Fu, Lianwu; Matalon, Sadis; Bebok, Zsuzsanna

2016-01-01

Synonymous mutations, such as I507-ATC→ATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), the most frequent disease-associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small-molecule drugs are being developed to correct ΔF508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on I507-ATT and I507-ATC ΔF508 CFTR biogenesis and function. C4 stabilized I507-ATT ΔF508 CFTR band B, but without considerable biochemical and functional correction. VX-809 biochemically corrected ∼10% of both of the variants, leading to stable, forskolin+3-isobutyl-1-methylxanthine (IBMX)-activated whole-cell currents in the presence of the corrector. Omitting VX-809 during whole-cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX-809. Treatment of cells with the C4+VX-809 combination resulted in enhanced rescue and 2-fold higher forskolin+IBMX–activated currents of both I507-ATT and I507-ATC ΔF508 CFTR, compared with VX-809 treatment alone. The lack of an effect of C4 on I507-ATC ΔF508 CFTR, but its additive effect in combination with VX-809, implies that C4 acted on VX-809–modified I507-ATC ΔF508 CFTR. Our results suggest that binding of C4 and VX-809 to ΔF508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.—Bali, V., Lazrak, A., Guroji, P., Fu, L., Matalon, S., Bebok, Z. A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator. PMID:26336913

Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR.

PubMed

Rubenstein, R C; Zeitlin, P L

2000-02-01

The most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), DeltaF508, is a trafficking mutant that has prolonged associations with molecular chaperones and is rapidly degraded, at least in part by the ubiquitin-proteasome system. Sodium 4-phenylbutyrate (4PBA) improves DeltaF508-CFTR trafficking and function in vitro in cystic fibrosis epithelial cells and in vivo. To further understand the mechanism of action of 4PBA, we tested the hypothesis that 4PBA modulates the targeting of DeltaF508-CFTR for ubiquitination and degradation by reducing the expression of Hsc70 in cystic fibrosis epithelial cells. IB3-1 cells (genotype DeltaF508/W1282X) that were treated with 0.05-5 mM 4PBA for 2 days in culture demonstrated a dose-dependent reduction in Hsc70 protein immunoreactivity and mRNA levels. Immunoprecipitation with Hsc70-specific antiserum demonstrated that Hsc70 and CFTR associated under control conditions and that treatment with 4PBA reduced these complexes. Levels of immunoreactive Hsp40, Hdj2, Hsp70, Hsp90, and calnexin were unaffected by 4PBA treatment. These data suggest that 4PBA may improve DeltaF508-CFTR trafficking by allowing a greater proportion of mutant CFTR to escape association with Hsc70.

Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508DEL-CFTR.

PubMed

Donaldson, Scott H; Solomon, George M; Zeitlin, Pamela L; Flume, Patrick A; Casey, Alicia; McCoy, Karen; Zemanick, Edith T; Mandagere, Arun; Troha, Janice M; Shoemaker, Steven A; Chmiel, James F; Taylor-Cousar, Jennifer L

2017-05-01

Cavosonstat (N91115), an orally bioavailable inhibitor of S-nitrosoglutathione reductase, promotes cystic fibrosis transmembrane conductance regulator (CFTR) maturation and plasma membrane stability, with a mechanism of action complementary to CFTR correctors and potentiators. A Phase I program evaluated pharmacokinetics, drug-drug interactions and safety of cavosonstat in healthy and cystic fibrosis (CF) subjects homozygous for F508del-CFTR. Exploratory outcomes included changes in sweat chloride in CF subjects. Cavosonstat was rapidly absorbed and demonstrated linear and predictable pharmacokinetics. Exposure was unaffected by a high-fat meal or rifampin-mediated effects on drug metabolism and transport. Cavosonstat was well tolerated, with no dose-limiting toxicities or significant safety findings. At the highest dose, significant reductions from baseline in sweat chloride were observed (-4.1mmol/L; P=0.032) at day 28. The favorable safety and clinical profile warrant further study of cavosonstat in CF. ClinicalTrials.gov Numbers: NCT02275936, NCT02013388, NCT02500667. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Curcumin/poly(2-methyl-2-oxazoline-b-tetrahydrofuran-b-2-methyl-2-oxazoline) formulation: An improved penetration and biological effect of curcumin in F508del-CFTR cell lines.

PubMed

Gonçalves, Cristine; Gomez, Jean-Pierre; Même, William; Rasolonjatovo, Bazoly; Gosset, David; Nedellec, Steven; Hulin, Philippe; Huin, Cécile; Le Gall, Tony; Montier, Tristan; Lehn, Pierre; Pichon, Chantal; Guégan, Philippe; Cheradame, Hervé; Midoux, Patrick

2017-08-01

Neutral amphiphilic triblock ABA copolymers are of great interest to solubilize hydrophobic drugs. We reported that a triblock ABA copolymer consisting of methyl-2-oxazoline (MeOx) and tetrahydrofuran (THF) (MeOx 6 -THF 19 -MeOx 6 ) (TBCP2) can solubilize curcumin (Cur) a very hydrophobic molecule exhibiting multiple therapeutic effects but whose insolubility and low stability in water is a major drawback for clinical applications. Here, we provide evidences by flow cytometry and confocal microscopy that Cur penetration in normal and ΔF508-CFTR human airway epithelial cell lines is facilitated by TBCP2. When used on ΔF508-CFTR cell lines, the Cur/TBCP2 formulation promotes the restoration of the expression of the CFTR protein in the plasma membrane. Furthermore, patch-clamp and MQAE fluorescence experiments show that this effect is associated with a correction of a Cl - selective current at the membrane surface of F508del-CFTR cells. The results show the great potential of the neutral amphiphilic triblock copolymer MeOx 6 -THF 19 -MeOx 6 as carrier for curcumin in a Cystic Fibrosis context. We anticipate that other MeOx n -THF m -MeOx n copolymers could have similar behaviours for other highly insoluble therapeutic drugs or cosmetic active ingredients. Copyright © 2017 Elsevier B.V. All rights reserved.

Local mechanical properties of Alloy 82/182 dissimilar weld joint between SA508 Gr.1a and F316 SS at RT and 320 °C

NASA Astrophysics Data System (ADS)

Kim, Jin Weon; Lee, Kyoungsoo; Kim, Jong Sung; Byun, Thak Sang

2009-02-01

The distributions of mechanical and microstructural properties were investigated for the dissimilar metal weld joints between SA508 Gr.1a ferritic steel and F316 austenitic stainless steel with Alloy 82/182 filler metal using small-size tensile specimens. The material properties varied significantly in different zones while those were relatively uniform within each material. In particular, significant gradient of the mechanical properties were observed near the both heat-affected zones (HAZs) of F316 SS and SA508 Gr.1a. Thus, the yield stress (YS) was under-matched with respect to the both HAZs, although, the YS of the weld metal was over-matched with respect to both base metals. The minimum ductility occurred in the HAZ of SA508 Gr.1a at both test temperatures. The plastic instability stress also varied considerably across the weld joints, with minimum values occurring in the SA508 Gr.1a base metal at RT and in the HAZ of F316 SS at 320 °C. The transmission electron micrographs showed that the strengthening in the HAZ of F316 SS was attributed to the strain hardening, induced by a strain mismatch between the weldment and the base metal, which was evidenced by high dislocation density in the HAZ of F316 SS.

Local mechanical properties of Alloy 82/182 dissimilar weld joint between SA508 Gr.1a and F316 SS at RT and 320C

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

Byun, Thak Sang; Kim, Jin Weon

2009-01-01

This paper presents the variations of local mechanical and microstructural properties in dissimilar metal weld joints consisting of the SA508 Gr.1a ferritic steel, Alloy 82/182 filler metal, and F316 austenitic stainless steel. Flat or round tensile specimens and transmission electron microscopy disks were taken from the base metals, welds, and heat-affected zones (HAZ) of the joints and tested at room temperature (RT) and/or at 320 C. The tensile test results indicated that the mechanical property was relatively uniform within each material zone, but varied considerably between different zones. Further, significant variations were observed both in the austenitic HAZ of F316more » SS and in the ferritic HAZ of SA508 Gr.1a. The yield stress (YS) of the weld metal was under-matched with respect to the HAZs of SA508 Gr.1a and F316 SS by 0.78 to 0.92, although the YS was over-matched with respect to both base metals. The minimum ductility occurred in the HAZ of SA508 Gr.1 at both test temperatures. The plastic instability stress also varied considerably in the weld joints, with minimum values occurring in the SA508 Gr.1a base metal at RT and in the HAZ of F316 SS at 320 C, suggesting that the probability of ductile failure caused by a unstable deformation at the Alloy 82/182 buttering layer is low. Within the HAZ of SA508 Gr.1a, the gradient of the YS and ultimate tensile strength (UTS) was significant, primarily because of the different microstructures produced by the phase transformation during the welding process. The increment of YS was unexpectedly high in the HAZ of F316 SS, which was explained by the strain hardening induced by a strain mismatch between the weldment and the base metal. This was confirmed by the transmission electron micrographs showing high dislocation density in the HAZ.« less

Altered intestinal bile salt biotransformation in a cystic fibrosis (Cftr-/-) mouse model with hepato-biliary pathology.

PubMed

Bodewes, Frank A J A; van der Wulp, Mariëtte Y M; Beharry, Satti; Doktorova, Marcela; Havinga, Rick; Boverhof, Renze; James Phillips, M; Durie, Peter R; Verkade, Henkjan J

2015-07-01

Cftr(-/-tm1Unc) mice develop progressive hepato-biliary pathology. We hypothesize that this liver pathology is related to alterations in biliary bile hydrophobicity and bile salt metabolism in Cftr(-/-tm1Unc) mice. We determined bile production, biliary and fecal bile salt- and lipid compositions and fecal bacterial composition of C57BL/6J Cftr(-/-tm1Unc) and control mice. We found no differences between the total biliary bile salt or lipid concentrations of Cftr(-/-) and controls. Compared to controls, Cftr(-/-) mice had a ~30% higher bile production and a low bile hydrophobicity, related to a ~7 fold higher concentration of the choleretic and hydrophilic bile salt ursocholate. These findings coexisted with a significantly smaller quantity of fecal Bacteroides bacteria. Liver pathology in Cftr(-/-tm1Unc) is not related to increased bile hydrophobicity. Cftr(-/-) mice do however display a biliary phenotype characterized by increased bile production and decreased biliary hydrophobicity. Our findings suggest Cftr dependent, alterations in intestinal bacterial biotransformation of bile salts. Copyright © 2014. Published by Elsevier B.V.

12 CFR 508.1 - Scope.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Scope. 508.1 Section 508.1 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.1 Scope. The rules in this part apply to hearings, which are exempt...

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

PubMed Central

Ren, Hong Yu; Grove, Diane E.; De La Rosa, Oxana; Houck, Scott A.; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J.; Cyr, Douglas M.

2013-01-01

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR. PMID:23924900

VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.

PubMed

Ren, Hong Yu; Grove, Diane E; De La Rosa, Oxana; Houck, Scott A; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J; Cyr, Douglas M

2013-10-01

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

Potentiator Ivacaftor Abrogates Pharmacological Correction of ΔF508 CFTR in Cystic Fibrosis

PubMed Central

Cholon, Deborah M.; Quinney, Nancy L.; Fulcher, M. Leslie; Esther, Charles R.; Das, Jhuma; Dokholyan, Nikolay V.; Randell, Scott H.; Boucher, Richard C.; Gentzsch, Martina

2014-01-01

Cystic Fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). Newly developed “correctors” such as lumacaftor (VX-809) that improve CFTR maturation and trafficking and “potentiators” such as ivacaftor (VX-770) that enhance channel activity may provide important advances in CF therapy. Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation (G551D) that affects only channel activity, a single compound is not sufficient to treat patients with the more common CFTR mutation, ΔF508. Thus, patients with ΔF508 will likely require treatment with both correctors and potentiators to achieve clinical benefit. However, whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro, the impact of chronic therapy has not been established. In studies of human primary airway epithelial cells, we found that both acute and chronic treatment with VX-770 improved CFTR function in cells with the G551D mutation, consistent with clinical studies. In contrast, chronic VX-770 administration caused a dose-dependent reversal of VX-809-mediated CFTR correction in ΔF508 homozygous cultures. This result reflected the destabilization of corrected ΔF508 CFTR by VX-770, dramatically increasing its turnover rate. Chronic VX-770 treatment also reduced mature wild-type CFTR levels and function. These findings demonstrate that chronic treatment with CFTR potentiators and correctors may have unexpected effects that cannot be predicted from short-term studies. Combining of these drugs to maximize rescue of ΔF508 CFTR may require changes in dosing and/or development of new potentiator compounds that do not interfere with CFTR stability. PMID:25101886

Robust method for TALEN-edited correction of pF508del in patient-specific induced pluripotent stem cells.

PubMed

Camarasa, María Vicenta; Gálvez, Víctor Miguel

2016-02-09

Cystic fibrosis is one of the most frequent inherited rare diseases, caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. Apart from symptomatic treatments, therapeutic protocols for curing the disease have not yet been established. The regeneration of genetically corrected, disease-free epithelia in cystic fibrosis patients is envisioned by designing a stem cell/genetic therapy in which patient-derived pluripotent stem cells are genetically corrected, from which target tissues are derived. In this framework, we present an efficient method for seamless correction of pF508del mutation in patient-specific induced pluripotent stem cells by gene edited homologous recombination. Gene edition has been performed by transcription activator-like effector nucleases and a homologous recombination donor vector which contains a PiggyBac transposon-based double selectable marker cassette.This new method has been designed to partially avoid xenobiotics from the culture system, improve cell culture efficiency and genome stability by using a robust culture system method, and optimize timings. Overall, once the pluripotent cells have been amplified for the first nucleofection, the procedure can be completed in 69 days, and can be easily adapted to edit and change any gene of interest.

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

PubMed Central

Rubenstein, R C; Egan, M E; Zeitlin, P L

1997-01-01

The most common cystic fibrosis transmembrane conductance regulator mutation, delta F508-CFTR, is a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We hypothesize that a known transcriptional regulator, sodium 4-phenylbutyrate (4PBA), will enable a greater fraction of delta F508-CFTR to escape degradation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (delta F508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (delta F508/W1282X) were exposed to 4PBA for up to 7 d in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of delta F508-CFTR. These data indicate that 4PBA is a promising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying the delta F508 mutation. PMID:9366560

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

PubMed

Rubenstein, R C; Egan, M E; Zeitlin, P L

1997-11-15

The most common cystic fibrosis transmembrane conductance regulator mutation, delta F508-CFTR, is a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We hypothesize that a known transcriptional regulator, sodium 4-phenylbutyrate (4PBA), will enable a greater fraction of delta F508-CFTR to escape degradation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (delta F508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (delta F508/W1282X) were exposed to 4PBA for up to 7 d in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of delta F508-CFTR. These data indicate that 4PBA is a promising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying the delta F508 mutation.

Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.

PubMed

Wainwright, Claire E; Elborn, J Stuart; Ramsey, Bonnie W; Marigowda, Gautham; Huang, Xiaohong; Cipolli, Marco; Colombo, Carla; Davies, Jane C; De Boeck, Kris; Flume, Patrick A; Konstan, Michael W; McColley, Susanna A; McCoy, Karen; McKone, Edward F; Munck, Anne; Ratjen, Felix; Rowe, Steven M; Waltz, David; Boyle, Michael P

2015-07-16

Cystic fibrosis is a life-limiting disease that is caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) protein activity. Phe508del is the most common CFTR mutation. We conducted two phase 3, randomized, double-blind, placebo-controlled studies that were designed to assess the effects of lumacaftor (VX-809), a CFTR corrector, in combination with ivacaftor (VX-770), a CFTR potentiator, in patients 12 years of age or older who had cystic fibrosis and were homozygous for the Phe508del CFTR mutation. In both studies, patients were randomly assigned to receive either lumacaftor (600 mg once daily or 400 mg every 12 hours) in combination with ivacaftor (250 mg every 12 hours) or matched placebo for 24 weeks. The primary end point was the absolute change from baseline in the percentage of predicted forced expiratory volume in 1 second (FEV1) at week 24. A total of 1108 patients underwent randomization and received study drug. The mean baseline FEV1 was 61% of the predicted value. In both studies, there were significant improvements in the primary end point in both lumacaftor-ivacaftor dose groups; the difference between active treatment and placebo with respect to the mean absolute improvement in the percentage of predicted FEV1 ranged from 2.6 to 4.0 percentage points (P<0.001), which corresponded to a mean relative treatment difference of 4.3 to 6.7% (P<0.001). Pooled analyses showed that the rate of pulmonary exacerbations was 30 to 39% lower in the lumacaftor-ivacaftor groups than in the placebo group; the rate of events leading to hospitalization or the use of intravenous antibiotics was lower in the lumacaftor-ivacaftor groups as well. The incidence of adverse events was generally similar in the lumacaftor-ivacaftor and placebo groups. The rate of discontinuation due to an adverse event was 4.2% among patients who received lumacaftor-ivacaftor versus 1.6% among those who received placebo. These data show that lumacaftor in

Cystic fibrosis Δf508 mutation screening in Brazilian women with altered fertility.

PubMed

Brunoro, G V F; Wolfgramm, E V; Louro, I D; Degasperi, I I; Busatto, V C W; Perrone, A M S; Batitucci, M C P

2011-10-01

Cystic Fibrosis (CF) is an autosomal recessive disease, caused by mutations in the Cystic Fibrosis Transmembrane Regulator gene (CFTR). The most frequent mutation in CF is ΔF508. The disease is clinically characterized by elevated concentrations of sweat chlorides and abnormally thick mucus. It affects organs such as lung, pancreas, gastrointestinal and reproductive tract. Women with CF commonly present delayed puberty and amenorrhea due to malnutrition. Our objective was to screen the presence of ΔF508 mutation in 24 women with altered fertility. Nine of these women presented reduced fertility without a known cause, four showed polycystic ovaries and two had early menopause. One woman with early menopause was a carrier of the ΔF508 mutation. Our study demonstrates that it is possible that the frequency of CF mutations among patients with altered fertility may be higher than expected. Previous data showed that fibrocystic women can show reduced fertility, maternal mortality associated with pregnancy and increased incidence of spontaneous abortion. We therefore recommend that women with reduced fertility undertake genetic tests for a better evaluation of pregnancy risks and clinical monitoring.

Cystic fibrosis in the Basque country: high frequency of mutation delta F508 in patients of Basque origin.

PubMed Central

Casals, T; Vázquez, C; Lázaro, C; Girbau, E; Giménez, F J; Estivill, X

1992-01-01

The Basque population is one of the oldest populations of Europe. It has been suggested that the Basques arose from a population established in western Europe during the late Paleolithic Age. The Basque language (Euskera) is a supposedly pre-Indo-European language that originates from the first settlers of Europe. The variable distribution of the major cystic fibrosis (CF) mutation (delta F508 deletion) in Europe, with higher frequencies of the mutation in northern Europe and lower frequencies in southern Europe, has suggested that the delta F508 mutation was spread by early farmers migrating from the Middle East during the Neolithic period. We have studied 45 CF families from the Basque Country, where the incidence of CF is approximately 1/4,500. The birthplaces of the parents and grandparents have been traced and are distributed according to their origin as Basque or Mixed Basque. The frequency of the delta F508 mutation in the chromosomes of Basque origin is 87%, compared with 58% in those of Mixed Basque origin. The analysis of haplotypes, both with markers closely linked to the CF gene and with intragenic markers, suggests that the delta F508 mutation was not spread by the Indo-European invasions but was already present in Europe more than 10,000 years ago, during the Paleolithic period. PMID:1370875

Non-native Conformers of Cystic Fibrosis Transmembrane Conductance Regulator NBD1 Are Recognized by Hsp27 and Conjugated to SUMO-2 for Degradation.

PubMed

Gong, Xiaoyan; Ahner, Annette; Roldan, Ariel; Lukacs, Gergely L; Thibodeau, Patrick H; Frizzell, Raymond A

2016-01-22

A newly identified pathway for selective degradation of the common mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), F508del, is initiated by binding of the small heat shock protein, Hsp27. Hsp27 collaborates with Ubc9, the E2 enzyme for protein SUMOylation, to selectively degrade F508del CFTR via the SUMO-targeted ubiquitin E3 ligase, RNF4 (RING finger protein 4) (1). Here, we ask what properties of CFTR are sensed by the Hsp27-Ubc9 pathway by examining the ability of NBD1 (locus of the F508del mutation) to mimic the disposal of full-length (FL) CFTR. Similar to FL CFTR, F508del NBD1 expression was reduced 50-60% by Hsp27; it interacted preferentially with the mutant and was modified primarily by SUMO-2. Mutation of the consensus SUMOylation site, Lys(447), obviated Hsp27-mediated F508del NBD1 SUMOylation and degradation. As for FL CFTR and NBD1 in vivo, SUMO modification using purified components in vitro was greater for F508del NBD1 versus WT and for the SUMO-2 paralog. Several findings indicated that Hsp27-Ubc9 targets the SUMOylation of a transitional, non-native conformation of F508del NBD1: (a) its modification decreased as [ATP] increased, reflecting stabilization of the nucleotide-binding domain by ligand binding; (b) a temperature-induced increase in intrinsic fluorescence, which reflects formation of a transitional NBD1 conformation, was followed by its SUMO modification; and (c) introduction of solubilizing or revertant mutations to stabilize F508del NBD1 reduced its SUMO modification. These findings indicate that the Hsp27-Ubc9 pathway recognizes a non-native conformation of mutant NBD1, which leads to its SUMO-2 conjugation and degradation by the ubiquitin-proteasome system. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

ERp29 Regulates ΔF508 and Wild-type Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking to the Plasma Membrane in Cystic Fibrosis (CF) and Non-CF Epithelial Cells*

PubMed Central

Suaud, Laurence; Miller, Katelyn; Alvey, Lora; Yan, Wusheng; Robay, Amal; Kebler, Catherine; Kreindler, James L.; Guttentag, Susan; Hubbard, Michael J.; Rubenstein, Ronald C.

2011-01-01

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o− WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells. PMID:21525008

ERp29 regulates DeltaF508 and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) trafficking to the plasma membrane in cystic fibrosis (CF) and non-CF epithelial cells.

PubMed

Suaud, Laurence; Miller, Katelyn; Alvey, Lora; Yan, Wusheng; Robay, Amal; Kebler, Catherine; Kreindler, James L; Guttentag, Susan; Hubbard, Michael J; Rubenstein, Ronald C

2011-06-17

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Synergy-based small-molecule screen using a human lung epithelial cell line yields ΔF508-CFTR correctors that augment VX-809 maximal efficacy.

PubMed

Phuan, Puay-Wah; Veit, Guido; Tan, Joseph; Roldan, Ariel; Finkbeiner, Walter E; Lukacs, Gergely L; Verkman, A S

2014-07-01

The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors. Copyright © 2014 by The

Human Parvovirus B19 NS1 Protein Aggravates Liver Injury in NZB/W F1 Mice

PubMed Central

Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Hsu, Huai-Sheng; Tzang, Bor-Show; Hsu, Tsai-Ching

2013-01-01

Human parvovirus B19 (B19) has been associated with a variety of diseases. However, the influence of B19 viral proteins on hepatic injury in SLE is still obscure. To elucidate the effects of B19 viral proteins on livers in SLE, recombinant B19 NS1, VP1u or VP2 proteins were injected subcutaneously into NZB/W F1 mice, respectively. Significant expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected in NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Markedly hepatocyte disarray and lymphocyte infiltration were observed in livers from NZB/WF 1 mice receiving B19 NS1 as compared to those mice receiving PBS. Additionally, significant increases of Tumor Necrosis Factor –α (TNF-α), TNF-α receptor, IκB kinase –α (IKK-α), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor-kappa B (NF-κB) were detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Accordingly, significant increases of matrix metalloproteinase-9 (MMP9) and U-plasminogen activator (uPA) were also detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Contrarily, no significant variation on livers from NZB/W F1 mice receiving B19 VP1u or VP2 was observed as compared to those mice receiving PBS. These findings firstly demonstrated the aggravated effects of B19 NS1 but not VP1u or VP2 protein on hepatic injury and provide a clue in understanding the role of B19 NS1 on hepatic injury in SLE. PMID:23555760

Evidence against Resveratrol as a viable therapy for the rescue of defective ΔF508 CFTR

PubMed Central

Jai, Ying; Shah, Kalpit; Bridges, Robert J.; Bradbury, Neil A.

2015-01-01

BACKGROUND Resveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations. METHODS Cells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers. RESULTS Consistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol CONCLUSIONS High concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents. PMID:26342647

Proinflammatory effect of sodium 4-phenylbutyrate in deltaF508-cystic fibrosis transmembrane conductance regulator lung epithelial cells: involvement of extracellular signal-regulated protein kinase 1/2 and c-Jun-NH2-terminal kinase signaling.

PubMed

Roque, Telma; Boncoeur, Emilie; Saint-Criq, Vinciane; Bonvin, Elise; Clement, Annick; Tabary, Olivier; Jacquot, Jacky

2008-09-01

Sodium 4-phenylbutyrate (4-PBA) has attracted a great deal of attention in cystic fibrosis (CF) pathology due to its capacity to traffic DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) to the cell membrane and restore CFTR chloride function at the plasma membrane of CF lung cells in vitro and in vivo. Using two different DeltaF508-CFTR lung epithelial cell lines (CFBE41o- and IB3-1 cells, characterized with DeltaF508-homozygous and heterozygous genotype, respectively) in vitro, 4-PBA induced an increase of proinflammatory cytokine interleukin (IL)-8 production in a concentration-dependent manner. This 4-PBA-induced IL-8 production was associated with a strong reduction of proteasome and nuclear factor-kappaB transcriptional activities in the two DeltaF508-CFTR lung cells either in a resting state or after tumor necrosis factor-alpha stimulation. In contrast, a strong increase of activator protein-1 transcriptional activity was observed. The inhibition of extracellular signal-regulated protein kinase 1/2 (ERK1/2) by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) and c-Jun-NH(2)-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by anthra[1,9-cd] pyrazol-6 (2H)-one (SP600125), respectively, was associated with a reduction (2-3.5-fold) of IL-8 production in both DeltaF508-CFTR lung cell lines treated with 4-PBA. No significant change of IL-8 production was observed after an inhibition of p38 MAPK with 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190). Therefore, we suggest that inhibition of both ERK1/2 and JNK signaling may be a means to strongly reduce 4-PBA-induced IL-8 production in combination with 4-PBA treatment to restore CFTR Cl(-) channel function in lung epithelial cells of patients with CF.

The effect of pea albumin 1F on glucose metabolism in mice.

PubMed

Dun, Xin-Peng; Li, Fa-Fang; Wang, Jian-He; Chen, Zheng-Wang

2008-06-01

Pea albumin 1F (PA1F), a plant peptide isolated from pea seeds, can dramatically increase blood glucose concentration by subcutaneous injection with a dosage of 5 or 10 microg/g (body weight) in normal and type II diabetic mice (KK/upj-Ay). The voltage-dependent anion channel 1 (VDAC-1) has been identified as the PA1F binding protein from mice pancreatic cell membrane, which may be involved in the regulation of enhancing blood glucose in response to PA1F binding. The results clearly show that peptide-signaling molecules from plants can affect mammalian physiological functions, especially, in association with glucose metabolism.

Introducing False EUR and False EUR exchange rates

NASA Astrophysics Data System (ADS)

Ausloos, M.; Ivanova, K.

2000-10-01

The Euro ( EUR) is a new currency introduced by the European Community. Its exchange rate is very puzzling. We have invented a false Euro ( FEUR) dating back to 1993 and have derived the exchange rates of the FEUR with respect to currencies not belonging to the EUR, i.e., DKK, CHF, JPY and USD. This allows us to search for correlations between the fluctuations preexisting to the introduction of EUR and present ones in such financial data. The detrended fluctuation analysis ( DFA) statistical method is used. This leads to assume a power-law behavior, i.e., a scaling hypothesis, through an exponent α. The latter has demonstrated its usefulness for the investigations of long-range power-law correlations in several types of financial sequences. Our findings show that the α exponent interestingly characterizes fractional Brownian motion of the currency exchange rates between EUR and DKK over a 25 day interval, and usual Brownian motion otherwise and for the three other investigated exchange rates. We can devise an investment strategy based on the localα technique and obtain appreciable gains for the time being.

Activation of deltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX.

PubMed

Andersson, C; Roomans, G M

2000-05-01

The cellular basis of cystic fibrosis (CF) is a defect in a cyclic adenosine monophosphate (cAMP)-activated chloride channel (CF transmembrane conductance regulator) in epithelial cells that leads to decreased chloride ion transport and impaired water transport across the cell membrane. This study investigated whether it was possible to activate the defective chloride channel in cystic fibrosis respiratory epithelial cells with 4-phenylbutyrate (4PBA), genistein and 8-cyclopentyl-1,3-dipropylxanthine (CPX). The CF bronchial epithelial cell line CFBE41o-, which expresses the deltaF508 mutation, was treated with these agents and loss of Cl-, indicating Cl- efflux, measured by X-ray microanalysis. 8-bromo-cAMP alone did not induce Cl- efflux in CFBE41o- cells, but after incubation with 4PBA a significant efflux of Cl- occurred. Stimulation of cells with a combination of genistein and cAMP also induced Cl- efflux, whereas a combination of pretreatment with 4PBA and a combined stimulation with genistein and cAMP induced an even larger Cl- efflux. Cl- efflux could also be stimulated by CPX, but this effect was not enhanced by 4PBA pretreatment. The deltaF508 mutation leads to impaired processing of the cystic fibrosis transmembrane conductance regulator. The increased efflux of chloride after 4-phenylbutyrate treatment can be explained by the fact that 4-phenylbutyrate allows the deltaF508 cystic fibrosis transmembrane conductance regulator to escape degradation and to be transported to the cell surface. Genistein and 8-cyclopentyl-1,3-dipropylxanthine act by stimulating chloride ion efflux by increasing the probability of the cystic fibrosis transmembrane conductance regulator being open. The combination of 4-phenylbutyrate and genistein may be useful in a potential pharmacological therapy for cystic fibrosis patients with the deltaF508 mutation.

Lumacaftor/ivacaftor, a novel agent for the treatment of cystic fibrosis patients who are homozygous for the F580del CFTR mutation.

PubMed

Bulloch, Marilyn N; Hanna, Cameron; Giovane, Richard

2017-10-01

Cystic Fibrosis (CF) is an autosomal recessive disease affecting up to 90,000 people worldwide. Approximately 73% of patients are homozygous for the F508del cystic fibrosis transmembrane conductance regulator [CFTR] mutation. Traditionally treatment has only included supportive care. Therefore, there is a need for safe and effective novel therapies targeting the underlying molecular defects seen with CF. Areas covered: In 2016, the Food and Drug Administration and the European Commission approved LUM/IVA (Orkambi), a CFTR modulator that includes both a CFTR corrector and potentiator, for CF patients homozygous for the F508del CFTR mutation. This article reviews the pharmacologic features, clinical efficacy, and safety of LUM/IVA and summarize the available pre-clinical and clinical data of LUM/IVA use. Expert commentary: LUM/IVA showed modest, but significant improvements from baseline in percent predicted FEV 1 (ppFEV 1 ) as well as a reduction in pulmonary exacerbations by 35% It was shown to be safe for short- and long-term use. Currently, LUM/IVA is the only oral agent in its class available and represents a milestone the development of therapies for the management of CF. Nonetheless, pharmacoeconomic data are necessary to justify its high cost before is use becomes standard of care.

CFTR DeltaF508 mutation detection from dried blood samples in the first trimester of pregnancy: a possible routine prenatal screening strategy for cystic fibrosis?

PubMed

Konialis, Christopher P; Hagnefelt, Birgitta; Kazamia, Constantina; Karapanou, Sophia; Pangalos, Constantinos

2007-01-01

The implementation and evaluation of a proposed wide-scale prenatal screening strategy, based on DNA isolated from dried blood spots in the first trimester of pregnancy, for the early detection of pregnancies at risk for cystic fibrosis (CF). The screening was performed in conjunction with routine biochemical marker screening for Down's syndrome risk in the first trimester of pregnancy. DNA was isolated from 1,233 dried blood spots and analyzed for the presence of the CF transmembrane regulator DeltaF508 mutation. Women carriers were offered and accepted the option for additional full testing of their partners in order to assess the risk for the fetus. All 1,233 samples were successfully analyzed, identifying 23 DeltaF508 carriers, corresponding to a DeltaF508 carrier rate of approximately 1/55 (1.8%). All partners of the women carriers were further tested without revealing any need for further prenatal testing in this group. This study reveals the relatively high frequency of the DeltaF508 CF mutation in the Greek population. More importantly, we demonstrate that the proposed prenatal screening strategy, based on the ease and cost-effectiveness of the analysis for the detection of a single common mutation, can be considered as a feasible and practical approach for wide-scale prenatal screening for CF, following the sequential model. It is applied early on in pregnancy, allowing for the timely management of families at risk for the corresponding genetic disorders. Finally, it can easily be extended to include screening for other common genetic disorders in specific population groups.

32 CFR 508.1 - Utilization of Army bands.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 3 2011-07-01 2009-07-01 true Utilization of Army bands. 508.1 Section 508.1 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY AID OF CIVIL AUTHORITIES AND... Secretary of Defense. The authority to determine whether the use of an Army band at a public gathering is...

Exacerbating Effects of Human Parvovirus B19 NS1 on Liver Fibrosis in NZB/W F1 Mice

PubMed Central

Hsu, Tsai-Ching; Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Tzang, Bor-Show

2013-01-01

Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19) is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling. PMID:23840852

Establishment of the Ph. Eur. erythropoietin chemical reference substance batch 1.

PubMed

Burns, C; Bristow, A F; Buchheit, K H; Daas, A; Wierer, M; Costanzo, A

2015-01-01

The Erythropoietin (EPO) European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 3 was calibrated in 2006 by in vivo bioassay and was used as a reference preparation for these assays as well as for the physicochemical methods in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). In order to avoid the frequent replacement of this standard and thus reduce the use of animals, a new EPO Chemical Reference Substance (CRS) was established to be used solely for the physicochemical methods. Here we report the outcome of a collaborative study aimed at demonstrating the suitability of the candidate CRS (cCRS) as a reference for the physicochemical methods in the Ph. Eur. monograph. Results from the study demonstrated that for the physicochemical methods currently required in the monograph (capillary zone electrophoresis (CZE), polyacrylamide gel electrophoresis (PAGE)/immunoblotting and peptide mapping), the cCRS is essentially identical to the existing BRP. However, data also indicated that, for the physicochemical methods under consideration for inclusion in a revised monograph (test for oxidised forms and glycan mapping), the suitability of the cCRS as a reference needs to be confirmed with additional work. Further to completion of the study, the Ph. Eur. Commission adopted the cCRS as "Erythropoietin for physicochemical tests CRS batch 1" to be used for CZE, PAGE/immunoblotting and peptide mapping.

Carcinogenicity of acrylamide in B6C3F(1) mice and F344/N rats from a 2-year drinking water exposure.

PubMed

Beland, Frederick A; Mellick, Paul W; Olson, Greg R; Mendoza, Maria C B; Marques, M Matilde; Doerge, Daniel R

2013-01-01

Acrylamide is a component of roasted coffee and certain baked and fried carbohydrate-rich foods prepared at high temperatures. We have assessed the carcinogenicity of acrylamide in male and female B6C3F(1) mice and F344/N rats administered 0, 0.0875, 0.175, 0.35, or 0.70mM acrylamide in the drinking water ad libitum for 2 years. Acrylamide caused significant dose-related decreasing trends in the body weights of F344/N rats. Acrylamide administration resulted in significant dose-related decreasing trends in survival in both sexes of B6C3F(1) mice and in female F344/N rats. Histopathological analyses indicated significant dose-related increases in Harderian gland and lung tumors in male and female B6C3F(1) mice. Male B6C3F(1) mice also had a significantly increased incidence of forestomach tumors, while female B6C3F(1) mice had significant dose-related increases in mammary gland, ovary, and skin tumors. In male and female F344/N rats, there were significant increases in thyroid tumors. Male F344/N rats also had significant dose-related increases in testes, heart, and pancreas tumors, while female F344 rats demonstrated significant increases in clitoral gland, mammary gland, oral cavity, and skin tumors. These results, combined with previous mechanistic studies, provide strong support for the concept that acrylamide is activated to a carcinogen through metabolism to glycidamide. Published by Elsevier Ltd.

A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in deltaF508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function.

PubMed

Rubenstein, R C; Zeitlin, P L

1998-02-01

Sodium 4-phenylbutyrate (Buphenyl, 4PBA) is a new FDA approved drug for management of urea cycle disorders. We have previously presented data suggesting that 4PBA, at clinically achievable concentrations, induces CFTR channel function on the plasma membrane of deltaF508-expressing cystic fibrosis (CF) airway epithelial cells in vitro (Rubenstein, R. C., and P. L. Zeitlin, 1997. J. Clin. Invest. 100:2457-2463). We hypothesized that 4PBA would induce epithelial CFTR function in vivo in individuals homozygous for deltaF508-CFTR. A randomized, double-blind, placebo-controlled trial in 18 deltaF508-homozygous patients with CF was performed with the maximum approved adult dose of 4PBA, 19 grams p.o. divided t.i.d., given for 1 wk. Nasal potential difference (NPD) response patterns and sweat chloride concentrations were determined before and after study drug treatment, and 4PBA and metabolites were assayed in plasma and urine at the end of study drug treatment. Subjects in the 4PBA group demonstrated small, but statistically significant improvements of the NPD response to perfusion of an isoproterenol/amiloride/chloride-free solution; this measure reflects epithelial CFTR function and is highly discriminatory between patients with and without CF. Subjects who had received 4PBA did not demonstrate significantly reduced sweat chloride concentrations or alterations in the amiloride-sensitive NPD. Side effects due to drug therapy were minimal and comparable in the two groups. These data are consistent with 4PBA therapy inducing CFTR function in the nasal epithelia of deltaF508-homozygous CF patients.

Cyclophilin D Promotes Brain Mitochondrial F1FO ATP Synthase Dysfunction in Aging Mice

PubMed Central

Gauba, Esha; Guo, Lan; Du, Heng

2017-01-01

Brain aging is the known strongest risk factor for Alzheimer’s disease (AD). In recent years, mitochondrial deficits have been proposed to be a common mechanism linking brain aging to AD. Therefore, to elucidate the causative mechanisms of mitochondrial dysfunction in aging brains is of paramount importance for our understanding of the pathogenesis of AD, in particular its sporadic form. Cyclophilin D (CypD) is a specific mitochondrial protein. Recent studies have shown that F1FO ATP synthase oligomycin sensitivity conferring protein (OSCP) is a binding partner of CypD. The interaction of CypD with OSCP modulates F1FO ATP synthase function and mediates mitochondrial permeability transition pore (mPTP) opening. Here, we have found that increased CypD expression, enhanced CypD/OSCP interaction, and selective loss of OSCP are prominent brain mitochondrial changes in aging mice. Along with these changes, brain mitochondria from the aging mice demonstrated decreased F1FO ATP synthase activity and defective F1FO complex coupling. In contrast, CypD deficient mice exhibited substantially mitigated brain mitochondrial F1FO ATP synthase dysfunction with relatively preserved mitochondrial function during aging. Interestingly, the aging-related OSCP loss was also dramatically attenuated by CypD depletion. Therefore, the simplest interpretation of this study is that CypD promotes F1FO ATP synthase dysfunction and the resultant mitochondrial deficits in aging brains. In addition, in view of CypD and F1FO ATP synthase alterations seen in AD brains, the results further suggest that CypD-mediated F1FO ATP synthase deregulation is a shared mechanism linking mitochondrial deficits in brain aging and AD. PMID:27834780

Cyclophilin D Promotes Brain Mitochondrial F1FO ATP Synthase Dysfunction in Aging Mice.

PubMed

Gauba, Esha; Guo, Lan; Du, Heng

2017-01-01

Brain aging is the known strongest risk factor for Alzheimer's disease (AD). In recent years, mitochondrial deficits have been proposed to be a common mechanism linking brain aging to AD. Therefore, to elucidate the causative mechanisms of mitochondrial dysfunction in aging brains is of paramount importance for our understanding of the pathogenesis of AD, in particular its sporadic form. Cyclophilin D (CypD) is a specific mitochondrial protein. Recent studies have shown that F1FO ATP synthase oligomycin sensitivity conferring protein (OSCP) is a binding partner of CypD. The interaction of CypD with OSCP modulates F1FO ATP synthase function and mediates mitochondrial permeability transition pore (mPTP) opening. Here, we have found that increased CypD expression, enhanced CypD/OSCP interaction, and selective loss of OSCP are prominent brain mitochondrial changes in aging mice. Along with these changes, brain mitochondria from the aging mice demonstrated decreased F1FO ATP synthase activity and defective F1FO complex coupling. In contrast, CypD deficient mice exhibited substantially mitigated brain mitochondrial F1FO ATP synthase dysfunction with relatively preserved mitochondrial function during aging. Interestingly, the aging-related OSCP loss was also dramatically attenuated by CypD depletion. Therefore, the simplest interpretation of this study is that CypD promotes F1FO ATP synthase dysfunction and the resultant mitochondrial deficits in aging brains. In addition, in view of CypD and F1FO ATP synthase alterations seen in AD brains, the results further suggest that CypD-mediated F1FO ATP synthase deregulation is a shared mechanism linking mitochondrial deficits in brain aging and AD.

16Oxygen irradiation enhances cued fear memory in B6D2F1 mice

NASA Astrophysics Data System (ADS)

Raber, Jacob; Marzulla, Tessa; Kronenberg, Amy; Turker, Mitchell S.

2015-11-01

The space radiation environment includes energetic charged particles that may impact cognitive performance. We assessed the effects of 16O ion irradiation on cognitive performance of C57BL/6J × DBA/2J F1 (B6D2F1) mice at OHSU (Portland, OR) one month following irradiation at Brookhaven National Laboratory (BNL, Upton, NY). Hippocampus-dependent contextual fear memory and hippocampus-independent cued fear memory of B6D2F1 mice were tested. 16O ion exposure enhanced cued fear memory. This effect showed a bell-shaped dose response curve. Cued fear memory was significantly stronger in mice irradiated with 16O ions at a dose of 0.4 or 0.8 Gy than in sham-irradiated mice or following irradiation at 1.6 Gy. In contrast to cued fear memory, contextual fear memory was not affected following 16O ion irradiation at the doses used in this study. These data indicate that the amygdala might be particularly susceptible to effects of 16O ion exposure.

Forecasting the Long-Term Clinical and Economic Outcomes of Lumacaftor/Ivacaftor in Cystic Fibrosis Patients with Homozygous phe508del Mutation.

PubMed

Dilokthornsakul, Piyameth; Patidar, Mausam; Campbell, Jonathan D

2017-12-01

To forecast lifetime outcomes and cost of lumacaftor/ivacaftor combination therapy in patients with cystic fibrosis (CF) with homozygous phe508del mutation from the US payer perspective. A lifetime Markov model was developed from a US payer perspective. The model included five health states: 1) mild lung disease (percent predicted forced expiratory volume in 1 second [FEV 1 ] >70%), 2) moderate lung disease (40% ≤ FEV 1 ≤ 70%), 3) severe lung disease (FEV 1 < 40%), 4) lung transplantation, and 5) death. All inputs were derived from published literature. We estimated lumacaftor/ivacaftor's improvement in outcomes compared with a non-CF referent population as well as CF-specific mortality estimates. Lumacaftor/ivacaftor was associated with additional 2.91 life-years (95% credible interval 2.55-3.56) and additional 2.42 quality-adjusted life-years (QALYs) (95% credible interval 2.10-2.98). Lumacaftor/ivacaftor was associated with improvements in survival and QALYs equivalent to 27.6% and 20.7%, respectively, for the survival and QALY gaps between CF usual care and their non-CF peers. The incremental lifetime cost was $2,632,249. Lumacaftor/ivacaftor increased life-years and QALYs in CF patients with the homozygous phe508del mutation and moved morbidity and mortality closer to that of their non-CF peers but it came with higher cost. Copyright © 2017 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.

Pou4f1 and Pou4f2 Are Dispensable for the Long-Term Survival of Adult Retinal Ganglion Cells in Mice

PubMed Central

Huang, Liang; Hu, Fang; Xie, Xiaoling; Harder, Jeffery; Fernandes, Kimberly; Zeng, Xiang-yun; Libby, Richard; Gan, Lin

2014-01-01

Purpose To investigate the role of Pou4f1 and Pou4f2 in the survival of adult retinal ganglion cells (RGCs). Methods Conditional alleles of Pou4f1 and Pou4f2 were generated (Pou4f1loxP and Pou4f2loxP respectively) for the removal of Pou4f1 and Pou4f2 in adult retinas. A tamoxifen-inducible Cre was used to delete Pou4f1 and Pou4f2 in adult mice and retinal sections and flat mounts were subjected to immunohistochemistry to confirm the deletion of both alleles and to quantify the changes in the number of RGCs and other retinal neurons. To determine the effect of loss of Pou4f1 and Pou4f2 on RGC survival after axonal injury, controlled optic nerve crush (CONC) was performed and RGC death was assessed. Results Pou4f1 and Pou4f2 were ablated two weeks after tamoxifen treatment. Retinal interneurons and Müller glial cells are not affected by the ablation of Pou4f1 or Pou4f2 or both. Although the deletion of both Pou4f1 and Pou4f2 slightly delays the death of RGCs at 3 days post-CONC in adult mice, it does not affect the cell death progress afterwards. Moreoever, deletion of Pou4f1 or Pou4f2 or both has no impact on the long-term viability of RGCs at up to 6 months post-tamoxifen treatment. Conclusion Pou4f1 and Pou4f2 are involved in the acute response to damage to RGCs but are dispensable for the long-term survival of adult RGC in mice. PMID:24736625

Nuclear Countermeasure Activity of TP508 Linked to Restoration of Endothelial Function and Acceleration of DNA Repair

PubMed Central

Olszewska-Pazdrak, Barbara; McVicar, Scott D.; Rayavara, Kempaiah; Moya, Stephanie M.; Kantara, Carla; Gammarano, Chris; Olszewska, Paulina; Fuller, Gerald M.; Sower, Laurie E.; Carney, Darrell H.

2016-01-01

There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation. PMID:27388041

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.

PubMed

Guissart, C; Dubucs, C; Raynal, C; Girardet, A; Tran Mau Them, F; Debant, V; Rouzier, C; Boureau-Wirth, A; Haquet, E; Puechberty, J; Bieth, E; Dupin Deguine, D; Khau Van Kien, P; Brechard, M P; Pritchard, V; Koenig, M; Claustres, M; Vincent, M C

2017-03-01

Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Targeted Metabolomics Reveals Early Dominant Optic Atrophy Signature in Optic Nerves of Opa1delTTAG/+ Mice.

PubMed

Chao de la Barca, Juan Manuel; Simard, Gilles; Sarzi, Emmanuelle; Chaumette, Tanguy; Rousseau, Guillaume; Chupin, Stéphanie; Gadras, Cédric; Tessier, Lydie; Ferré, Marc; Chevrollier, Arnaud; Desquiret-Dumas, Valérie; Gueguen, Naïg; Leruez, Stéphanie; Verny, Christophe; Miléa, Dan; Bonneau, Dominique; Amati-Bonneau, Patrizia; Procaccio, Vincent; Hamel, Christian; Lenaers, Guy; Reynier, Pascal; Prunier-Mirebeau, Delphine

2017-02-01

Dominant optic atrophy (MIM No. 165500) is a blinding condition related to mutations in OPA1, a gene encoding a large GTPase involved in mitochondrial inner membrane dynamics. Although several mouse models mimicking the disease have been developed, the pathophysiological mechanisms responsible for retinal ganglion cell degeneration remain poorly understood. Using a targeted metabolomic approach, we measured the concentrations of 188 metabolites in nine tissues, that is, brain, three types of skeletal muscle, heart, liver, retina, optic nerve, and plasma in symptomatic 11-month-old Opa1delTTAG/+ mice. Significant metabolic signatures were found only in the optic nerve and plasma of female mice. The optic nerve signature was characterized by altered concentrations of phospholipids, amino acids, acylcarnitines, and carnosine, whereas the plasma signature showed decreased concentrations of amino acids and sarcosine associated with increased concentrations of several phospholipids. In contrast, the investigation of 3-month-old presymptomatic Opa1delTTAG/+ mice showed no specific plasma signature but revealed a significant optic nerve signature in both sexes, although with a sex effect. The Opa1delTTAG/+ versus wild-type optic nerve signature was characterized by the decreased concentrations of 10 sphingomyelins and 10 lysophosphatidylcholines, suggestive of myelin sheath alteration, and by alteration in the concentrations of metabolites involved in neuroprotection, such as dimethylarginine, carnitine, spermine, spermidine, carnosine, and glutamate, suggesting a concomitant axonal metabolic dysfunction. Our comprehensive metabolomic investigations revealed in symptomatic as well as in presymptomatic Opa1delTTAG/+ mice, a specific sensitiveness of the optic nerve to Opa1 insufficiency, opening new routes for protective therapeutic strategies.

Brain energy metabolism and neuroinflammation in ageing APP/PS1-21 mice using longitudinal 18F-FDG and 18F-DPA-714 PET imaging.

PubMed

Takkinen, Jatta S; López-Picón, Francisco R; Al Majidi, Rana; Eskola, Olli; Krzyczmonik, Anna; Keller, Thomas; Löyttyniemi, Eliisa; Solin, Olof; Rinne, Juha O; Haaparanta-Solin, Merja

2017-08-01

Preclinical animal model studies of brain energy metabolism and neuroinflammation in Alzheimer's disease have produced conflicting results, hampering both the elucidation of the underlying disease mechanism and the development of effective Alzheimer's disease therapies. Here, we aimed to quantify the relationship between brain energy metabolism and neuroinflammation in the APP/PS1-21 transgenic mouse model of Alzheimer's disease using longitudinal in vivo 18 F-FDG and 18 F-DPA-714) PET imaging and ex vivo brain autoradiography. APP/PS1-21 (TG, n = 9) and wild type control mice (WT, n = 9) were studied longitudinally every third month from age 6 to 15 months with 18 F-FDG and 18 F-DPA-714 with a one-week interval between the scans. Additional TG (n = 52) and WT (n = 29) mice were used for ex vivo studies. In vivo, the 18 F-FDG SUVs were lower and the 18 F-DPA-714 binding ratios relative to the cerebellum were higher in the TG mouse cortex and hippocampus than in WT mice at age 12 to 15 months ( p < 0.05). The ex vivo cerebellum binding ratios supported the results of the in vivo 18 F-DPA-714 studies but not the 18 F-FDG studies. This longitudinal PET study demonstrated decreased energy metabolism and increased inflammation in the brains of APP/PS1-21 mice compared to WT mice.

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice.

PubMed

Chen, Mei-Shu; Lin, Hua-Kuo; Chiu, Hsun; Lee, Don-Ching; Chung, Yu-Fen; Chiu, Ing-Ming

2015-03-01

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B-GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (-540 to +31). Using the fresh brain sections of F1B-GFP transgenic mice, we found that the F1B-GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B-GFP(+) cells existed in the brains of F1B-GFP transgenic mice. We demonstrated that one population of F1B-GFP(+) cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B-GFP(+) cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B-GFP(+) cells and tyrosine hydroxylase indicated that a subpopulation of F1B-GFP(+) -neuronal cells was dopaminergic neurons. Importantly, these F1B-GFP(+) /TH(+) cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B-GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. © 2014 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.

Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2.

PubMed

Rosser, Meredith F N; Grove, Diane E; Chen, Liling; Cyr, Douglas M

2008-11-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRDeltaF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

Two Small Molecules Restore Stability to a Subpopulation of the Cystic Fibrosis Transmembrane Conductance Regulator with the Predominant Disease-causing Mutation.

PubMed

Meng, Xin; Wang, Yiting; Wang, Xiaomeng; Wrennall, Joe A; Rimington, Tracy L; Li, Hongyu; Cai, Zhiwei; Ford, Robert C; Sheppard, David N

2017-03-03

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl - channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl - channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopic CFTR Cl - currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wild-type CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified full-length F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl - channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl - currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl - channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative disposition of (R)-(+)-pulegone in B6C3F1 mice and F344 rats.

PubMed

Chen, L-J; Lebetkin, E H; Burka, L T

2003-07-01

Pulegone is a monoterpene ketone that is usually associated with the herb pennyroyal but is also found in the essential oils from many other mint species. It is the major constituent of pennyroyal oil. Pennyroyal is used as a flavoring and fragrance and as an herbal medicine to induce menstruation and abortion. A disposition study of 14C-pulegone in B6C3F1 mice and F344 rats has been conducted at doses from 0.8 to 80 mg/kg. Mice excrete 85 to 100% of the dose in 24 h. Rats excrete only 59 to 81% of the administered radioactivity in the same time, primarily in urine and feces, with a trace in respired air. Consequently, tissue concentrations are lower in mice than in rats. Male rats tend to have higher tissue concentrations, especially in kidney, than female rats have, but this sex difference is not seen in mice. The residual radioactivity at 24 h demonstrates potential for accumulation of pulegone-derived material in several tissues following multiple doses. The metabolic profile is complex in both species, with at least three pathways involving hydroxylation, reduction, or conjugation with glutathione as first steps. Mercapturic acid pathway metabolites were detected in bile in mice and both bile and urine in rats.

Differences in the metabolism and disposition of inhaled (3H)benzene by F344/N rats and B6C3F1 mice

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

Sabourin, P.J.; Bechtold, W.E.; Birnbaum, L.S.

1988-06-15

Benzene is a potent hematotoxin and has been shown to cause leukemia in man. Chronic toxicity studies indicate that B6C3F1 mice are more susceptible than F334/N rats to benzene toxicity. The purpose of the studies presented in this paper was to determine if there were metabolic differences between F344/N rats and B6C3F1 mice which might be responsible for this increased susceptibility. Metabolites of benzene in blood, liver, lung, and bone marrow were measured during and following a 6-hr 50 ppm exposure to benzene vapor. Hydroquinone glucuronide, hydroquinone, and muconic acid, which reflect pathways leading to potential toxic metabolites of benzene,more » were present in much greater concentrations in the mouse than in rat tissues. Phenylsulfate, a detoxified metabolite, and an unknown water-soluble metabolite were present in approximately equal concentrations in these two species. These results indicate that the proportion of benzene metabolized via pathways leading to the formation of potentially toxic metabolites as opposed to detoxification pathways was much higher in B6C3F1 mice than in F344 rats, which may explain the higher susceptibility of mice to benzene-induced hematotoxicity and carcinogenicity.« less

Toxicology and carcinogenesis studies of acrylamide (CASRN 79-06-1) in F344/N rats and B6C3F1 mice (feed and drinking water studies).

PubMed

2012-07-01

Acrylamide, a water-soluble α,β-unsaturated amide, is a contaminant in baked and fried starchy foods, including french fries, potato chips, and bread, as a result of Maillard reactions involving asparagine and reducing sugars. Additional sources of acrylamide exposure include cigarettes, laboratory procedures involving polyacrylamide gels, and various occupations (e.g, monomer production and polymerization processes). Acrylamide is carcinogenic in experimental animals. To obtain data for developing quantitative risk assessments for dietary exposures to acrylamide, the Food and Drug Administration nominated acrylamide for an in-depth toxicological evaluation by the National Toxicology Program. As part of this evaluation, male and female B6C3F1/Nctr (C57BL/6N x C3H/HeN MTV-) mice and male and female F344/N Nctr rats were exposed to acrylamide (at least 99.4% pure) in drinking water for 2 years. 2-WEEK STUDY IN RATS: Groups of four male and four female F344/N rats were administered 0, 0.14, 0.35, 0.70, 1.41, 3.52, or 7.03 mM acrylamide in the drinking water (0, 10, 25, 50, 100, 250, or 500 ppm acrylamide) or 0.0, 7.4, 18.5, 37, 74, 185, or 370 mg acrylamide per kg diet for 14 days. One male rat administered 7.03 mM acrylamide in the drinking water died on day 14. Male and female rats receiving 7.03 mM acrylamide weighed 56% and 64% of controls, respectively. Male and female rats fed 370 mg acrylamide per kg diet weighed 74% and 83% of controls, respectively. Female rats receiving 3.52 mM acrylamide in drinking water and male rats fed 185 mg acrylamide per kg diet weighed 85% and 89% of controls, respectively. Rats receiving 7.03 mM acrylamide in drinking water or 370 mg acrylamide per kg diet exhibited hind-leg paralysis on day 14. Mild to moderate dilatation of the urinary bladder was observed in all rats given 370 mg acrylamide per kg diet, and in three of four male rats and all four female rats given 7.03 mM acrylamide in drinking water, and in one of four male

Inheritance of steroid-independent male sexual behavior in male offspring of B6D2F1 mice.

PubMed

McInnis, Christine M; Bonthuis, Paul J; Rissman, Emilie F; Park, Jin Ho

2016-04-01

The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrates steroid-independent male sexual behavior (identified as "maters"), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones. Recent studies have revealed several proteins that play a significant factor in regulating steroid-independent male sexual behavior in B6D2F1 male mice, including amyloid precursor protein (APP), tau, and synaptophysin. The specific goals of our study were to determine whether steroid-independent male sexual behavior was a heritable trait by determining if it was dependent upon the behavioral phenotype of the B6D2F1 sire, and whether the differential expression of APP, tau, and synaptophysin in the medial preoptic area found in the B6D2F1 sires that did and did not mate after gonadectomy was similar to those found in their male offspring. After adult B6D2F1 male mice were bred with C57BL/6J female mice, they and their male offspring (BXB1) were orchidectomized and identified as either maters or "non-maters". A significant proportion of the BXB1 maters was sired only from B6D2F1 maters, indicating that the steroid-independent male sexual behavior behavioral phenotype of the B6D2F1 hybrid males, when crossed with C57BL/6J female mice, is inherited by their male offspring. Additionally, APP, tau, and synaptophysin were elevated in in the medial preoptic area in both the B6D2F1 and BXB1 maters relative to the B6D2F1 and BXB1 non-maters, respectively, suggesting a potential genetic mechanism for the inheritance of steroid-independent male sexual behavior. Copyright

Inheritance of steroid-independent male sexual behavior in male offspring of B6D2F1 mice

PubMed Central

McInnis, Christine M.; Bonthuis, Paul J.; Rissman, Emilie F.; Park, Jin Ho

2016-01-01

The importance of gonadal steroids in modulating male sexual behavior is well established. Individual differences in male sexual behavior, independent of gonadal steroids, are prevalent across a wide range of species, including man. However, the genetic mechanisms underlying steroid-independent male sexual behavior are poorly understood. A high proportion of B6D2F1 hybrid male mice demonstrate steroid-independent male sexual behavior (identified as “maters”), providing a mouse model that opens up avenues of investigation into the mechanisms regulating male sexual behavior in the absence of gonadal hormones. Recent studies have revealed several proteins that play a significant factor in regulating steroid-independent male sexual behavior in B6D2F1 male mice, including amyloid precursor protein (APP), tau, and synaptophysin. The specific goals of our study were to determine whether steroid-independent male sexual behavior was a heritable trait by determining if it was dependent upon the behavioral phenotype of the B6D2F1 sire, and whether the differential expression of APP, tau, and synaptophysin in the medial preoptic area found in the B6D2F1 sires that did and did not mate after gonadectomy was similar to those found in their male offspring. After adult B6D2F1 male mice were bred with C57BL/6J female mice, they and their male offspring (BXB1) were orchidectomized and identified as either maters or “non-maters.” A significant proportion of the BXB1 maters were sired only from B6D2F1 maters, indicating that the steroid-independent male sexual behavior behavioral phenotype of the B6D2F1 hybrid males, when crossed with C57BL/6J female mice, is inherited by their male offspring. Additionally, APP, tau, and synaptophysin were elevated in in the medial preoptic area in both the B6D2F1 and BXB1 maters relative to the B6D2F1 and BXB1 non-maters, respectively, suggesting a potential genetic mechanism for the inheritance of steroid-independent male sexual behavior

Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

PubMed

Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

2004-05-01

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Correlation of the level of full-length CFTR transcript with pulmonary phenotype in patients carrying R117H and 1342-1,-2delAG mutations

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

Hamosh, A.; Cutting, G.R.; Oates, R.

The R117H mutation occurs on two chromosome backgrounds, one associated with a 7 thymidine tract (7T-R11H) in the splice-acceptor site of intron 8, the other with a 5 thymidine tract (5T-R117H). We examined exon 9 splicing efficiency in 5 patients of genotype R117H/{delta}F508 and one carrying 1342-1,-2delAG{delta}F508, an obligate exon 9 slice site mutation. Four patients carried R117H on a 7T background -- three adult men with congenital bilateral absence of the vas deferens and one adolescent female with pancreatitis and borderline sweat chloride concentration. The patient with R117H on a 5T background had pancreatic sufficient CF (PS-CF). The 1342-1,-2delAGmore » patient has classic pancreatic insufficient CF (PI-CF). cDNA was synthesized from total RNA extracted from nasal epithlial cells and analyzed for CFTR splicing by 35 cycle PCR using primers in exon 7 and 11. The quantity of full length transcript derived from the R117H or {delta}F508 alleles was assessed by allele-specific oligonucleotide hybridization. While 91.4% of transcript from the 5T-R117H allele was full-length, only 42.2% of CFTR transcript from the 5T-R117H allele was full length. Since CBAVD patients have no lung disease and PS-CF patients do, this indicates that the threshold of developing CF lung disease is crossed when the amount of CFTR transcript bearing R117H is reduced by half. Interestingly, 17.1% of transcript derived from the 1342-1,-2delAG allele (or 8.6% of total CFTR transcript) was normal and full length. This suggests that up to 9% of full length wild-type CFTR transcript may be inadequate to escape the lung disease of CF and that a 9 thymidine tract followed by AAC (the result of the AG deletion) can be used as a splice donor with 2-9% efficiency.« less

High Precision Metrology on the Ultra-Lightweight W 50.8 cm f/1.25 Parabolic SHARPI Primary Mirror using a CGH Null Lens

NASA Technical Reports Server (NTRS)

Antonille, Scott

2004-01-01

For potential use on the SHARPI mission, Eastman Kodak has delivered a 50.8cm CA f/1.25 ultra-lightweight UV parabolic mirror with a surface figure error requirement of 6nm RMS. We address the challenges involved in verifying and mapping the surface error of this large lightweight mirror to +/-3nm using a diffractive CGH null lens. Of main concern is removal of large systematic errors resulting from surface deflections of the mirror due to gravity as well as smaller contributions from system misalignment and reference optic errors. We present our efforts to characterize these errors and remove their wavefront error contribution in post-processing as well as minimizing the uncertainty these calculations introduce. Data from Kodak and preliminary measurements from NASA Goddard will be included.

28Silicon Irradiation Impairs Contextual Fear Memory in B6D2F1 Mice.

PubMed

Raber, Jacob; Marzulla, Tessa; Stewart, Blair; Kronenberg, Amy; Turker, Mitchell S

2015-06-01

The space radiation environment consists of multiple species of charged particles, including (28)Si, (48)Ti and protons that may impact cognition, but their damaging effects have been poorly defined. In mouse studies, C57Bl6/J homozygous wild-type mice and genetic mutant mice on a C57Bl6/J background have typically been used for assessing effects of space radiation on cognition. In contrast, little is known about the radiation response of mice on a heterozygous background. Therefore, in the current study we tested the effects of (28)Si, (48)Ti and proton radiation on hippocampus-dependent contextual fear memory and hippocampus-independent cued fear memory in C57Bl6/J × DBA2/J F1 (B6D2F1) mice three months after irradiation. Contextual fear memory was impaired at a 1.6 Gy dose of (28)Si radiation, but not cued fear memory. (48)Ti or proton irradiation did not affect either type of memory. Based on earlier space radiation cognitive data in C57Bl6/J mice, these data highlight the importance of including different genetic backgrounds in studies aimed at assessing cognitive changes after exposure to space radiation.

Studies of styrene, styrene oxide and 4-hydroxystyrene toxicity in CYP2F2 knockout and CYP2F1 humanized mice support lack of human relevance for mouse lung tumors.

PubMed

Cruzan, G; Bus, J; Hotchkiss, J; Sura, R; Moore, C; Yost, G; Banton, M; Sarang, S

2013-06-01

Styrene (S) is lung tumorigenic in mice but not in rats. S and its alkene-oxidized metabolite styrene oxide (SO) were not lung toxic in CYP2F2(-/-) [knockout] mice, indicating S-induced mouse lung tumors are mediated through mouse-specific CYP2F2-generated ring-oxidized metabolite(s) in lung bronchioles. The human relevance of the CYP2F MOA was assessed by insertion of a human CYP2F1, 2A13, 2B6 transgene into CYP2F2(-/-) mice; CYP2F1 expression and activity were confirmed in the transgenic (TG) mice. No evidence of cytotoxicity or increased cell proliferation (BrdU labeling) was seen in TG mice treated with either S or SO (200mg/kg/day ip for 5days). In contrast to S and SO, 4HS (105mg/kg/day ip for 5days) increased BrdU labeling 5-10-fold in WT mice, <3-fold increase in KO mice and 2-4-fold in TG mice. The limited response of 4HS in KO and TG mice may result from intrinsic toxicity or from further metabolism; regardless of the MOA, these findings indicate that the CYP2F-mediated tumorigenic MOA in WT mice is not operative for S, SO, or for 4HS putatively derived from metabolism of S by CYP2F1 in humans, and thus S-induced mouse lung tumors are unlikely to be relevant to human risk. Copyright © 2013. Published by Elsevier Inc.

45 CFR 50.8 - Compliance.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Compliance. 50.8 Section 50.8 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION U.S. EXCHANGE VISITOR PROGRAM-REQUEST FOR WAIVER OF THE TWO-YEAR FOREIGN RESIDENCE REQUIREMENT § 50.8 Compliance. If an alien physician acquires H...

45 CFR 50.8 - Compliance.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Compliance. 50.8 Section 50.8 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION U.S. EXCHANGE VISITOR PROGRAM-REQUEST FOR WAIVER OF THE TWO-YEAR FOREIGN RESIDENCE REQUIREMENT § 50.8 Compliance. If an alien physician acquires H...

The effects of in utero bisphenol A exposure on ovarian follicle numbers and steroidogenesis in the F1 and F2 generations of mice.

PubMed

Mahalingam, Sharada; Ther, Laura; Gao, Liying; Wang, Wei; Ziv-Gal, Ayelet; Flaws, Jodi A

2017-12-01

Bisphenol A (BPA) is a commonly used plasticizer. Previous studies show that in utero exposure to BPA affects reproductive outcomes in the F1-F3 generations of mice. However, its multigenerational effects on ovarian histology and steroidogenesis over the reproductive lifespan are unknown. Thus, we tested the hypothesis that BPA has multigenerational effects on follicle numbers and steroidogenesis. Mice were exposed in utero to vehicle control or BPA (0.5, 20, and 50μg/kg/day). Ovaries were collected for histological and gene expression analyses and sera were collected for hormone assays. In utero BPA exposure decreased preantral follicle numbers, cytochrome P450 aromatase mRNA levels, and estradiol levels in the F1 generation, whereas it decreased testosterone levels and altered steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage, 3β-hydroxysteroid dehydrogenase 1, and cytochrome P450 aromatase mRNA levels in the F2 generation. These data suggest that BPA has multigenerational effects on the ovary in mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Tolerance induction between two different strains of parental mice prevents graft-versus-host disease in haploidentical hematopoietic stem cell transplantation to F1 mice

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

Guo, Yixian; Zhang, Lanfang; Wan, Suigui

Highlights: • Injection of UVB-irradiated iDCs induces alloantigen tolerance. • This alloantigen tolerance may be associated regulatory T cell induction. • Tolerant mice serve as bone marrow donors reduces GVHD to their F1 recipients in allo-HSCT. • Tolerance is maintained in F1 recipients for long time post HSCT. - Abstract: Haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) has been employed worldwide in recent years and led to favorable outcome in a group of patients who do not have human leukocyte antigen (HLA)-matched donors. However, the high incidence of severe graft-versus-host disease (GVHD) is a major problem for Haplo-HSCT. In the currentmore » study, we performed a proof of concept mouse study to test whether induction of allogeneic tolerance between two different parental strains was able to attenuate GVHD in Haplo-HSCT to the F1 mice. We induced alloantigen tolerance in C3H mice (H-2k) using ultraviolet B (UVB) irradiated immature dendritic cells (iDCs) derived from the cultures of Balb/c bone marrow cells. Then, we performed Haplo-HSCT using tolerant C3H mice as donors to F1 mice (C3H × Balb/c). The results demonstrated that this approach markedly reduced GVHD-associated death and significantly prolonged the survival of recipient mice in contrast to the groups with donors (C3H mice) that received infusion of non-UVB-irradiated DCs. Further studies showed that there were enhanced Tregs in the tolerant mice and alloantigen-specific T cell response was skewed to more IL-10-producing T cells, suggesting that these regulatory T cells might have contributed to the attenuation of GVHD. This study suggests that it is a feasible approach to preventing GVHD in Haplo-HSCT in children by pre-induction of alloantigen tolerance between the two parents. This concept may also lead to more opportunities in cell-based immunotherapy for GVHD post Haplo-HSCT.« less

CARCINOGENICITY OF BROMODICHLOROMETHANE ADMINISTERED IN DRINKING WATER TO MALE F344/N RATS AND B6C3F1 MICE

EPA Science Inventory

A life-time exposure study was conducted to assess the carcinogenicity of bromodichloromethane (BDCM) administered in the drinking water to male F344/N rats and B6C3F1 mice. Mouse: Target concentrations of BDCM (dissolved in deionized water containing 0.25% emulphor) were 0.05, ...

45 CFR 162.508 - Enumeration System.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Enumeration System. 162.508 Section 162.508 Public... ADMINISTRATIVE REQUIREMENTS Standard Unique Health Identifier for Health Plans § 162.508 Enumeration System. The Enumeration System must do all of the following: (a) Assign a single, unique— (1) HPID to a health plan...

45 CFR 162.508 - Enumeration System.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Enumeration System. 162.508 Section 162.508 Public... ADMINISTRATIVE REQUIREMENTS Standard Unique Health Identifier for Health Plans § 162.508 Enumeration System. The Enumeration System must do all of the following: (a) Assign a single, unique— (1) HPID to a health plan...

45 CFR 162.508 - Enumeration System.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Enumeration System. 162.508 Section 162.508 Public... ADMINISTRATIVE REQUIREMENTS Standard Unique Health Identifier for Health Plans § 162.508 Enumeration System. The Enumeration System must do all of the following: (a) Assign a single, unique— (1) HPID to a health plan...

Targeting the Intracellular Environment in Cystic Fibrosis: Restoring Autophagy as a Novel Strategy to Circumvent the CFTR Defect

PubMed Central

Villella, Valeria Rachela; Esposito, Speranza; Bruscia, Emanuela M.; Maiuri, Maria Chiara; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

2013-01-01

Cystic fibrosis (CF) patients harboring the most common deletion mutation of the CF transmembrane conductance regulator (CFTR), F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane (PM)-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the PM even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1), a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the PM. We focus on the relationship between the improvement of peripheral proteostasis and CFTR PM stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent pre-clinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation. PMID:23346057

Functional genomic responses to cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR(delta508) in the lung.

PubMed

Xu, Yan; Liu, Cong; Clark, Jean C; Whitsett, Jeffrey A

2006-04-21

Cystic fibrosis (CF), a common lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that disturbs fluid homeostasis and host defense in target organs. The effects of CFTR and delta508-CFTR were assessed in transgenic mice that 1) lack CFTR expression (Cftr-/-); 2) express the human delta508 CFTR (CFTR(delta508)); 3) overexpress the normal human CFTR (CFTR(tg)) in respiratory epithelial cells. Genes were selected from Affymetrix Murine Gene-Chips analysis and subjected to functional classification, k-means clustering, promoter cis-elements/modules searching, literature mining, and pathway exploring. Genomic responses to Cftr-/- were not corrected by expression of CFTR(delta508). Genes regulating host defense, inflammation, fluid and electrolyte transport were similarly altered in Cftr-/- and CFTR(delta508) mice. CFTR(delta508) induced a primary disturbance in expression of genes regulating redox and antioxidant systems. Genomic responses to CFTR(tg) were modest and were not associated with lung pathology. CFTR(tg) and CFTR(delta508) induced genes encoding heat shock proteins and other chaperones but did not activate the endoplasmic reticulum-associated degradation pathway. RNAs encoding proteins that directly interact with CFTR were identified in each of the CFTR mouse models, supporting the hypothesis that CFTR functions within a multiprotein complex whose members interact at the level of protein-protein interactions and gene expression. Promoters of genes influenced by CFTR shared common regulatory elements, suggesting that their co-expression may be mediated by shared regulatory mechanisms. Genes and pathways involved in the response to CFTR may be of interest as modifiers of CF.

Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

PubMed Central

Ahner, Annette; Gong, Xiaoyan; Schmidt, Bela Z.; Peters, Kathryn W.; Rabeh, Wael M.; Thibodeau, Patrick H.; Lukacs, Gergely L.; Frizzell, Raymond A.

2013-01-01

Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein. PMID:23155000

SiglecF+Gr1hi eosinophils are a distinct subpopulation within the lungs of allergen-challenged mice

PubMed Central

Percopo, Caroline M.; Brenner, Todd A.; Ma, Michelle; Kraemer, Laura S.; Hakeem, Reem M. A.; Lee, James J.; Rosenberg, Helene F.

2017-01-01

Although eosinophils as a group are readily identified by their unique morphology and staining properties, flow cytometry provides an important means for identification of subgroups based on differential expression of distinct surface Ags. Here, we characterize an eosinophil subpopulation defined by high levels of expression of the neutrophil Ag Gr1 (CD45+CD11c−SiglecF+Gr1hi). SiglecF+Gr1hi eosinophils, distinct from the canonical SiglecF+Gr1− eosinophil population, were detected in allergen-challenged wild-type and granule protein-deficient (EPX−/− and MBP-1−/−) mice, but not in the eosinophil-deficient ΔdblGATA strain. In contrast to Gr1+ neutrophils, which express both cross-reacting Ags Ly6C and Ly6G, SiglecF+Gr1hi eosinophils from allergen-challenged lung tissue are uniquely Ly6G+. Although indistinguishable from the more-numerous SiglecF+Gr1− eosinophils under light microscopy, FACS-isolated populations revealed prominent differences in cytokine contents. The lymphocyte-targeting cytokines CXCL13 and IL-27 were identified only in the SiglecF+Gr1hi eosinophil population (at 3.9 and 4.8 pg/106 cells, respectively), as was the prominent proinflammatory mediator IL-13 (72 pg/106 cells). Interestingly, bone marrow-derived (SiglecF+), cultured eosinophils include a more substantial Gr1+ subpopulation (∼50%); Gr1+ bmEos includes primarily a single Ly6C+ and a smaller, double-positive (Ly6C+Ly6G+) population. Taken together, our findings characterize a distinct SiglecF+Gr1hi eosinophil subset in lungs of allergen-challenged, wild-type and granule protein-deficient mice. SiglecF+Gr1hi eosinophils from wild-type mice maintain a distinct subset of cytokines, including those active on B and T lymphocytes. These cytokines may facilitate eosinophil-mediated immunomodulatory responses in the allergen-challenged lung as well as in other distinct microenvironments. PMID:27531929

SiglecF+Gr1hi eosinophils are a distinct subpopulation within the lungs of allergen-challenged mice.

PubMed

Percopo, Caroline M; Brenner, Todd A; Ma, Michelle; Kraemer, Laura S; Hakeem, Reem M A; Lee, James J; Rosenberg, Helene F

2017-01-01

Although eosinophils as a group are readily identified by their unique morphology and staining properties, flow cytometry provides an important means for identification of subgroups based on differential expression of distinct surface Ags. Here, we characterize an eosinophil subpopulation defined by high levels of expression of the neutrophil Ag Gr1 (CD45 + CD11c - SiglecF + Gr1 hi ). SiglecF + Gr1 hi eosinophils, distinct from the canonical SiglecF + Gr1 - eosinophil population, were detected in allergen-challenged wild-type and granule protein-deficient (EPX -/- and MBP-1 -/- ) mice, but not in the eosinophil-deficient ΔdblGATA strain. In contrast to Gr1 + neutrophils, which express both cross-reacting Ags Ly6C and Ly6G, SiglecF + Gr1 hi eosinophils from allergen-challenged lung tissue are uniquely Ly6G + Although indistinguishable from the more-numerous SiglecF + Gr1 - eosinophils under light microscopy, FACS-isolated populations revealed prominent differences in cytokine contents. The lymphocyte-targeting cytokines CXCL13 and IL-27 were identified only in the SiglecF + Gr1 hi eosinophil population (at 3.9 and 4.8 pg/10 6 cells, respectively), as was the prominent proinflammatory mediator IL-13 (72 pg/10 6 cells). Interestingly, bone marrow-derived (SiglecF + ), cultured eosinophils include a more substantial Gr1 + subpopulation (∼50%); Gr1 + bmEos includes primarily a single Ly6C + and a smaller, double-positive (Ly6C + Ly6G + ) population. Taken together, our findings characterize a distinct SiglecF + Gr1 hi eosinophil subset in lungs of allergen-challenged, wild-type and granule protein-deficient mice. SiglecF + Gr1 hi eosinophils from wild-type mice maintain a distinct subset of cytokines, including those active on B and T lymphocytes. These cytokines may facilitate eosinophil-mediated immunomodulatory responses in the allergen-challenged lung as well as in other distinct microenvironments. © Society for Leukocyte Biology.

32 CFR 508.1 - Utilization of Army bands.

Code of Federal Regulations, 2010 CFR

2010-07-01

... PUBLIC RELATIONS COMPETITION WITH CIVILIAN BANDS § 508.1 Utilization of Army bands. (a) General... Secretary of Defense. The authority to determine whether the use of an Army band at a public gathering is... Forces, veterans, and patriotic organizations. (3) At public rallies and parades intended to stimulate...

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770

PubMed Central

Gentzsch, Martina; Ren, Hong Y.; Houck, Scott A.; Quinney, Nancy L.; Cholon, Deborah M.; Sopha, Pattarawut; Chaudhry, Imron G.; Das, Jhuma; Dokholyan, Nikolay V.; Randell, Scott H.

2016-01-01

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients. PMID:27402691

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770.

PubMed

Gentzsch, Martina; Ren, Hong Y; Houck, Scott A; Quinney, Nancy L; Cholon, Deborah M; Sopha, Pattarawut; Chaudhry, Imron G; Das, Jhuma; Dokholyan, Nikolay V; Randell, Scott H; Cyr, Douglas M

2016-09-01

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients. Copyright © 2016 the American Physiological Society.

A PLC-γ1-independent, RasGRP1-ERK dependent pathway drives lymphoproliferative disease in LAT-Y136F mutant mice

PubMed Central

Kortum, Robert L.; Rouquette-Jazdanian, Alexandre K.; Miyaji, Michihiko; Merrill, Robert K.; Markegard, Evan; Pinski, John M.; Wesselink, Amelia; Nath, Nandan N.; Alexander, Clayton P.; Li, Wenmei; Kedei, Noemi; Roose, Jeroen P.; Blumberg, Peter M.; Samelson, Lawrence E.; Sommers, Connie L.

2012-01-01

Mice expressing a germline mutation in the PLC-γ1 binding site of LAT (linker for activation of T cells) show progressive lymphoproliferation and ultimately die at 4–6 months of age. The hyper-activated T cells in these mice show defective TCR-induced calcium flux, but enhanced Ras/ERK activation that is critical for disease progression. Despite the loss of LAT-dependent PLC-γ1 binding and activation, genetic analysis revealed RasGRP1, and not Sos1 or Sos2, to be the major RasGEF responsible for ERK activation and the lymphoproliferative phenotype in these mice. Analysis of isolated CD4+ T cells from LAT-Y136F mice showed altered proximal TCR-dependent kinase signaling, which activated a Zap70- and LAT-independent pathway. Moreover, LAT-Y136F T cells showed ERK activation that was dependent on Lck and/or Fyn, PKCθ, and RasGRP1. These data demonstrate a novel route to Ras activation in vivo in a pathological setting. PMID:23209318

Correctors of the Major Cystic Fibrosis Mutant Interact through Membrane-Spanning Domains.

PubMed

Laselva, Onofrio; Molinski, Steven; Casavola, Valeria; Bear, Christine E

2018-06-01

The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2:F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Anchored PDE4 regulates chloride conductance in wild-type and ΔF508-CFTR human airway epithelia

PubMed Central

Blanchard, Elise; Zlock, Lorna; Lao, Anna; Mika, Delphine; Namkung, Wan; Xie, Moses; Scheitrum, Colleen; Gruenert, Dieter C.; Verkman, Alan S.; Finkbeiner, Walter E.; Conti, Marco; Richter, Wito

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impair its expression and/or chloride channel function. Here, we provide evidence that type 4 cyclic nucleotide phosphodiesterases (PDE4s) are critical regulators of the cAMP/PKA-dependent activation of CFTR in primary human bronchial epithelial cells. In non-CF cells, PDE4 inhibition increased CFTR activity under basal conditions (ΔISC 7.1 μA/cm2) and after isoproterenol stimulation (increased ΔISC from 13.9 to 21.0 μA/cm2) and slowed the return of stimulated CFTR activity to basal levels by >3-fold. In cells homozygous for ΔF508-CFTR, the most common mutation found in CF, PDE4 inhibition alone produced minimal channel activation. However, PDE4 inhibition strongly amplified the effects of CFTR correctors, drugs that increase expression and membrane localization of CFTR, and/or CFTR potentiators, drugs that increase channel gating, to reach ∼25% of the chloride conductance observed in non-CF cells. Biochemical studies indicate that PDE4s are anchored to CFTR and mediate a local regulation of channel function. Taken together, our results implicate PDE4 as an important determinant of CFTR activity in airway epithelia, and support the use of PDE4 inhibitors to potentiate the therapeutic benefits of CFTR correctors and potentiators.—Blanchard, E., Zlock, L., Lao, A., Mika, D., Namkung, W., Xie, M., Scheitrum, C., Gruenert, D.C., Verkman, A.S., Finkbeiner, W.E., Conti, M., Richter, W. Anchored PDE4 regulates chloride conductance in wild type and ΔF508-CFTR human airway epithelia. PMID:24200884

INDUCTION OF CYP1A1 AD CYP1B1 AND FORMATION OF DNA ADDUCTS IN C57BL/6, BALB/C, AND F1 MICE FOLLOWING IN UTERO EXPOSURE TO 3-METHYLCHOLANTHRENE

EPA Science Inventory

Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 x B6D2F1]F2 mice and Balb/c mice treated in utero with 3�m...

Reevaluation and Classification of Duodenal Lesions in B6C3F1 Mice and F344 Rats from 4 Studies of Hexavalent Chromium in Drinking Water.

PubMed

Cullen, John M; Ward, Jerrold M; Thompson, Chad M

2016-02-01

Thirteen-week and 2-year drinking water studies conducted by the National Toxicology Program (NTP) reported that hexavalent chromium (Cr(VI)) induced diffuse epithelial hyperplasia in the duodenum of B6C3F1 mice but not F344 rats. In the 2-year study, Cr(VI) exposure was additionally associated with duodenal adenomas and carcinomas in mice only. Subsequent 13-week Cr(VI) studies conducted by another group demonstrated non-neoplastic duodenal lesions in B6C3F1 mice similar to those of the NTP study as well as mild duodenal hyperplasia in F344 rats. Because intestinal lesions in mice are the basis for proposed safety standards for Cr(VI), and the histopathology data are relevant to the mode of action, consistency (an important Hill criterion for causality) was assessed across the aforementioned studies. Two veterinary pathologists applied uniform diagnostic criteria to the duodenal lesions in rats and mice from the 4 repeated-dose studies. Comparable non-neoplastic intestinal lesions were evident in mice and rats from all 4 studies; however, the incidence and severity of intestinal lesions were greater in mice than rats. These findings demonstrate consistency across studies and species and highlight the importance of standardized nomenclature for intestinal pathology. The differences in the severity of non-neoplastic lesions also likely contribute to the differential tumor response. © The Author(s) 2015.

Reevaluation and Classification of Duodenal Lesions in B6C3F1 Mice and F344 Rats from 4 Studies of Hexavalent Chromium in Drinking Water

PubMed Central

Cullen, John M.; Ward, Jerrold M.

2015-01-01

Thirteen-week and 2-year drinking water studies conducted by the National Toxicology Program (NTP) reported that hexavalent chromium (Cr(VI)) induced diffuse epithelial hyperplasia in the duodenum of B6C3F1 mice but not F344 rats. In the 2-year study, Cr(VI) exposure was additionally associated with duodenal adenomas and carcinomas in mice only. Subsequent 13-week Cr(VI) studies conducted by another group demonstrated non-neoplastic duodenal lesions in B6C3F1 mice similar to those of the NTP study as well as mild duodenal hyperplasia in F344 rats. Because intestinal lesions in mice are the basis for proposed safety standards for Cr(VI), and the histopathology data are relevant to the mode of action, consistency (an important Hill criterion for causality) was assessed across the aforementioned studies. Two veterinary pathologists applied uniform diagnostic criteria to the duodenal lesions in rats and mice from the 4 repeated-dose studies. Comparable non-neoplastic intestinal lesions were evident in mice and rats from all 4 studies; however, the incidence and severity of intestinal lesions were greater in mice than rats. These findings demonstrate consistency across studies and species and highlight the importance of standardized nomenclature for intestinal pathology. The differences in the severity of non-neoplastic lesions also likely contribute to the differential tumor response. PMID:26538584

Dissociation Between Brown Adipose Tissue 18F-FDG Uptake and Thermogenesis in Uncoupling Protein 1-Deficient Mice.

PubMed

Hankir, Mohammed K; Kranz, Mathias; Keipert, Susanne; Weiner, Juliane; Andreasen, Sille G; Kern, Matthias; Patt, Marianne; Klöting, Nora; Heiker, John T; Brust, Peter; Hesse, Swen; Jastroch, Martin; Fenske, Wiebke K

2017-07-01

18 F-FDG PET imaging is routinely used to investigate brown adipose tissue (BAT) thermogenesis, which requires mitochondrial uncoupling protein 1 (UCP1). It remains uncertain, however, whether BAT 18 F-FDG uptake is a reliable surrogate measure of UCP1-mediated heat production. Methods: UCP1 knockout (KO) and wild-type (WT) mice housed at thermoneutrality were treated with the selective β3 adrenergic receptor agonist CL 316, 243 and underwent metabolic cage, infrared thermal imaging and 18 F-FDG PET/MRI experiments. Primary brown adipocytes were additionally examined for their bioenergetics by extracellular flux analysis as well as their uptake of 2-deoxy- 3 H-glucose. Results: In response to CL 316, 243 treatments, oxygen consumption, and BAT thermogenesis were diminished in UCP1 KO mice, but BAT 18 F-FDG uptake was fully retained. Isolated UCP1 KO brown adipocytes exhibited defective induction of uncoupled respiration whereas their glycolytic flux and 2-deoxy- 3 H-glucose uptake rates were largely unaffected. Conclusion: Adrenergic stimulation can increase BAT 18 F-FDG uptake independently of UCP1 thermogenic function. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

40 CFR 35.508 - Funding period.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Funding period. 35.508 Section 35.508... ASSISTANCE Environmental Program Grants for Tribes Preparing An Application § 35.508 Funding period. The Regional Administrator and applicant may negotiate the length of the funding period for environmental...

40 CFR 35.508 - Funding period.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 1 2013-07-01 2013-07-01 false Funding period. 35.508 Section 35.508... ASSISTANCE Environmental Program Grants for Tribes Preparing An Application § 35.508 Funding period. The Regional Administrator and applicant may negotiate the length of the funding period for environmental...

40 CFR 35.508 - Funding period.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 1 2012-07-01 2012-07-01 false Funding period. 35.508 Section 35.508... ASSISTANCE Environmental Program Grants for Tribes Preparing An Application § 35.508 Funding period. The Regional Administrator and applicant may negotiate the length of the funding period for environmental...

40 CFR 35.508 - Funding period.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 1 2014-07-01 2014-07-01 false Funding period. 35.508 Section 35.508... ASSISTANCE Environmental Program Grants for Tribes Preparing An Application § 35.508 Funding period. The Regional Administrator and applicant may negotiate the length of the funding period for environmental...

40 CFR 35.508 - Funding period.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Funding period. 35.508 Section 35.508... ASSISTANCE Environmental Program Grants for Tribes Preparing An Application § 35.508 Funding period. The Regional Administrator and applicant may negotiate the length of the funding period for environmental...

47 CFR 54.508 - Technology plans.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 3 2010-10-01 2010-10-01 false Technology plans. 54.508 Section 54.508... SERVICE Universal Service Support for Schools and Libraries § 54.508 Technology plans. (a) Contents. The technology plans referred to in this subpart must include the following five elements: (1) A clear statement...

Tg.rasH2 Mice and not CByB6F1 Mice Should Be Used for 28-Day Dose Range Finding Studies Prior to 26-Week Tg.rasH2 Carcinogenicity Studies.

PubMed

Paranjpe, Madhav G; Belich, Jessica; Vidmar, Tom J; Elbekai, Reem H; McKeon, Marie; Brown, Caren

Our recent retrospective analysis of data, collected from 29 Tg.rasH2 mouse carcinogenicity studies, determined how successful the strategy of choosing the high dose for the 26-week studies was based on the estimated maximum tolerated dose (EMTD) derived from earlier 28-day dose range finding (DRF) studies conducted in CByB6F1 mice. Our analysis demonstrated that the high doses applied at EMTD in the 26-week Tg.rasH2 studies failed to detect carcinogenic effects. To investigate why the dose selection process failed in the 26-week carcinogenicity studies, the initial body weights, terminal body weights, body weight gains, food consumption, and mortality from the first 4 weeks of 26-week studies with Tg.rasH2 mice were compared with 28-day DRF studies conducted with CByB6F1 mice. Both the 26-week and the earlier respective 28-day studies were conducted with the exact same vehicle, test article, and similar dose levels. The analysis of our results further emphasizes that the EMTD and subsequent lower doses, determined on the basis of the 28-day studies in CByB6F1 mice, may not be an accurate strategy for selecting appropriate dose levels for the 26-week carcinogenicity studies in Tg.rasH2 mice. Based on the analysis presented in this article, we propose that the Tg.rasH2 mice and not the CByB6F1 mice should be used in future DRF studies. The Tg.rasH2 mice demonstrate more toxicity than the CByB6F1 mice, possibly because of their smaller size compared to CByB6F1 mice. Also, the Tg.rasH2 males appear to be more sensitive than the female Tg.rasH2 mice.

Establishment of the Ph. Eur. Hepatitis A virus RNA for NAT testing BRP batch 1.

PubMed

Chudy, M; Nübling, C M; Blümel, J; Daas, A; Costanzo, A

2017-01-01

Detection of viral contamination in plasma donations is critical to prevent transmission of infectious diseases. The European Pharmacopoeia (Ph. Eur.) monograph 1646 'Human plasma (pooled and treated for virus inactivation)', requires that plasma pools used for the manufacture of this product be tested, among others, for the presence of hepatitis A virus RNA by nucleic acid testing (NAT) using a positive control containing 100 International Units (IU) of hepatitis A virus (HAV) RNA per mL. To this end, the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) organised an international collaborative study under the aegis of the Biological Standardisation Programme, for the establishment of the 1 st Biological Reference Preparation (BRP) for HAV RNA for NAT testing. A freeze-dried candidate material was thus prepared and calibrated against the WHO 2 nd International Standard for HAV for NAT (00/562) in a study in which thirteen European and North American laboratories including Official Medicines Control Laboratories (OMCLs), manufacturers of plasma-derived products, producers of in vitro diagnostic kits and a blood transfusion centre participated. Based on the outcome of the study, an HAV RNA content of 40 000 IU/vial (corresponding approximately to 4.6 log 10 IU/vial) was assigned to the BRP, which was adopted by the Ph. Eur. Commission in March 2016 as Ph. Eur. hepatitis A virus RNA for NAT testing BRP batch 1.

Prenatal exposure to an environmentally relevant phthalate mixture disrupts reproduction in F1 female mice

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

Zhou, Changqing; Gao, Liying; Flaws, Jodi A., E-ma

Phthalates are used in a large variety of products, such as building materials, medical devices, and personal care products. Most previous studies on the toxicity of phthalates have focused on single phthalates, but it is also important to study the effects of phthalate mixtures because humans are exposed to phthalate mixtures. Thus, we tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture adversely affects female reproduction in mice. To test this hypothesis, pregnant CD-1 dams were orally dosed with vehicle (tocopherol-stripped corn oil) or a phthalate mixture (20 and 200 μg/kg/day, 200 and 500 mg/kg/day) daily frommore » gestational day 10 to birth. The mixture was based on the composition of phthalates detected in urine samples from pregnant women in Illinois. The mixture included 35% diethyl phthalate, 21% di(2-ethylhexyl) phthalate, 15% dibutyl phthalate, 15% diisononyl phthalate, 8% diisobutyl phthalate, and 5% benzylbutyl phthalate. Female mice born to the exposed dams were subjected to tissue collections and fertility tests at different ages. Our results indicate that prenatal exposure to the phthalate mixture significantly increased uterine weight and decreased anogenital distance on postnatal days 8 and 60, induced cystic ovaries at 13 months, disrupted estrous cyclicity, reduced fertility-related indices, and caused some breeding complications at 3, 6, and 9 months of age. Collectively, our data suggest that prenatal exposure to an environmentally relevant phthalate mixture disrupts aspects of female reproduction in mice. - Highlights: • Prenatal exposure to a phthalate mixture disrupts F1 estrous cyclicity. • Prenatal exposure to a phthalate mixture induces F1 ovarian cysts. • Prenatal exposure to a phthalate mixture decreases F1 female fertility-related indices. • Prenatal exposure to a phthalate mixture induces F1 breeding complications.« less

Uncaria rhynchophylla ameliorates cognitive deficits induced by D-galactose in mice.

PubMed

Xian, Yan-Fang; Lin, Zhi-Xiu; Zhao, Ming; Mao, Qing-Qiu; Ip, Siu-Po; Che, Chun-Tao

2011-12-01

The stem with hooks of Uncaria rhynchophylla is a component herb of many traditional formulae for the treatment of neurodegenerative diseases. However, scientific evidence of the efficacy of Uncaria rhynchophylla in the treatment of Alzheimer's disease (AD) in animal models is lacking. Thus, in the present study, we investigated whether the 70 % aqueous ethanol extract of Uncaria rhynchophylla (EUR) could protect against D-galactose (D-gal)-induced cognitive deficits in mice. Mice were given a subcutaneous injection of D-gal (50 mg/kg) and orally administered EUR (100, 200, or 400 mg/kg) daily for 8 weeks. The effect of EUR on D-gal-induced cognitive deficits was evaluated by measuring behavioral and neurochemical parameters of AD and the antioxidant status of brain tissue. The results showed that EUR (200 or 400 mg/kg) significantly increased exploratory behavior (assessed by an open-field test) and improved spatial learning and memory function (assessed by the Morris water maze test) in D-gal-treated mice. In addition, EUR (200 or 400 mg/kg) significantly increased the levels of acetylcholine and glutathione and decreased the activity of acetylcholinesterase and the level of malondialdehyde in the brains of D-gal-treated mice. These results indicate that EUR ameliorates cognitive deficits induced by D-gal in mice, and that this action may be mediated, at least in part, by the inhibition of acetylcholinesterase activity and the enhancement of the antioxidant status of brain tissue. © Georg Thieme Verlag KG Stuttgart · New York.

Recombinant V antigen protects mice against pneumonic and bubonic plague caused by F1-capsule-positive and -negative strains of Yersinia pestis.

PubMed

Anderson, G W; Leary, S E; Williamson, E D; Titball, R W; Welkos, S L; Worsham, P L; Friedlander, A M

1996-11-01

The purified recombinant V antigen from Yersinia pestis, expressed in Escherichia coli and adsorbed to aluminum hydroxide, an adjuvant approved for human use, was used to immunize outbred Hsd:ND4 mice subcutaneously. Immunization protected mice from lethal bubonic and pneumonic plague caused by CO92, a wild-type F1+ strain, or by the isogenic F1- strain C12. This work demonstrates that a subunit plague vaccine formulated for human use provides significant protection against bubonic plague caused by an F1- strain (C12) or against substantial aerosol challenges from either F1+ (CO92) or F1-(C12) Y. pestis.

Collaborative study for the establishment of the Ph. Eur. Hepatitis E virus RNA for NAT testing biological reference preparation batch 1.

PubMed

Baylis, S A; Terao, E; Blümel, J; Hanschmann, K-M O

2017-01-01

A new European Pharmacopoeia (Ph. Eur.) biological reference preparation (BRP) had to be established further to the decision to include nucleic acid testing (NAT) for the detection of hepatitis E virus (HEV) RNA in the monograph Human plasma (pooled and treated for virus inactivation) (1646). To this purpose, an international collaborative study was launched in the framework of the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the Commission of the European Union (EU). The study was run in conjunction with the establishment of the 1 st World Health Organization (WHO) international reference panel (IRP) for hepatitis E virus RNA genotypes (8578/13). Twenty-three laboratories used in-house developed and commercially available assays to calibrate a lyophilised candidate BRP prepared from a HEV 3f strain positive human plasma against the 1 st WHO International Standard (IS) for HEV RNA (6329/10). Results from quantitative and qualitative assays were in good agreement and were combined to calculate an assigned potency. Real-time stability studies indicated that the candidate BRP is very stable at lower temperatures and is thus suitable for long-term use. Based on these results, in February 2016, the Ph. Eur. Commission adopted the candidate material as the hepatitis E virus RNA for NAT testing BRP batch 1, with an assigned unitage of 2.1 × 10 4 IU/vial (4.32 log 10 IU/vial).

Assessment of safety and efficacy of long-term treatment with combination lumacaftor and ivacaftor therapy in patients with cystic fibrosis homozygous for the F508del-CFTR mutation (PROGRESS): a phase 3, extension study.

PubMed

Konstan, Michael W; McKone, Edward F; Moss, Richard B; Marigowda, Gautham; Tian, Simon; Waltz, David; Huang, Xiaohong; Lubarsky, Barry; Rubin, Jaime; Millar, Stefanie J; Pasta, David J; Mayer-Hamblett, Nicole; Goss, Christopher H; Morgan, Wayne; Sawicki, Gregory S

2017-02-01

The 24-week safety and efficacy of lumacaftor/ivacaftor combination therapy was shown in two randomised controlled trials (RCTs)-TRAFFIC and TRANSPORT-in patients with cystic fibrosis who were aged 12 years or older and homozygous for the F508del-CFTR mutation. We aimed to assess the long-term safety and efficacy of extended lumacaftor/ivacaftor therapy in this group of patients in PROGRESS, the long-term extension of TRAFFIC and TRANSPORT. PROGRESS was a phase 3, parallel-group, multicentre, 96-week study of patients who completed TRAFFIC or TRANSPORT in 191 sites in 15 countries. Patients were eligible if they were at least 12 years old with cystic fibrosis and homozygous for the F508del-CFTR mutation. Exclusion criteria included any comorbidity or laboratory abnormality that, in the opinion of the investigator, might confound the results of the study or pose an additional risk in administering the study drug to the participant, history of drug intolerance, and history of poor compliance with the study drug. Patients who previously received active treatment in TRANSPORT or TRAFFIC remained on the same dose in PROGRESS. Patients who had received placebo in TRANSPORT or TRAFFIC were randomly assigned (1:1) to receive lumacaftor (400 mg every 12 h)/ivacaftor (250 mg every 12 h) or lumacaftor (600 mg once daily)/ivacaftor (250 mg every 12 h). The primary outcome was to assess the long-term safety of combined therapy. The estimated annual rate of decline in percent predicted FEV 1 (ppFEV 1 ) in treated patients was compared with that of a matched registry cohort. Efficacy analyses were based on modified intention-to-treat, such that data were included for all patients who were randomly assigned and received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01931839. Between Oct 24, 2013, and April 7, 2016, 1030 patients from the TRANSPORT and TRAFFIC studies enrolled in PROGRESS, and 1029 received at least one dose of study

Hexavalent Chromium Is Carcinogenic to F344/N Rats and B6C3F1 Mice after Chronic Oral Exposure

PubMed Central

Stout, Matthew D.; Herbert, Ronald A.; Kissling, Grace E.; Collins, Bradley J.; Travlos, Gregory S.; Witt, Kristine L.; Melnick, Ronald L.; Abdo, Kamal M.; Malarkey, David E.; Hooth, Michelle J.

2009-01-01

Background Hexavalent chromium [Cr(VI)] is a human carcinogen after inhalation exposure. Humans also ingest Cr(VI) from contaminated drinking water and soil; however, limited data exist on the oral toxicity and carcinogenicity of Cr(VI). Objective We characterized the chronic oral toxicity and carcinogenicity of Cr(VI) in rodents. Methods The National Toxicology Program (NTP) conducted 2-year drinking water studies of Cr(VI) (as sodium dichromate dihydrate) in male and female F344/N rats and B6C3F1 mice. Results Cr(VI) exposure resulted in increased incidences of rare neoplasms of the squamous epithelium that lines the oral cavity (oral mucosa and tongue) in male and female rats, and of the epithelium lining the small intestine in male and female mice. Cr(VI) exposure did not affect survival but resulted in reduced mean body weights and water consumption, due at least in part to poor palatability of the dosed water. Cr(VI) exposure resulted in transient microcytic hypochromic anemia in rats and microcytosis in mice. Nonneoplastic lesions included diffuse epithelial hyperplasia in the duodenum and jejunum of mice and histiocytic cell infiltration in the duodenum, liver, and mesenteric and pancreatic lymph nodes of rats and mice. Conclusions Cr(VI) was carcinogenic after administration in drinking water to male and female rats and mice. PMID:19479012

30 CFR 77.508-1 - Lightning arresters; wires entering buildings.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Lightning arresters; wires entering buildings... OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.508-1 Lightning arresters; wires entering buildings. Lightning arresters protecting exposed telephone wires entering buildings shall be provided at...

30 CFR 77.508-1 - Lightning arresters; wires entering buildings.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Lightning arresters; wires entering buildings... OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.508-1 Lightning arresters; wires entering buildings. Lightning arresters protecting exposed telephone wires entering buildings shall be provided at...

30 CFR 77.508-1 - Lightning arresters; wires entering buildings.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Lightning arresters; wires entering buildings... OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.508-1 Lightning arresters; wires entering buildings. Lightning arresters protecting exposed telephone wires entering buildings shall be provided at...

30 CFR 77.508-1 - Lightning arresters; wires entering buildings.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Lightning arresters; wires entering buildings... OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.508-1 Lightning arresters; wires entering buildings. Lightning arresters protecting exposed telephone wires entering buildings shall be provided at...

30 CFR 77.508-1 - Lightning arresters; wires entering buildings.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Lightning arresters; wires entering buildings... OF UNDERGROUND COAL MINES Electrical Equipment-General § 77.508-1 Lightning arresters; wires entering buildings. Lightning arresters protecting exposed telephone wires entering buildings shall be provided at...

9 CFR 93.508 - Articles accompanying swine.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Articles accompanying swine. 93.508 Section 93.508 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF... FOR MEANS OF CONVEYANCE AND SHIPPING CONTAINERS Swine § 93.508 Articles accompanying swine. No litter...

9 CFR 93.508 - Articles accompanying swine.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Articles accompanying swine. 93.508 Section 93.508 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF... FOR MEANS OF CONVEYANCE AND SHIPPING CONTAINERS Swine § 93.508 Articles accompanying swine. No litter...

9 CFR 93.508 - Articles accompanying swine.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Articles accompanying swine. 93.508 Section 93.508 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF... FOR MEANS OF CONVEYANCE AND SHIPPING CONTAINERS Swine § 93.508 Articles accompanying swine. No litter...

9 CFR 93.508 - Articles accompanying swine.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Articles accompanying swine. 93.508 Section 93.508 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF... FOR MEANS OF CONVEYANCE AND SHIPPING CONTAINERS Swine § 93.508 Articles accompanying swine. No litter...

9 CFR 93.508 - Articles accompanying swine.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Articles accompanying swine. 93.508 Section 93.508 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF... FOR MEANS OF CONVEYANCE AND SHIPPING CONTAINERS Swine § 93.508 Articles accompanying swine. No litter...

48 CFR 9.508 - Examples.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Examples. 9.508 Section 9... CONTRACTOR QUALIFICATIONS Organizational and Consultant Conflicts of Interest 9.508 Examples. The examples in... officer apply the general rules in 9.505 to individual contract situations. (a) Company A agrees to...

45 CFR 73.735-508 - Other prohibitions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 1 2014-10-01 2014-10-01 false Other prohibitions. 73.735-508 Section 73.735-508 Public Welfare Department of Health and Human Services GENERAL ADMINISTRATION STANDARDS OF CONDUCT Gifts, Entertainment, and Favors § 73.735-508 Other prohibitions. Employees shall avoid any action whether or not...

45 CFR 73.735-508 - Other prohibitions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 1 2012-10-01 2012-10-01 false Other prohibitions. 73.735-508 Section 73.735-508 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION STANDARDS OF CONDUCT Gifts, Entertainment, and Favors § 73.735-508 Other prohibitions. Employees shall avoid any action whether or not...

45 CFR 73.735-508 - Other prohibitions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 1 2013-10-01 2013-10-01 false Other prohibitions. 73.735-508 Section 73.735-508 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION STANDARDS OF CONDUCT Gifts, Entertainment, and Favors § 73.735-508 Other prohibitions. Employees shall avoid any action whether or not...

63. G.F.H., photographer July 30, 1932 DEL NORTE COUNTY, SECTION ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

63. G.F.H., photographer July 30, 1932 DEL NORTE COUNTY, SECTION A, HIGHWAY 1. 1-DN A #124, STA. 164=00 SHOWING DRAINAGE CONDITIONS, G.F.H., 7-30-32. - Redwood National & State Parks Roads, California coast from Crescent City to Trinidad, Crescent City, Del Norte County, CA

Toxicology and carcinogenesis studies of methylphenidate hydrochloride (Cas No. 298-59-9) in F344/N rats and B6C3F1 mice (feed studies). Technical report series

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

NONE

1995-07-01

Toxicology and carcinogenicity studies were conducted by administration of methylphenidate hydrochloride in feed to groups of 70 F344/N rats of each sex at doses of 0, 100, 500, or 1,0000 ppm and to groups of 70 B6C3F1 mice of each sex at doses of 0, 50, 250, or 500 ppm. Under the conditions of these 2-year feed studies, there was no evidence of carcinogenic activity of methylphenidate hydrochloride in male or female F344/N rats receiving 100, 500, or 1,000 ppm. There was some evidence of carcinogenic activity in male and female B6C3F1 mice, based on the occurrence of hepatocellular neoplasms.more » Treatment of female rats with methylphenidate hydrochloride was associated with a decrease in the incidence of mammary gland fibroadenomas. Administration of methylphenidate hydrochloride to male and female mice resulted in increased incidence of eosinophilic foci in the liver.« less

Are EUR and GBP different words for the same currency?

NASA Astrophysics Data System (ADS)

Ivanova, K.; Ausloos, M.

2002-05-01

The British Pound (GBP) is not part of the Euro (EUR) monetary system. In order to find out arguments on whether GBP should join the EUR or not correlations are calculated between GBP exchange rates with respect to various currencies: USD, JPY, CHF, DKK, the currencies forming EUR and a reconstructed EUR for the time interval from 1993 till June 30, 2000. The distribution of fluctuations of the exchange rates is Gaussian for the central part of the distribution, but has fat tails for the large size fluctuations. Within the Detrended Fluctuation Analysis (DFA) statistical method the power law behavior describing the root-mean-square deviation from a linear trend of the exchange rate fluctuations is obtained as a function of time for the time interval of interest. The time-dependent exponent evolution of the exchange rate fluctuations is given. Statistical considerations imply that the GBP is already behaving as a true EUR.

Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

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

Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao

Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made itmore » more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.« less

Widespread Over-Expression of the X Chromosome in Sterile F1 Hybrid Mice

PubMed Central

Good, Jeffrey M.; Giger, Thomas; Dean, Matthew D.; Nachman, Michael W.

2010-01-01

The X chromosome often plays a central role in hybrid male sterility between species, but it is unclear if this reflects underlying regulatory incompatibilities. Here we combine phenotypic data with genome-wide expression data to directly associate aberrant expression patterns with hybrid male sterility between two species of mice. We used a reciprocal cross in which F1 males are sterile in one direction and fertile in the other direction, allowing us to associate expression differences with sterility rather than with other hybrid phenotypes. We found evidence of extensive over-expression of the X chromosome during spermatogenesis in sterile but not in fertile F1 hybrid males. Over-expression was most pronounced in genes that are normally expressed after meiosis, consistent with an X chromosome-wide disruption of expression during the later stages of spermatogenesis. This pattern was not a simple consequence of faster evolutionary divergence on the X chromosome, because X-linked expression was highly conserved between the two species. Thus, transcriptional regulation of the X chromosome during spermatogenesis appears particularly sensitive to evolutionary divergence between species. Overall, these data provide evidence for an underlying regulatory basis to reproductive isolation in house mice and underscore the importance of transcriptional regulation of the X chromosome to the evolution of hybrid male sterility. PMID:20941395

Lactobacillus paracasei GMNL-32 exerts a therapeutic effect on cardiac abnormalities in NZB/W F1 mice

PubMed Central

Rajendran, Peramaiyan; Tzang, Bor-Show; Yeh, Yu-Lan; Shen, Chia-Yao; Chen, Ray-Jade; Ho, Tsung-Jung; Vijaya Padma, Viswanadha

2017-01-01

Systemic lupus erythematosus (SLE) is a disease that mostly affects women. Accelerated atherosclerosis is a high-risk factor associated with SLE patients. SLE associated with cardiovascular disease is one of the most important causes of death. In this study, we demonstrated that Lactobacillus paracasei GMNL-32 (GMNL-32), a probiotic species, exhibits anti-fibrosis and anti-apoptotic effects on the cardiac tissue of NZB/WF1 mice. Female NZB/W F1 mice, a well-known and commonly used lupus-prone mouse strain, were treated with or without GMNL-32 administration for 12 weeks. Oral administration of GMNL-32 to NZB/WF1 mice significantly increased the ventricular thickness when compared to that of NZB/WF1 mice. Administration of GMNL-32 significantly attenuated the cardiac cell apoptosis that was observed in exacerbate levels in the control NZB/WF1 mice. Further, the cellular morphology that was slightly distorted in the NZB/WF1 was effectively alleviated in the treatment group mice. In addition, GMNL-32 reduced the level of Fas death receptor-related pathway of apoptosis signaling and enhanced anti-apoptotic proteins. These results indicate that GMNL-32 exhibit an effective protective effect on cardiac cells of SLE mice. Thus, GMNL-32 may be a potential therapeutic strategy against SLE associated arthrosclerosis. PMID:28934296

The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating

PubMed Central

Cui, Liying; Aleksandrov, Luba; Hou, Yue-Xian; Gentzsch, Martina; Chen, Jey-Hsin; Riordan, John R; Aleksandrov, Andrei A

2006-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function. PMID:16484308

30 CFR 75.508 - Map of electrical system.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Map of electrical system. 75.508 Section 75.508... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Electrical Equipment-General § 75.508 Map of electrical system. [Statutory Provisions] The location and the electrical rating of all stationary electric...

30 CFR 75.508 - Map of electrical system.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Map of electrical system. 75.508 Section 75.508... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Electrical Equipment-General § 75.508 Map of electrical system. [Statutory Provisions] The location and the electrical rating of all stationary electric...

30 CFR 75.508 - Map of electrical system.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Map of electrical system. 75.508 Section 75.508... MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Electrical Equipment-General § 75.508 Map of electrical system. [Statutory Provisions] The location and the electrical rating of all stationary electric...

Effect of lactational exposure to tributyltin chloride on innate immunodefenses in the F1 generation in mice.

PubMed

Kimura, Kimiko; Kobayashi, Kazuo; Naito, Hiroyuki; Suzuki, Yoshihiko; Sugita-Konishi, Yoshiko

2005-06-01

We examined the effect of lactational exposure to tributyltin on innate immunodefenses in the F1 generation using in vivo and in vitro experiments. Pregnant C57BL/6 mice were given drinking water containing 0, 15, or 50 microg/ml of tributyltin chloride (TBTCl) from parturition to weaning. At weaning time, offspring were inoculated with Escherichia coli K-12, and bacterial clearances from the peritoneal cavity and spleen were examined. In vivo infection experiments indicated that bacterial clearance was significantly depressed in offspring breast-fed by dams exposed to 15 microg/ml of TBTCl (15 ppm F1), but not in offspring by dams exposed to 50 microg/ml of TBTCl (50 ppm F1). In vitro functional assays revealed that the killing activity of neutrophils decreased significantly in 15 ppm F1, but not in 50 ppm F1. We suggest that lactational exposure to TBT impairs innate immunodefenses in the F1 generation against non-pathogenic bacterial infection.

Gestational Exposure to Bisphenol A Affects the Function and Proteome Profile of F1 Spermatozoa in Adult Mice.

PubMed

Rahman, Md Saidur; Kwon, Woo-Sung; Karmakar, Polash Chandra; Yoon, Sung-Jae; Ryu, Buom-Yong; Pang, Myung-Geol

2017-02-01

Maternal exposure to the endocrine disruptor bisphenol A (BPA) has been linked to offspring reproductive abnormalities. However, exactly how BPA affects offspring fertility remains poorly understood. The aim of the present study was to evaluate the effects of gestational BPA exposure on sperm function, fertility, and proteome profile of F1 spermatozoa in adult mice. Pregnant CD-1 mice (F0) were gavaged with BPA at three different doses (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on embryonic days 7 to 14. We investigated the function, fertility, and related processes of F1 spermatozoa at postnatal day 120. We also evaluated protein profiles of F1 spermatozoa to monitor their functional affiliation to disease. BPA inhibited sperm count, motility parameters, and intracellular ATP levels in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased protein kinase A (PKA) activity and tyrosine phosphorylation in spermatozoa. We also found that BPA compromised average litter size. Proteins differentially expressed in spermatozoa from BPA treatment groups are known to play a critical role in ATP generation, oxidative stress response, fertility, and in the pathogenesis of several diseases. Our study provides mechanistic support for the hypothesis that gestational exposure to BPA alters sperm function and fertility via down-regulation of tyrosine phosphorylation through a PKA-dependent mechanism. In addition, we anticipate that the BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa. Citation: Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. 2017. Gestational exposure to bisphenol-A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 125:238-245; http://dx.doi.org/10.1289/EHP378.

Long-term Records of Trace Metal Elements in Core Sediments: Anthropogenic Impacts in The Eure River Watershed

NASA Astrophysics Data System (ADS)

Gardes, T.; Debret, M.; Copard, Y.; Patault, E.; Deloffre, J.; Marcotte, S.; Develle, A. L.; Sabatier, P.; Chaumillon, E.; Coulombier, T.; Revillon, S.; Nizou, J.; Laberdesque, Y.; Koltalo, F.

2017-12-01

The Martot Dam is located in the Eure River Watershed (Normandy, France), few hundred meters upstream the Eure-Seine Rivers confluence. In the context of the European Water Framework Directive (2000/60/EC), the French Authorities planned to remove this dam in 2017. Nevertheless, impacts of the removal remain poorly studied. Classically, dam blocked sedimentary transfers downstream, but here, sediments are not blocked behind the dam but stored three hundred meters upstream in a hydraulic annex, called the Martot Pond. Furthermore, this pond is submitted to the tidal flow from the Seine Estuary despite the Martot Dam. The aim of the study is to evaluate the dam removal impacts on sedimentary transfers and re-suspension of contaminated sediments stored in the Martot Pond and the Eure River's channel. Concerning past transfers and sediments accumulation in the Eure River Watershed, sedimentary archives have been cored, before dam removal, at the Martot Pond and the Les Damps Pond (located 10km upstream the latter). Dating of sedimentary cores for both ponds indicates a sedimentation rate around 1 cm y-1. Trace metal elements quantification showed a wide metallic contamination with highest concentrations evidenced during the 1950-1960's (As: 13-22 mg kg-1; Cd: 40-55 mg kg-1; Cr: 170-210 mg kg-1; Cu: 400-490 mg kg-1; Hg: 2.3 mg kg-1; Mn: 1,280-2,200 mg kg-1; Ni: 64-75 mg kg-1; Zn: 905-990 mg kg-1) and the 1990-2000's (Cr: 95-215 mg kg-1; Ni: 100 mg kg-1; Pb: 670-855 mg kg-1). These variations of concentrations along cores can be associated with industrial past of the Eure River Watershed and sources of contamination can be identified. Thereby, Zn, Ni or Hg contamination could be associated with wastes of battery factory released in the Eure River during the economic recovery, while Pb contamination is linked to the activities of a cathode-ray tubes factory. Metals quantification in core materials highlighted anthropogenic impacts in the Eure River Watershed. These

46 CFR 122.508 - Wearing of life jackets.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Wearing of life jackets. 122.508 Section 122.508... Preparations for Emergencies § 122.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including, but not limited to: (1...

46 CFR 122.508 - Wearing of life jackets.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 4 2011-10-01 2011-10-01 false Wearing of life jackets. 122.508 Section 122.508... Preparations for Emergencies § 122.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including, but not limited to: (1...

46 CFR 122.508 - Wearing of life jackets.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 4 2013-10-01 2013-10-01 false Wearing of life jackets. 122.508 Section 122.508... Preparations for Emergencies § 122.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including, but not limited to: (1...

46 CFR 122.508 - Wearing of life jackets.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 4 2014-10-01 2014-10-01 false Wearing of life jackets. 122.508 Section 122.508... Preparations for Emergencies § 122.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including, but not limited to: (1...

46 CFR 122.508 - Wearing of life jackets.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 4 2012-10-01 2012-10-01 false Wearing of life jackets. 122.508 Section 122.508... Preparations for Emergencies § 122.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including, but not limited to: (1...

NTP toxicity studies of sodium dichromate dihydrate (CAS No. 7789-12-0) administered in drinking water to male and female F344/N rats and B6C3F1 mice and male BALB/c and am3-C57BL/6 mice.

PubMed

Bucher, John R

2007-01-01

Sodium dichromate dihydrate is one of a number of inorganic compounds containing hexavalent chromium (CR VI) found in drinking water supplies as a contaminant resulting from various industrial processes including electroplating operations, leather tanning, and textile manufacturing. Because of the lack of adequate experimental data on the toxicity and carcinogenicity of hexavalent chromium ingested orally, and because hexavalent chromium has been found in human drinking water supplies, the California Congressional delegation and the California Environmental Protection Agency nominated hexavalent chromium to the NTP for study. In study 1, male and female F344/N rats and B6C3F1 mice were exposed to sodium dichromate dihydrate (greater than 99% pure) in drinking water for 3 months. In study 2, sodium dichromate dihydrate was administered in drinking water to male B6C3F1, BALB/c, and am3-C57BL/6 mice for 3 months. Genetic toxicology studies were conducted in Salmonella typhimurium, Escherichia coli, and mouse peripheral blood erythrocytes. In study 1, groups of 10 male and 10 female F344/N rats and B6C3F1 mice were given drinking water containing 0, 62.5, 125, 250, 500, or 1,000 mg sodium dichromate dihydrate/L for 3 months (equivalent to average daily doses of approximately 5, 10, 17, 32, or 60 mg sodium dichromate dihydrate/kg body weight to rats and 9, 15, 26, 45, or 80 mg/kg to mice). On a molecular weight basis, these doses are equivalent to approximately 1.7, 3.5, 5.9, 11.2, and 20.9 mg hexavalent chromium/kg body weight per day to rats and 3.1, 5.2, 9.1, 15.7, and 27.9 mg/kg per day to mice. Additional groups of 10 rats per sex were exposed to the same concentrations of sodium dichromate dihydrate for 4 weeks. All rats and mice survived to the end of the study. Reduced body weights occurred in 500 and 1,000 mg/L male rats, 1,000 mg/L female rats, and in male and female mice exposed to 125 mg/L or greater. Water consumption by male and female rats exposed to 250

Disposition of [14C]N,N-dimethyl-p-toluidine in F344 rats and B6C3F1 mice.

PubMed

Dix, Kelly J; Ghanbari, Katayoon; Hedtke-Weber, Briana M

2007-05-15

N,N-Dimethyl-p-toluidine (DMPT) is used as a polymerization accelerator, in industrial glues, and as an intermediate in dye and pesticide synthesis. There is potential for human exposure to DMPT. The disposition of oral and intravenous (i.v.) doses of [14C]DMPT in F344 rats and B6C3F1 mice was investigated. A single i.v. (2.5 mg/kg) or oral (2.5, 25, or 250 mg/kg) dose of [14C]DMPT (1-25 microCi) was administered in an aqueous vehicle to male rats and mice. The 25-mg/kg oral dose was administered to females to investigate possible gender differences in disposition. However, no striking gender differences were observed. Since toxicity studies conducted elsewhere used a corn oil vehicle, the 250-mg/kg oral dose also was administered in corn oil to male rats; disposition was not dependent on vehicle. Excreta (through 24 h) and tissues collected at sacrifice were analyzed for total radioactivity. Dose-dependent differences in toxicity and disposition were observed. Toxicity at the 250-mg/kg oral dose to male mice was consistent with acute renal failure. At the same dose, male rats exhibited clinical signs of toxicity through 12 h but were clinically normal by 24 h. At lower oral doses, [14C]DMPT-derived radioactivity was well absorbed and rapidly excreted, primarily in urine.

Evaluation of 6-([18F] fluoroacetamido)-1-hexanoic-anilide (18F-FAHA) as imaging probe in tumor xenograft mice model

NASA Astrophysics Data System (ADS)

Li, Fiona; Cho, Sung Ju; Yu, Lihai; Hudson, Robert H. E.; Luyt, Leonard G.; Pin, Christopher L.; Kovacs, Michael S.; Koropatnick, James; Lee, Ting-Yim

2016-03-01

Alteration in genetic expression is as important as gene mutation in cancer development and proliferation. Epigenetic changes affect gene expression without altering the DNA sequence. Histone deacetylase (HDAC), an enzyme facilitating histone remodelling, can lead to silencing of tumor suppressor genes making HDAC inhibitors viable anticancer drugs against tumors with increased activity of the enzyme. In this study we evaluated 18F-fluroacetamido-1-hexanoicanilide (18F-FAHA), an artificial HDAC substrate, as imaging probe of HDAC activity of human tumor xenografts in immunocompromised host mice. Human breast and melanoma cell lines, MDA-MB-468 and MDA-MB-435 respectively, known to overexpress HDAC activity were xenografted into immunocompromised mice and HDAC activity was imaged using 18F-FAHA. The melanoma group was treated with saline, SAHA (suberoylanilide hydroxamic acid, an approved anticancer HDAC inhibitor) in DMSO, or DMSO as positive control. Tracer kinetic modelling and SUV were used to estimate HDAC activity from dynamic PET data. Both breast tumor and melanoma group showed great variability in binding rate constant (BRC) of 18F-FAHA suggesting highly variable inter- and intra-tumoral HDAC activity. For the SAHA treated melanoma group, HDAC activity, as monitored by BRC of 18F-FAHA, decreased more than the two (positive and negative) control groups but not tumor growth. Our preliminary study showed that noninvasive PET imaging with 18F-FAHA has the potential to identify patients for whom treatment with HDAC inhibitors are appropriate, to assess the effectiveness of that treatment as an early marker of target reduction, and also eliminate the need for invasive tissue biopsy to individualize treatment.

Cystic fibrosis transmembrane conductance regulator mutations at a referral center for cystic fibrosis*

PubMed Central

Coutinho, Cyntia Arivabeni de Araújo Correia; Marson, Fernando Augusto de Lima; Ribeiro, Antônio Fernando; Ribeiro, José Dirceu; Bertuzzo, Carmen Silvia

2013-01-01

OBJECTIVE: To determine the frequency of six mutations (F508del, G542X, G551D, R553X, R1162X, and N1303K) in patients with cystic fibrosis (CF) diagnosed, at a referral center, on the basis of abnormal results in two determinations of sweat sodium and chloride concentrations. METHODS: This was a cross-sectional study involving 70 patients with CF. The mean age of the patients was 12.38 ± 9.00 years, 51.43% were female, and 94.29% were White. Mutation screening was performed with polymerase chain reaction (for F508del), followed by enzymatic digestion (for other mutations). Clinical analysis was performed on the basis of gender, age, ethnicity, pulmonary/gastrointestinal symptoms, and Shwachman-Kulczycki (SK) score. RESULTS: All of the patients showed pulmonary symptoms, and 8 had no gastrointestinal symptoms. On the basis of the SK scores, CF was determined to be mild, moderate, and severe in 22 (42.3%), 17 (32.7%), and 13 (25.0%) of the patients, respectively. There was no association between F508del mutation and disease severity by SK score. Of the 140 alleles analyzed, F508del mutation was identified in 70 (50%). Other mutations (G542X, G551D, R553X, R1162X, and N1303K) were identified in 12 (7.93%) of the alleles studied. In F508del homozygous patients with severe disease, the OR was 0.124 (95% CI: 0.005-0.826). CONCLUSIONS: In 50% of the alleles studied, the molecular diagnosis of CF was confirmed by identifying a single mutation (F508del). If we consider the analysis of the six most common mutations in the Brazilian population (including F508del), the molecular diagnosis was confirmed in 58.57% of the alleles studied. PMID:24310628

Cystic fibrosis transmembrane conductance regulator mutations at a referral center for cystic fibrosis.

PubMed

Coutinho, Cyntia Arivabeni de Araújo Correia; Marson, Fernando Augusto de Lima; Ribeiro, Antônio Fernando; Ribeiro, José Dirceu; Bertuzzo, Carmen Silvia

2013-01-01

To determine the frequency of six mutations (F508del, G542X, G551D, R553X, R1162X, and N1303K) in patients with cystic fibrosis (CF) diagnosed, at a referral center, on the basis of abnormal results in two determinations of sweat sodium and chloride concentrations. This was a cross-sectional study involving 70 patients with CF. The mean age of the patients was 12.38 ± 9.00 years, 51.43% were female, and 94.29% were White. Mutation screening was performed with polymerase chain reaction (for F508del), followed by enzymatic digestion (for other mutations). Clinical analysis was performed on the basis of gender, age, ethnicity, pulmonary/gastrointestinal symptoms, and Shwachman-Kulczycki (SK) score. All of the patients showed pulmonary symptoms, and 8 had no gastrointestinal symptoms. On the basis of the SK scores, CF was determined to be mild, moderate, and severe in 22 (42.3%), 17 (32.7%), and 13 (25.0%) of the patients, respectively. There was no association between F508del mutation and disease severity by SK score. Of the 140 alleles analyzed, F508del mutation was identified in 70 (50%). Other mutations (G542X, G551D, R553X, R1162X, and N1303K) were identified in 12 (7.93%) of the alleles studied. In F508del homozygous patients with severe disease, the OR was 0.124 (95% CI: 0.005-0.826). In 50% of the alleles studied, the molecular diagnosis of CF was confirmed by identifying a single mutation (F508del). If we consider the analysis of the six most common mutations in the Brazilian population (including F508del), the molecular diagnosis was confirmed in 58.57% of the alleles studied.

40 CFR 89.508 - Test procedures.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 20 2014-07-01 2013-07-01 true Test procedures. 89.508 Section 89.508... Test procedures. (a)(1) For nonroad engines subject to the provisions of this subpart, the prescribed test procedures are the nonroad engine 8-mode test procedure as described in subpart E of this part...

Respiratory syncytial virus infection disrupts monolayer integrity and function in cystic fibrosis airway cells.

PubMed

Kong, Michele; Maeng, Patrick; Hong, Jeong; Szczesniak, Rhonda; Sorscher, Eric; Sullender, Wayne; Clancy, John Paul

2013-09-19

Respiratory Syncytial Virus (RSV) infection is a common contributor to pulmonary symptoms in children with cystic fibrosis (CF). Here we examined RSV infection in immortalized bronchial epithelial cells (CFBE41o-) expressing wild-type (wt) or F508del cystic fibrosis transmembrane conductance regulator (CFTR), for monolayer integrity and RSV replication. CFBE41o- monolayers expressing wt or F508del CFTR were grown on permeable supports and inoculated with RSV A2 strain. Control experiments utilized UV-inactivated RSV and heat-killed RSV. Monolayer resistance and RSV production was monitored for up to six days post-infection. Within 24 h, a progressive decrease in monolayer resistance was observed in RSV infected F508del CFBE41o- cells, while the monolayer integrity of RSV infected wt CFTR CFBE41o- cells remained stable. RSV replication was necessary to disrupt F508del CFBE41o- monolayers as UV-irradiated and heat killed RSV had no effect on monolayer integrity, with an earlier and much more pronounced peak in RSV titer noted in F508del relative to wt CFTR-expressing cells. RSV infection of wt CFBE41o- monolayers also resulted in blunting of CFTR response. These findings identify an enhanced sensitivity of CFBE41o- cells expressing F508del CFTR to RSV infection, replication and monolayer disruption independent of the cellular immune response, and provide a novel mechanism by which cystic fibrosis airway epithelia are susceptible to RSV-dependent injury.

LMW Heparin Prevents Increased Kidney Expression of Proinflammatory Mediators in (NZBxNZW)F1 Mice

PubMed Central

Kanapathippillai, Premasany; Rekvig, Ole Petter; Fenton, Kristin Andreassen

2013-01-01

We have previously demonstrated that continuous infusion of low molecular weight (LMW) heparin delays autoantibody production and development of lupus nephritis in (NZBxNZW)F1 (B/W) mice. In this study we investigated the effect of LMW heparin on renal cytokine and chemokine expression and on nucleosome-mediated activation of nucleosome-specific splenocytes. Total mRNA extracted from kidneys of heparin-treated or -untreated B/W mice was analysed by qPCR for the expression of several cytokines, chemokines, and Toll-like receptors. Splenocytes taken from B/W mice were stimulated with nucleosomes with or without the presence of heparin. Splenocyte cell proliferation as thymidine incorporation and the expression of costimulatory molecules and cell activation markers were measured. Heparin treatment of B/W mice reduced the in vivo expression of CCR2, IL1β, and TLR7 compared to untreated B/W mice. Nucleosome-induced cell proliferation of splenocytes was not influenced by heparin. The expression of CD80, CD86, CD69, CD25, CTLA-4, and TLR 2, 7, 8, and 9 was upregulated upon stimulation by nucleosomes, irrespective of whether heparin was added to the cell culture or not. In conclusion, treatment with heparin lowers the kidney expression of proinflammatory mediators in B/W mice but does not affect nucleosomal activation of splenocytes. PMID:24151519

Pathology of Serially Sacrificed Female B6C3F1 Mice Continuously Exposed to Very Low-Dose-Rate Gamma Rays.

PubMed

Tanaka, I B; Komura, J; Tanaka, S

2017-03-01

We have previously reported on life span shortening as well as increased incidence rates in several neoplasms in B6C3F1 mice that were continuously exposed to 21 mGy/day of gamma rays for 400 days. To clarify whether the life shortening was due to early appearance of neoplasms (shortened latency) or increased promotion/progression, 8-week-old female specific-pathogen-free B6C3F1 mice were gamma-ray irradiated at a low dose rate of 20 mGy/day for 400 days. At 100 days postirradiation, 60-90 mice were sacrificed, and thereafter every 100 days alongside the age-matched nonirradiated controls, for 700 days. Additional groups were allowed to live out their natural life span. Pathological examination was performed on all mice to identify lesions, non-neoplastic and neoplastic, as well as to determine the cause of death. Body weights were significantly increased in irradiated mice from sacrifice days 200-500. Incidence rates for spontaneously occurring non-neoplastic lesions, such as adrenal subcapsular cell hyperplasia, fatty degeneration of the liver, atrophy and tubulostromal hyperplasia of the ovaries, were significantly increased in irradiated mice. Significantly increased incidence rates with no shortening of latency periods were observed in irradiated mice for malignant lymphomas, hepatocellular adenomas/carcinomas, bronchioloalveolar adenomas, harderian gland adenoma/adenocarcinoma. Shortened latencies with significantly increased incidence rates were observed for adrenal subcapsular cell adenomas and ovarian neoplasms (tubulostromal adenoma, granulosa cell tumors) in irradiated mice. Life span shortening in mice exposed to 20 mGy/day was mostly due to malignant lymphomas. Multiple primary neoplasms were significantly increased in mice exposed to 20 mGy/day from sacrifice days 400-700 and in the life span group. Our results confirm that continuous low-dose-rate gamma-ray irradiation of female B6C3F1 mice causes both cancer induction (shortened latency) and

Systemic administration of thrombin peptide TP508 enhances VEGF-stimulated angiogenesis and attenuates effects of chronic hypoxia

PubMed Central

Olszewska-Pazdrak, Barbara; Carney, Darrell H.

2015-01-01

Revascularization of chronic wounds and ischemic tissue is attenuated by endothelial dysfunction and the inability of angiogenic factors to stimulate angiogenesis. We recently showed that TP508, a nonproteolytic thrombin peptide, increases perfusion and NO-dependent vasodilation in hearts with chronic ischemia and stimulates NO production by endothelial cells. In this study, we investigated systemic in vivo effects of TP508 on VEGF-stimulated angiogenesis in vitro using aortic explants in normoxic and hypoxic conditions. Mice were injected with saline or TP508 and 24h later aortas were removed and cultured to quantify endothelial sprouting. TP508 injection increased endothelial sprouting and potentiated the in vitro response to VEGF. Exposure of control explants to hypoxia inhibited basal and VEGF-stimulated endothelial cell sprouting. This effect of hypoxia was significantly prevented by TP508 injection. Thus, TP508 systemic administration increases responsiveness of aortic endothelial cells to VEGF and diminishes the effect of chronic hypoxia on endothelial cell sprouting. Studies using human endothelial cells in culture suggest that protective effects of TP508 during hypoxia may involve stimulation of endothelial cell NO production. These data suggest potential clinical benefit of using a combination of systemic TP508 and local VEGF as a therapy for revascularization of ischemic tissue. PMID:23594718

Methyl isobutyl ketone-induced hepatocellular carcinogenesis in B6C3F1 mice: A constitutive androstane receptor (CAR)-mediated mode of action.

PubMed

Hughes, B J; Thomas, J; Lynch, A M; Borghoff, S J; Green, S; Mensing, T; Sarang, S S; LeBaron, M J

2016-11-01

In a National Toxicology Program (NTP) chronic inhalation study with methyl isobutyl ketone (MIBK), increases in hepatocellular adenomas and hepatocellular adenomas and carcinomas (combined) were observed in male and female B6C3F 1 mice at 1800 ppm. A DNA reactive Mode-of-Action (MOA) for this liver tumor response is not supported by the evidence as MIBK and its major metabolites lack genotoxicity in both in vitro and in vivo studies. Constitutive androstane receptor (CAR) nuclear receptor-mediated activation has been hypothesized as the MOA for MIBK-induced mouse liver tumorigenesis. To further investigate the MOA for MIBK-induced murine liver tumors, male and female B6C3F1, C57BL/6, and CAR/PXR Knockout (KO) mice were exposed to either 0 or 1800 ppm MIBK for 6 h/day, 5 days/week for a total of 10 days. On day 1, mice were implanted with osmotic mini-pumps containing 5-Bromo-2-deoxyuridine (BrdU) 1 h following exposure and humanely euthanized 1-3 h following the final exposure. B6C3F 1 and C57BL/6 mice had statistically significant increases in liver weights compared to controls that corresponded with hepatocellular hypertrophy and increased mitotic figures. Hepatocellular proliferation data indicated induction of S-phase DNA synthesis in B6C3F 1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to control, and no increase was observed in MIBK exposed CAR/PXR KO mice. Liver gene expression changes indicated a maximally-induced Cyp2b10 (CAR-associated) transcript and a slight increase in Cyp3a11(PXR-associated) transcript in B6C3F 1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to controls, but not in Cyp1a1 (AhR-associated) or Cyp4a10 (PPAR-α-associated) transcripts. CAR/PXR KO mice exposed to 1800 ppm MIBK showed no evidence of activation of AhR, CAR, PXR or PPAR-α nuclear receptors via their associated transcripts. MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the CAR and

NTP technical report on toxicity studies of cadmium oxide (Cas No. 1306-19-0) administered by inhalation to F344/N rats and B6C3f1 mice. Toxicity report series

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

NONE

1995-03-01

Three thousand tons of cadmium are imported or produced annually in the United States, and approximately 90% of this is cadmium oxide. Cadmium oxide is used in batteries, electroplating baths, pigments, plastics, synthetic products, and a variety of other materials. Cadmium oxide was nominated for study by the National Cancer Institute because of its widespread use and to obtain toxicity and carcinogenicity information. This report describes toxicity studies of cadmium oxide aerosol in F344/N rats and B6C3F1 mice, including sperm motility and vaginal cytology evaluations, and developmental toxicity studies of cadmium oxide aerosol in Sprague-Dawley rats and Swiss (CD-1) mice.more » Genetic toxicology studies were done in Salmonella typhimurium and B6C3F1 mice erythrocytes.« less

Gestational Exposure to Bisphenol A Affects the Function and Proteome Profile of F1 Spermatozoa in Adult Mice

PubMed Central

Rahman, Md Saidur; Kwon, Woo-Sung; Karmakar, Polash Chandra; Yoon, Sung-Jae; Ryu, Buom-Yong; Pang, Myung-Geol

2016-01-01

Background: Maternal exposure to the endocrine disruptor bisphenol A (BPA) has been linked to offspring reproductive abnormalities. However, exactly how BPA affects offspring fertility remains poorly understood. Objectives: The aim of the present study was to evaluate the effects of gestational BPA exposure on sperm function, fertility, and proteome profile of F1 spermatozoa in adult mice. Methods: Pregnant CD-1 mice (F0) were gavaged with BPA at three different doses (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on embryonic days 7 to 14. We investigated the function, fertility, and related processes of F1 spermatozoa at postnatal day 120. We also evaluated protein profiles of F1 spermatozoa to monitor their functional affiliation to disease. Results: BPA inhibited sperm count, motility parameters, and intracellular ATP levels in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased protein kinase A (PKA) activity and tyrosine phosphorylation in spermatozoa. We also found that BPA compromised average litter size. Proteins differentially expressed in spermatozoa from BPA treatment groups are known to play a critical role in ATP generation, oxidative stress response, fertility, and in the pathogenesis of several diseases. Conclusions: Our study provides mechanistic support for the hypothesis that gestational exposure to BPA alters sperm function and fertility via down-regulation of tyrosine phosphorylation through a PKA-dependent mechanism. In addition, we anticipate that the BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa. Citation: Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. 2017. Gestational exposure to bisphenol-A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 125:238–245; http://dx.doi.org/10.1289/EHP378 PMID:27384531

Editor's Highlight: Complete Attenuation of Mouse Lung Cell Proliferation and Tumorigenicity in CYP2F2 Knockout and CYP2F1 Humanized Mice Exposed to Inhaled Styrene for up to 2 Years Supports a Lack of Human Relevance.

PubMed

Cruzan, George; Bus, James S; Banton, Marcy I; Sarang, Satinder S; Waites, Robbie; Layko, Debra B; Raymond, James; Dodd, Darol; Andersen, Melvin E

2017-10-01

Styrene is a mouse-specific lung carcinogen, and short-term mode of action studies have demonstrated that cytotoxicity and/or cell proliferation, and genomic changes are dependent on CYP2F2 metabolism. The current study examined histopathology, cell proliferation, and genomic changes in CD-1, C57BL/6 (WT), CYP2F2(-/-) (KO), and CYP2F2(-/-) (CYP2F1, 2B6, 2A13-transgene) (TG; humanized) mice following exposure for up to 104 weeks to 0- or 120-ppm styrene vapor. Five mice per treatment group were sacrificed at 1, 26, 52, and 78 weeks. Additional 50 mice per treatment group were followed until death or 104 weeks of exposure. Cytotoxicity was present in the terminal bronchioles of some CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Hyperplasia in the terminal bronchioles was present in CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Increased cell proliferation, measured by KI-67 staining, occurred in CD-1 and WT mice exposed to styrene for 1 week, but not after 26, 52, or 78 weeks, nor in KO or TG mice. Styrene increased the incidence of bronchioloalveolar adenomas and carcinomas in CD-1 mice. No increase in lung tumors was found in WT despite clear evidence of lung toxicity, or, KO or TG mice. The absence of preneoplastic lesions and tumorigenicity in KO and TG mice indicates that mouse-specific CYP2F2 metabolism is responsible for both the short-term and chronic toxicity and tumorigenicity of styrene, and activation of styrene by CYP2F2 is a rodent MOA that is neither quantitatively or qualitatively relevant to humans. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

42 CFR 403.508 - Limitations.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 2 2014-10-01 2014-10-01 false Limitations. 403.508 Section 403.508 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS SPECIAL PROGRAMS AND PROJECTS Beneficiary Counseling and Assistance Grants § 403.508 Limitations. (a) Use...

42 CFR 403.508 - Limitations.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 2 2013-10-01 2013-10-01 false Limitations. 403.508 Section 403.508 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS SPECIAL PROGRAMS AND PROJECTS Beneficiary Counseling and Assistance Grants § 403.508 Limitations. (a) Use...

42 CFR 403.508 - Limitations.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 2 2010-10-01 2010-10-01 false Limitations. 403.508 Section 403.508 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS SPECIAL PROGRAMS AND PROJECTS Beneficiary Counseling and Assistance Grants § 403.508 Limitations. (a) Use...

42 CFR 403.508 - Limitations.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 2 2012-10-01 2012-10-01 false Limitations. 403.508 Section 403.508 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS SPECIAL PROGRAMS AND PROJECTS Beneficiary Counseling and Assistance Grants § 403.508 Limitations. (a) Use...

42 CFR 403.508 - Limitations.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 2 2011-10-01 2011-10-01 false Limitations. 403.508 Section 403.508 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS SPECIAL PROGRAMS AND PROJECTS Beneficiary Counseling and Assistance Grants § 403.508 Limitations. (a) Use...

Structure and Dynamics of NBD1 from CFTR Characterized Using Crystallography and Hydrogen/Deuterium Exchange Mass Spectrometry

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

Lewis, H.A.; Wang, C.; Zhao, X.

2012-04-30

The {Delta}F508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and {Delta}F508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because {Delta}F508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and {Delta}F508 constructs, and the {Delta}F508 structure contained additional mutationsmore » that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide {sup 1}H/{sup 2}H exchange rates in matched F508 and {Delta}F508 constructs reveal that {Delta}F508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the {Delta}F508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-{Delta}F508 structures but completely solvent exposed in all {Delta}F508 structures. These results reinforce the importance of the perturbation {Delta}F508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that

NTP Toxicology and Carcinogenesis Studies of 1,4-Dichlorobenzene (CAS No. 106-46-7) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

PubMed

1987-01-01

1,4-Dichlorobenzene is commonly used as a space deodorant in toilets and for moth control. Because of its extensive production and use and the absence of carcinogenicity data, carcinogenesis studies were conducted by administering 1,4-dichlorobenzene (greater than 99% pure) in corn oil by gavage (5 days per week) to male F344/N rats at doses of 0, 150, or 300 mg/kg and to female F344/N rats and male and female B6C3F1 mice at doses of 0, 300, or 600 mg/kg per day for 2 years (50 animals per group). Fourteen-day and 13-week studies were performed to characterize the toxicity, identify affected sites, and set doses for the 2-year studies. Clinical chemistry and hematologic studies were performed during the 13-week studies to assess the effects of 1,4-dichlorobenzene on the liver, kidney, and hematopoietic system and to assess whether the compound produced hepatic porphyria. Two 13-week studies were performed in rats. In the first study, rats were dosed with 300-1,500 mg/kg 1,4-dichlorobenzene. Because histologic changes were observed in the kidney of male rats at all doses, a second 13-week study was performed at doses of 38-600 mg/kg. In the 13-week studies, survival was decreased in groups of male rats given 1,200 or 1,500 mg/kg and in female rats given 1,500 mg/kg. Weight gain was decreased in male rats receiving doses of 300 mg/kg or more and in female rats given doses of 1,200 or 1,500 mg/kg. Doses of 1,200 or 1,500 mg/kg produced degeneration and necrosis of hepatocytes, hypoplasia of the bone marrow, lymphoid depletion of the spleen and thymus, and epithelial necrosis of the nasal turbinates in male and female rats. Renal tubular cell degeneration was observed in male rats receiving 300 mg/kg or more in the first study, but only slight changes were seen at 300 mg/kg in the second study. Liver weight to brain weight ratios were increased at 900 mg/kg or more for both male and female rats. The kidney weight to brain weight ratio was increased in male rats

NTP toxicity studies of dimethylaminopropyl chloride, hydrochloride (CAS No. 5407-04-5) administered by Gavage to F344/N rats and B6C3F1 mice.

PubMed

Abdo, Km

2007-07-01

Dimethylaminopropyl chloride, hydrochloride is used primarily as an industrial and research organic chemical intermediate acting as an alkylating reagent in Grignard and other types of reactions. It is also used as a pharmaceutical intermediate for the synthesis of many types of drugs, as an agricultural chemical intermediate, as a photographic chemical intermediate, and as a biochemical reagent for enzyme and other studies. Human occupational or other accidental exposure can occur by inhalation, ingestion, or skin absorption. Male and female F344/N rats and B6C3F1 mice received dimethylaminopropyl chloride, hydrochloride (greater than 99% pure) in water by gavage for 2 weeks or 3 months. Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes. In the 2-week toxicity studies, groups of five male and five female F344/N rats and B6C3F1 mice were administered doses of 0, 6.25, 12.5, 25, 50, or 100 mg dimethylaminopropyl chloride, hydrochloride/kg body weight in deionized water by gavage, 5 days per week for 16 days. All dosed male and female rats and mice survived until the end of the 2-week study; one vehicle control female mouse died early. Mean body weights of all dosed groups of rats and mice were similar to those of the vehicle control groups. No gross or microscopic lesions were considered related to dimethylaminopropyl chloride, hydrochloride administration. In the 3-month toxicity studies, groups of 10 male and 10 female F344/N rats and B6C3F1 mice were administered doses of 0, 6.25, 12.5, 25, 50, or 100 mg/kg in deionized water by gavage, 5 days per week for 3 months. One male rat in the 50 mg/kg group died during week 12 of the study, and one female mouse in the 100 mg/kg group died during week 9 and another during week 13. The final mean body weights of 50 mg/kg male rats and 50 mg/kg female mice were significantly less than those of the vehicle controls. Possible chemical-related clinical findings in rats

12 CFR 508.8 - Default.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Default. 508.8 Section 508.8 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.8 Default. If the subject individual fails to file a petition for a...

Cirrhosis could be associated with severe mutations of the cystic fibrosis gene

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

Lenaerts, C.; Piussan, C.; Soto, B.

1994-09-01

Previous studies failed to demonstrate a genetic predisposition to liver disease in cystic fibrosis. In order to characterize patients with cirrhosis defined on the basis of either hepatosplenomegaly, portal hypertension or liver biopsy, we analyzed a total number of 110 cirrhotic CF patients from different CF centers in France. Of them, 71 are males, which is not different from the overall CF french population. All but 2 are pancreatic insufficient. A history of meconium ileus {plus_minus} meconium ileus equivalent seems to be a risk factor for cirrhosis since these complications are present in 29% of the cirrhotic patients vs. 19%more » in the non-cirrhotic population (p = 0.03). This confirms our previous data in a postmortem study. Genotype analysis was performed in all the patients. {Delta}F508 represents 70% of the identified mutations with a higher proportion of {Delta}F508 +/+ in the cirrhotic than in the non-cirrhotic population (52% vs. 42%, p=0.003), 35% {Delta}F508 +/- vs. 42% and 13% {Delta}F508 -/- vs. 16%. Sixty percent of the other mutations associated with cirrhosis are identified, usually in {Delta}F508 +/- and include 1303 N-K, 542 G-X, 1078 del T, 1282 W-X, 1313 Q-X, 827 E-X, 1061 G-R, 1301 N-H, 14 K-X, 1717-1 G-A, 1918 delGC, 2183 A-G, 2184 delA, 405+1 G-A, 507 {Delta}l, 574 delA, 621+1 G-T, 85 G-E and 1303 N-K/other, 227 L-R/other. None of the cirrhotic patients bear one of the dominant missense mutations regarded as mild with respect to pancreatic function (117 R-H, 334 R-W, 347 R-P, 455 A-E, 574 P-H) or both the {Delta}F508 and the 5512 G-A mutations associated with a decreased risk of meconium ileus. Cirrhosis could thus be linked to the presence of 2 of the severe mutations of the CF gene associated with pancreatic insufficiency.« less

NTP Toxicology and Carcinogenesis Studies of Benzene (CAS No. 71-43-2) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

PubMed

1986-04-01

Benzene ranks 16th in production volume for chemicals produced in the United States, with approximately 9.9 billion pounds being produced in 1984, 9.1 billion pounds in 1983, and 7.8 billion pounds in 1982. This simplest aromatic chemical in used in the synthesis of styrene (polystyrene plastics and synthetic rubber), phenol (phenolic resins), cyclohexane (nylon), aniline, maleic anhydride (polyester resins), alkylbenzenes (detergents), chlorobenzenes, and other products used in the production of drugs, dyes, insecticides, and plastics. Benzene, along with other light, high-octane aromatic hydrocarbons, such as toluene and xylenes, is a component of motor gasoline. Benzene is also used as a solvent, but for most applications, it has been replaced by less hazardous solvents. During the 17-week studies, groups of 10 or 15 male and female F344/N rats and B6C3F1 mice were gavaged 5 days per week with benzene in corn oil (5 ml/kg) at doses of 0 to 600 mg/kg. No benzene-related deaths occurred; in rats that received benzene, final mean body weights were 14%-22% lower compared with vehicle controls and in mice, slight dose-related reductions were observed (less than 10% differences). Doses for the 2-year studies were selected based on clinical observations (tremors in higher dosed mice), on clinical pathologic findings (lymphoid depletion in rats and leukopenia in mice), and on body weight effects. Two-year toxicology and carcinogenesis studies of benzene (greater than 99.7% pure) were conducted in groups of 50 F344/N rats and 50 B6C3F1 mice of each sex and for each dose. Doses of 0, 50, 100, or 200 mg/kg body weight benzene in corn oil (5 ml/kg) were administered by gavage to male rats, 5 days per week, for 103 weeks. Doses of 0, 25, 50, or 100 mg/kg benzene in corn oil were administered by gavage to female rats and to male and female mice for 103 weeks. Ten additional animals in each of the 16 groups were killed at 12 months and necropsies were performed. Hematologic

Learn About Section 508

EPA Pesticide Factsheets

Find out about EPA's commitment to making its websites and other electronic and information technology (EIT) products accessible, learn what Section 508 of the Rehabilitation Act covers and why, and how to make your EIT 508 compliant.

Carcinogenicity of glycidamide in B6C3F1 mice and F344/N rats from a two-year drinking water exposure.

PubMed

Beland, Frederick A; Olson, Greg R; Mendoza, Maria C B; Marques, M Matilde; Doerge, Daniel R

2015-12-01

Acrylamide is a contaminant in baked and fried starchy foods, roasted coffee, and cigarette smoke. Previously we reported that acrylamide is a multi-organ carcinogen in B6C3F1 mice and F344/N rats, and hypothesized that acrylamide is activated to an ultimate carcinogen through metabolism to the epoxide glycidamide. We have now examined the carcinogenic effects of glycidamide administered at 0, 0.0875, 0.175, 0.35 and 0.70 mM in drinking water to the same strains of rodents for two years. In male and female mice, there were significant increases in tumors of the Harderian gland, lung, forestomach, and skin. Female mice also had an increased incidence of tumors of the mammary gland and ovary. In male and female rats, there were significant increases in thyroid gland and oral cavity neoplasms and mononuclear cell leukemia. Male rats also had increases in tumors of the epididymis/testes and heart, while female rats demonstrated increases in tumors of the mammary gland, clitoral gland, and forestomach. A similar spectrum of tumors was obtained in mice and rats administered acrylamide. These data indicate that, under the conditions of these bioassays, acrylamide is efficiently metabolized to glycidamide and that the carcinogenic activity of acrylamide is due to its conversion into glycidamide. Published by Elsevier Ltd.

Hmga2 promotes the development of myelofibrosis in Jak2V617F knockin mice by enhancing TGF-β1 and Cxcl12 pathways.

PubMed

Dutta, Avik; Hutchison, Robert E; Mohi, Golam

2017-08-17

Myelofibrosis (MF) is a devastating blood disorder. The JAK2V617F mutation has been detected in ∼50% cases of MF. Elevated expression of high-mobility group AT hook 2 (HMGA2) has also been frequently observed in patients with MF. Interestingly, upregulation of HMGA2 expression has been found in association with the JAK2V617F mutation in significant cases of MF. However, the contribution of HMGA2 in the pathogenesis of MF remains elusive. To determine the effects of concurrent expression of HMGA2 and JAK2V617F mutation in hematopoiesis, we transduced bone marrow cells from Jak2 V617F knockin mice with lentivirus expressing Hmga2 and performed bone marrow transplantation. Expression of Hmga2 enhanced megakaryopoiesis, increased extramedullary hematopoiesis, and accelerated the development of MF in mice expressing Jak2 V617F Mechanistically, the data show that expression of Hmga2 enhances the activation of transforming growth factor-β1 (TGF-β1) and Cxcl12 pathways in mice expressing Jak2 V617F In addition, expression of Hmga2 causes upregulation of Fzd2, Ifi27l2a, and TGF-β receptor 2. Forced expression of Cxcl12, Fzd2, or Ifi27l2a increases megakaryocytic differentiation and proliferation in the bone marrow of Jak2 V617F mice, whereas TGF-β1 or Cxcl12 stimulation induces collagen deposition in the bone marrow mesenchymal stromal cells. Together, these findings demonstrate that expression of Hmga2 cooperates with Jak2 V617F in the pathogenesis of MF. © 2017 by The American Society of Hematology.

Upregulation of estrogen receptor expression in the uterus of ovariectomized B6C3F1 mice and Ishikawa cells treated with bromoethane

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

Aoyama, Hiroaki; Couse, John F.; Hewitt, Sylvia C.

2005-12-15

In a 2-year NTP bioassay, Bromoethane (BE) was found to induce endometrial neoplasms in the uterus of B6C3F1 mice [; ]. In women, hormonal influences, such as 'unopposed' estrogenic stimulus, have been implicated as important etiologic factors in uterine cancer. BE, however, does not affect the serum concentrations of sex hormones in female B6C3F1 mice [] and the mechanism of BE-induced uterine carcinogenesis still remains unclear. In the present study, we examined the estrogenic effects of BE on the uterus of ovariectomized B6C3F1 mice and on Ishikawa cells. Groups of 6 mice were given daily s.c. injections of 0, 100,more » 500 or 1000 mg BE/kg for 3 consecutive days. Mice treated with 17{beta}-estradiol served as positive controls. Mice were necropsied 24 h after the final injection, and uteri were weighed and examined histologically and immunohistochemically along with the vagina. Changes observed in the estrogen-treated mice included increased uterine weights, edema and inflammation of the endometrium, increased epithelial layers of the uterine and vaginal lumens and keratinization of the vaginal epithelium. In the BE-treated mice, no such changes occurred; however, immunohistochemical staining of the uterus revealed a significant increase in immunoexpression of the estrogen receptor alpha (ER{alpha}) in the two higher dose groups. Analysis of mRNA also showed slightly increased uterine ER{alpha} expression in these groups. Upregulated expression of ER{alpha} was confirmed in BE-treated Ishikawa cells, in which Western blotting analyses identified an intense signal at approximately 66 kDa, which is consistent with ER{alpha}. These data suggest that upregulated expression of ER{alpha} may be important in the induction of endometrial neoplasms in BE-treated mice.« less

Cystic fibrosis transmembrane conductance regulator (CFTR) allelic variants relate to shifts in faecal microbiota of cystic fibrosis patients.

PubMed

Schippa, Serena; Iebba, Valerio; Santangelo, Floriana; Gagliardi, Antonella; De Biase, Riccardo Valerio; Stamato, Antonella; Bertasi, Serenella; Lucarelli, Marco; Conte, Maria Pia; Quattrucci, Serena

2013-01-01

In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced. This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

PubMed Central

Santangelo, Floriana; Gagliardi, Antonella; De Biase, Riccardo Valerio; Stamato, Antonella; Bertasi, Serenella; Lucarelli, Marco

2013-01-01

Introduction In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age. Methods Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure. Results Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced. Conclusions This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a ‘systemic disease’, linking the lung and the gut in a joined axis. PMID:23613805

48 CFR 3045.508 - Physical inventories.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 48 Federal Acquisition Regulations System 7 2011-10-01 2011-10-01 false Physical inventories. 3045.508 Section 3045.508 Federal Acquisition Regulations System DEPARTMENT OF HOMELAND SECURITY, HOMELAND... Property in the Possession of Contractors 3045.508 Physical inventories. ...

48 CFR 3045.508 - Physical inventories.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Physical inventories. 3045.508 Section 3045.508 Federal Acquisition Regulations System DEPARTMENT OF HOMELAND SECURITY, HOMELAND... Property in the Possession of Contractors 3045.508 Physical inventories. ...

13 CFR 134.508 - What is the standard of review?

Code of Federal Regulations, 2010 CFR

2010-01-01

... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false What is the standard of review? 134.508 Section 134.508 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION RULES OF... Service-Disabled Veteran Owned Small Business Concern Protests § 134.508 What is the standard of review...

5 CFR 293.508 - Type of folder to be used.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 5 Administrative Personnel 1 2012-01-01 2012-01-01 false Type of folder to be used. 293.508 Section 293.508 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PERSONNEL RECORDS Employee Medical File System Records § 293.508 Type of folder to be used. Each agency must...

5 CFR 293.508 - Type of folder to be used.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Type of folder to be used. 293.508 Section 293.508 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PERSONNEL RECORDS Employee Medical File System Records § 293.508 Type of folder to be used. Each agency must...

5 CFR 293.508 - Type of folder to be used.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 5 Administrative Personnel 1 2014-01-01 2014-01-01 false Type of folder to be used. 293.508 Section 293.508 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PERSONNEL RECORDS Employee Medical File System Records § 293.508 Type of folder to be used. Each agency must...

5 CFR 293.508 - Type of folder to be used.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 5 Administrative Personnel 1 2013-01-01 2013-01-01 false Type of folder to be used. 293.508 Section 293.508 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PERSONNEL RECORDS Employee Medical File System Records § 293.508 Type of folder to be used. Each agency must...

5 CFR 293.508 - Type of folder to be used.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 5 Administrative Personnel 1 2011-01-01 2011-01-01 false Type of folder to be used. 293.508 Section 293.508 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS PERSONNEL RECORDS Employee Medical File System Records § 293.508 Type of folder to be used. Each agency must...

Protection of mice from fatal bubonic and pneumonic plague by passive immunization with monoclonal antibodies against the F1 protein of Yersinia pestis.

PubMed

Anderson, G W; Worsham, P L; Bolt, C R; Andrews, G P; Welkos, S L; Friedlander, A M; Burans, J P

1997-04-01

Monoclonal antibodies (MAbs) to the fraction 1 (F1) protein of Yersinia pestis protected mice against fatal pneumonic as well as bubonic plague from wild-type F1+ organisms. The rare isolation of a virulent F1- isolate from surviving animals supports earlier studies suggesting that improved vaccines should consist of immunogens to protect against F1- variants. The high degree of protection with IgG MAb suggests that secretory IgA is not required for protection from pneumonic plague.

48 CFR 1845.508 - Physical inventories.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Physical inventories. 1845.508 Section 1845.508 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND SPACE... Contractors 1845.508 Physical inventories. NASA contractors shall reconcile inventories with the official...

Chandra Observations of the Field Containing HESS J1616-508

NASA Astrophysics Data System (ADS)

Hare, Jeremy; Kargaltsev, Oleg; Pavlov, George G.; Rangelov, Blagoy; Volkov, Igor

2017-06-01

We report the results of three Chandra observations covering most of the extent of the TeV γ-ray source HESS J1616-508 and a search for a lower-energy counterpart to this source. We detect 56 X-ray sources, 37 of which have counterparts at lower frequencies, including a young massive star cluster, but none of them appear to be a particularly promising counterpart to the TeV source. The brightest X-ray source, CXOU J161423.4-505738, with a flux F 0.5-7 keV ≈ 5 × 10-13 erg cm-2 s-1, has a hard spectrum that is well fit by a power-law model with a photon index Γ = 0.2 ± 0.3 and is a likely intermediate polar CV candidate. No counterparts of this source were detected at other wavelengths. CVs are not known to produce extended TeV emission, and the source is also largely offset (19‧) from HESS J1616-508, making them unlikely to be associated. We have also set an upper limit on the X-ray flux of PSR J1614-5048 in the 0.5-8 keV band (F 0.5-8 keV < 5 × 10-15 erg cm-2 s-1 at a 90% confidence level). This makes PSR J1614-5048 one of the least X-ray-efficient pulsars known, with an X-ray efficiency {η }0.5{--8{keV}}={L}0.5{--8{keV}}/\\dot{E}< 2× {10}-5. We find no evidence supporting the association between the pulsar and the TeV source. We rule out a number of X-ray sources as possible counterparts to the TeV emission and do not find a plausible counterpart among the other sources. Lastly, we discuss the possible relation of PSR J1617-5055 to HESS J1616-508 in light of the new observations.

50 CFR 600.508 - Fishing operations.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Fishing operations. 600.508 Section 600.508 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE MAGNUSON-STEVENS ACT PROVISIONS Foreign Fishing § 600.508 Fishing...

Genistein modulation of streptozotocin diabetes in male B6C3F1 mice can be induced by diet

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

Guo, Tai L., E-mail: tlguo1@uga.edu; Wang, Yunbiao; Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102

Diet and phytoestrogens affect the development and progression of diabetes. The objective of the present study was to determine if oral exposure to phytoestrogen genistein (GE) by gavage changed blood glucose levels (BGL) through immunomodulation in streptozotocin (STZ)-induced diabetic male B6C3F1 mice fed with three different diets. These three diets were: NTP-2000 diet (NTP), soy- and alfalfa-free 5K96 diet (SOF) and high fat diet (HFD) with 60% of kcal from fat, primarily rendered fat of swine. The dosing regimen for STZ consisted of three 100 mg/kg doses (i.p.): the first dose was administered at approximately 2 weeks following the initiationmore » of daily GE (20 mg/kg) gavage, and the second dose was on day 19 following the first dose, and the third dose was on day 57 following the first dose. In mice on the NTP diet, GE treatment decreased BGL with statistical significances observed on days 33 and 82 following the first STZ injection. In mice fed the HFD diet, GE treatment produced a significant decrease and a significant increase in BGL on days 15 and 89 following the first STZ injection, respectively. In mice fed the SOF diet, GE treatment had no significant effects on BGL. Although GE treatment affected phenotypic distributions of both splenocytes (T cells, B cells, natural killer cells and neutrophils) and thymocytes (CD4/CD8 and CD44/CD25), and their mitochondrial transmembrane potential and generation of reactive oxygen species, indicators of cell death (possibly apoptosis), GE modulation of neutrophils was more consistent with its diabetogenic or anti-diabetic potentials. The differential effects of GE on BGL in male B6C3F1 mice fed with three different diets with varied phytoestrogen contents suggest that the estrogenic properties of this compound may contribute to its modulation of diabetes. - Highlights: • Diets affected streptozotocin-induced diabetes in male B6C3F1 mice. • Genistein modulation of streptozotocin diabetes can be induced by

Genetic basis of HDL variation in 129/SvImJ and C57BL/6J mice: importance of testing candidate genes in targeted mutant mice.

PubMed

Su, Zhiguang; Wang, Xiaosong; Tsaih, Shirng-Wern; Zhang, Aihong; Cox, Allison; Sheehan, Susan; Paigen, Beverly

2009-01-01

To evaluate the effect of genetic background on high-density lipoprotein cholesterol (HDL) levels in Soat1(-/-) mice, we backcrossed sterol O-acyltransferase 1 (Soat1)(-/-) mice, originally reported to have elevated HDL levels, to C57BL/6 mice and constructed a congenic strain with only a small region (3.3Mb) of 129 alleles, specifically excluding the nearby apolipoprotein A-II (Apoa2) gene from 129. HDL levels in these Soat1(-/-) mice were no different from C57BL/6, indicating that the passenger gene Apoa2 caused the previously reported elevation of HDL in these Soat1(-/-) mice. Because many knockouts are made in strain 129 and then subsequently backcrossed into C57BL/6, it is important to identify quantitative trait loci (QTL) that differ between 129 and C57BL/6 so that one can guard against effects ascribed to a knockout but really caused by a passenger gene from 129. To provide such data, we generated 528 F(2) progeny from an intercross of 129S1/SvImJ and C57BL/6 and measured HDL concentrations in F(2) animals first fed chow and then atherogenic diet. A genome wide scan using 508 single-nucleotide polymorphisms (SNPs) identified 19 QTL, 2 of which were male specific and 2 were female specific. Using comparative genomics and haplotype analysis, we narrowed QTL on chromosomes 3, 5, 8, 17, and 18 to 0.5, 6.3, 2.6, 1.1, and 0.6 Mb, respectively. These data will serve as a reference for any effort to test the impact of candidate genes on HDL using a knockout strategy.

24 CFR 965.508 - Individual relief.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Individual relief. 965.508 Section 965.508 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued... PHA-OWNED OR LEASED PROJECTS-GENERAL PROVISIONS Resident Allowances for Utilities § 965.508 Individual...

47 CFR 54.508 - Technology plans.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 3 2013-10-01 2013-10-01 false Technology plans. 54.508 Section 54.508... SERVICE Universal Service Support for Schools and Libraries § 54.508 Technology plans. (a) Applicants must develop a technology plan when requesting discounts for internal connections and basic maintenance for...

Immunotoxicological Profile of Chloroform in Female B6c3f1 Mice When Administered In Drinking Water

EPA Science Inventory

Chloroform can be formed as a disinfection by-product during water chlorination, one of the primary modalities for purifying municipal water supplies for human consumption. The goal of this study was to characterize the immunotoxic effects of chloroform in female B6C3F1 mice when...

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

PubMed

Tosco, A; De Gregorio, F; Esposito, S; De Stefano, D; Sana, I; Ferrari, E; Sepe, A; Salvadori, L; Buonpensiero, P; Di Pasqua, A; Grassia, R; Leone, C A; Guido, S; De Rosa, G; Lusa, S; Bona, G; Stoll, G; Maiuri, M C; Mehta, A; Kroemer, G; Maiuri, L; Raia, V

2016-08-01

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts 'on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

12 CFR 508.11 - Relevant considerations.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Relevant considerations. 508.11 Section 508.11 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.11 Relevant considerations. (a) In determining whether...

7 CFR 1207.508 - USDA costs.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 10 2011-01-01 2011-01-01 false USDA costs. 1207.508 Section 1207.508 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... Rules and Regulations General § 1207.508 USDA costs. Pursuant to § 1207.341 of the Plan the Board shall...

7 CFR 1207.508 - USDA costs.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 10 2013-01-01 2013-01-01 false USDA costs. 1207.508 Section 1207.508 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... Rules and Regulations General § 1207.508 USDA costs. Pursuant to § 1207.341 of the Plan the Board shall...

7 CFR 1207.508 - USDA costs.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 10 2014-01-01 2014-01-01 false USDA costs. 1207.508 Section 1207.508 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... Rules and Regulations General § 1207.508 USDA costs. Pursuant to § 1207.341 of the Plan the Board shall...

7 CFR 1207.508 - USDA costs.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 10 2010-01-01 2010-01-01 false USDA costs. 1207.508 Section 1207.508 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... Rules and Regulations General § 1207.508 USDA costs. Pursuant to § 1207.341 of the Plan the Board shall...

7 CFR 1207.508 - USDA costs.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 10 2012-01-01 2012-01-01 false USDA costs. 1207.508 Section 1207.508 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... Rules and Regulations General § 1207.508 USDA costs. Pursuant to § 1207.341 of the Plan the Board shall...

45 CFR 160.508 - Authority of the ALJ.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Authority of the ALJ. 160.508 Section 160.508 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES ADMINISTRATIVE DATA STANDARDS AND RELATED... scope and timing of documentary discovery as permitted by this subpart; (8) Regulate the course of the...

40 CFR 90.508 - Test procedures.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 20 2014-07-01 2013-07-01 true Test procedures. 90.508 Section 90.508....508 Test procedures. (a) For nonroad engines subject to the provisions of this subpart, the prescribed test procedures are the appropriate small SI engine test procedures as described in subpart E of this...

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

PubMed

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-10-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

Immunization of Mice by BCG Formulated HCV Core Protein Elicited Higher Th1-Oriented Responses Compared to Pluronic-F127 Copolymer

PubMed Central

Yazdanian, Maryam; Memarnejadian, Arash; Mahdavi, Mehdi; Sadat, Seyed Mehdi; Motevali, Fatemeh; Vahabpour, Rouhollah; Khanahmad, Hossein; Siadat, Seyed Davar; Aghasadeghi, Mohammad Reza; Roohvand, Farzin

2013-01-01

Background A supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses. Objectives To evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses. Materials and Methods Expression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune responses (Proliferation, In vivo CTL and IFN-γ/IL-4 ELISpot assays against a strong and dominant H2-d restricted, CD8+-epitopic peptide, core 39-48; RRGPRLGVRA of HCVcp) were compared in each group of immunized animals. Results Expression and purification of core protein around the expected size (21 kDa) was confirmed by Western blotting. The HCVcp + BCG vaccinated mice showed significantly higher lymphocyte proliferation and IFN-γ production but lower levels of cell lysis (45% versus 62% in CTL assay) than the HCVcp+F127 immunized animals. “Besides, total anti-core IgG and IgG1 levels were significantly higher in HCVcp + F127 immunized mice as compared to HCVcp + BCG vaccinated animals, indicating relatively higher efficacy of F127 for the stimulation of humoral and Th2-oriented immune responses”. Conclusions Results showed that HCVcp + BCG induced a moderate CTL and mixed Th1/Th2 immune responses with higher levels of

Ntp report on the toxicity studies of ethylbenzene in f344/n rats and b6c3f1 mice (inhalation studies). Report for 29 March-30 June 1988

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

Chan, P.

1992-03-01

Inhalation toxicology studies of ethylbenzene (99% pure) were conducted by exposing groups of F344/N rats and B6C3F1 mice of each sex to ethylbenzene vapor at chamber concentrations of 0 to 1000 ppm, 6 hours per day, 5 days per week for 13 weeks. No rats or mice died during the 13-week exposure. Body weight gains were slightly lower in the high dose groups of male and female rats, but the differences were not statistically significant. Absolute and relative kidney, liver, and lung weights were increased in the exposed rats, while weight increases occurred only in the livers of exposed mice.more » No changes were observed in the evaluation of sperm or vaginal cytology in rats or mice. Ethylbenzene was not mutagenic in Salmonella and did not induce chromosomal aberrations or sister chromatid exchanges in Chinese hamster ovary (CHO) cells in vitro, though it did induce trifluorothymidine resistance in mouse lymphoma cells at the highest concentration tested. Micronuclei assays in peripheral blood of mice were negative.« less

Promotion of hepatic preneoplastic lesions in male B6C3F1 mice by unleaded gasoline.

PubMed Central

Standeven, A M; Wolf, D C; Goldsworthy, T L

1995-01-01

In previous studies, unleaded gasoline (UG) vapor was found to be a liver tumor promoter and hepatocarcinogen in female mice, but UG was not a hepatocarcinogen in male mice. However, UG vapor had similar transient mitogenic effects in nonlesioned liver of both male and female mice under the conditions of the cancer bioassay. We used an initiation-promotion protocol to determine whether UG vapor acts as a liver tumor promoter in male mice and to examine proliferative effects that may be critical to tumor development. Twelve-day-old male B6C3F1 mice were injected with N-nitrosodiethylamine (DEN; 5 mg/kg, intraperitoneally) or vehicle. Starting at 5-7 weeks of age, mice were exposed by inhalation 6 hr/day, 5 days/week for 16 weeks to 0 or 2046 ppm of PS-6 blend UG. UG treatment caused a significant 2.3-fold increase in the number of macroscopic hepatic masses in DEN-initiated mice, whereas no macroscopic masses were observed in non-initiated mice. Altered hepatic foci (AHF), which were predominantly basophilic in phenotype, were found almost exclusively in DEN-initiated mice. UG treatment significantly increased both the mean volume (threefold) and the volume fraction (twofold) of the AHF without increasing the number of AHF per unit area. UG also induced hepatic pentoxyresorufin-O-dealkylase (PROD) activity, a marker of CYP2B, by more than 12-fold over control with or without DEN cotreatment. To study hepatocyte proliferative effects of UG, we treated mice with 5-bromo-2'-deoxyuridine (BrdU) via osmotic pump for 3 days before necropsy and measured hepatocyte BrdU labeling index (LI) in AHF and nonlesioned liver.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1. PMID:7588481

Spatial reversal learning defect coincides with hypersynchronous telencephalic BOLD functional connectivity in APPNL-F/NL-F knock-in mice.

PubMed

Shah, Disha; Latif-Hernandez, Amira; De Strooper, Bart; Saito, Takashi; Saido, Takaomi; Verhoye, Marleen; D'Hooge, Rudi; Van der Linden, Annemie

2018-04-19

Amyloid pathology occurs early in Alzheimer's disease (AD), and has therefore been the focus of numerous studies. Transgenic mouse models have been instrumental to study amyloidosis, but observations might have been confounded by APP-overexpression artifacts. The current study investigated early functional defects in an APP knock-in mouse model, which allows assessing the effects of pathological amyloid-beta (Aβ) without interference of APP-artifacts. Female APP NL/NL knock-in mice of 3 and 7 months old were compared to age-matched APP NL-F/NL-F mice with increased Aβ42/40 ratio and initial Aβ-plaque deposition around 6 months of age. Spatial learning was examined using a Morris water maze protocol consisting of acquisition and reversal trials interleaved with reference memory tests. Functional connectivity (FC) of brain networks was assessed using resting-state functional MRI (rsfMRI). The Morris water maze data revealed that 3 months old APP NL-F/NL-F mice were unable to reach the same reference memory proficiency as APP NL/NL mice after reversal training. This cognitive defect in 3-month-old APP NL-F/NL-F mice coincided with hypersynchronous FC of the hippocampal, cingulate, caudate-putamen, and default-mode-like networks. The occurrence of these defects in APP NL-F/NL-F mice demonstrates that cognitive flexibility and synchronicity of telencephalic activity are specifically altered by early Aβ pathology without changes in APP neurochemistry.

EUReKA! A Conceptual Model of Emotion Understanding

PubMed Central

Castro, Vanessa L.; Cheng, Yanhua; Halberstadt, Amy G.; Grühn, Daniel

2015-01-01

The field of emotion understanding is replete with measures, yet lacks an integrated conceptual organizing structure. To identify and organize skills associated with the recognition and knowledge of emotions, and to highlight the focus of emotion understanding as localized in the self, in specific others, and in generalized others, we introduce the conceptual framework of Emotion Understanding in Recognition and Knowledge Abilities (EUReKA). We then categorize fifty-six existing methods of emotion understanding within this framework to highlight current gaps and future opportunities in assessing emotion understanding across the lifespan. We hope the EUReKA model provides a systematic and integrated framework for conceptualizing and measuring emotion understanding for future research. PMID:27594904

Two-hour methyl isocyanate inhalation and 90-day recovery study in B6C3F1 mice

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

Boorman, G.A.; Uraih, L.C.; Gupta, B.N.

1987-06-01

B6C3F1 mice were exposed by inhalation to 0, 3, 10, and 30 ppm methyl isocyanate for 2 hr followed by a 90-day recovery period. Sixteen of eight (20%) male mice in the 30 ppm group died following exposure. There were no other unscheduled deaths in the mice. Five mice/sex/group were examined at 2 hr or at 1, 3, 7, 14, 28, 49, or 91 days following exposure. Chemical-related changes were restricted to the respiratory system. At 30 ppm there were extensive necrosis and erosion of the respiratory and olfactory epithelium in the nasal cavity. Severe necrosis and epithelial erosion weremore » also found in the trachea and main bronchi. Regeneration of the mucosal epithelium occurred rapidly in the nasal cavity and airways. In the turbinates, mild incomplete olfactory epithelial regeneration persisted to day 91 in the male mice. Intraluminal fibrotic projections covered by respiratory epithelium and bronchial fibrosis were found in the major airways of the 30 ppm male and female mice by day 7. The intraluminal fibrosis persisted to day 91. In males with severe bronchial fibrosis, chronic alveolitis and atelectasis were found. In mice exposed to 3 or 10 ppm, persistent pulmonary changes were not found. These studies indicate that methyl isocyanate inhalation at or near lethal concentrations can cause persistent fibrosis of the major bronchi in mice.« less

NTP Toxicology and Carcinogenesis Studies of Dimethyl Methylphosphonate (CAS No. 756-79-6) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

PubMed

1987-11-01

Dimethyl methylphosphonate (98% pure) is one of four chemicals nominated by the U.S. Army for toxicology and carcinogenesis studies because it was being considered for use to simulate the physical and spectroscopic (but not the biologic) properties of anticholinesterase (nerve) agents. Dimethyl methylphosphonate is also used as a flame retardant, a preignition additive for gasoline, an antifoam agent, a plasticizer and stabilizer, a textile conditioner and antistatic agent, and an additive for solvents and low-temperature hydraulic fluids. The United States produces 0.2-2 million pounds (91,000-910,000 kg) of per year. Gavage was chosen as the route of administration for all four candidate "simulants" to mimic potential exposure. Experimental Design: Dimethyl methylphosphonate was administered in corn oil by gavage to male and female F344/N rats and B6C3F1 mice in single-administration, 15-day, and 13-week studies to obtain toxicity data, to establish dose levels for the 2-year studies, and to identify target tissues. Additional studies were also performed to determine toxicity to the reproductive system of male F344/N rats and B6C3F1 mice and to study the potential for genetic damage in bacteria, mammalian cells, and Drosophila. Single-Administration Studies: In the single-administration studies, dimethyl methylphosphonate was given to rats and mice at doses up to 6,810 mg/kg body weight. No compound-related deaths were seen in male or female rats or male mice; two high dose female mice died. Rats exhibited inactivity, unsteady gait, and prostration after dosing; mice were inactive after dosing. Fifteen-Day Studies: Rats and mice received doses of 0, 1,250, 2,500, 5,000, 10,000, or 15,000 mg/kg dimethyl methylphosphonate per day. Compound-related deaths occurred in the three highest dose groups of rats and the two highest dose groups of mice. Rats receiving doses of 2,500 mg/kg or higher were inactive and at 5,000 or 10,000 mg/kg had an unsteady gait after dosing

Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

PubMed

Romani, Luigina; Oikonomou, Vasilis; Moretti, Silvia; Iannitti, Rossana G; D'Adamo, Maria Cristina; Villella, Valeria R; Pariano, Marilena; Sforna, Luigi; Borghi, Monica; Bellet, Marina M; Fallarino, Francesca; Pallotta, Maria Teresa; Servillo, Giuseppe; Ferrari, Eleonora; Puccetti, Paolo; Kroemer, Guido; Pessia, Mauro; Maiuri, Luigi; Goldstein, Allan L; Garaci, Enrico

2017-05-01

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.

Thymosin α1 represents a potential potent single molecule-based therapy for cystic fibrosis

PubMed Central

Romani, Luigina; Oikonomou, Vasilis; Moretti, Silvia; Iannitti, Rossana G.; D’Adamo, Maria Cristina; Villella, Valeria R.; Pariano, Marilena; Sforna, Luigi; Borghi, Monica; Bellet, Marina M.; Fallarino, Francesca; Pallotta, Maria Teresa; Servillo, Giuseppe; Ferrari, Eleonora; Puccetti, Paolo; Kroemer, Guido; Pessia, Mauro; Maiuri, Luigi; Goldstein, Allan L.; Garaci, Enrico

2017-01-01

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride-channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF —which include impaired chloride permeability and persistent lung inflammation—a multidrug approach is required for efficacious CF therapy. To date, no individual, drug with pleiotropic beneficial effects for CF is available. Here we report on the ability of thymosin alpha 1 (Tα1)—a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent—to rectify the multiple tissue defects in CF mice as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology; namely, it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 offers a strong potential to be an efficacious single molecule-based therapeutic agent in CF. PMID:28394330

Thalidomide enhances both primary and secondary host resistances to Listeria monocytogenes infection by a neutrophil-related mechanism in female B6C3F1 mice

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

Guo, Tai L.; Chi, Rui P.; Karrow, Niel A.

2005-12-15

Previously, we have reported that thalidomide can modulate the immune responses in female B6C3F1 mice. Furthermore, thalidomide immunomodulation increased primary host resistance to intravenously infected Listeria monocytogenes. The present study was intended to evaluate the mechanisms underlying the enhanced host resistance to L. monocytogenes by focusing on the neutrophils. Female B6C3F1 mice were treated intraperitoneally with thalidomide (100 mg/kg) for 15 days. Exposure to thalidomide increased the numbers of neutrophils in the spleens and livers of L. monocytogenes-infected mice when compared to the L. monocytogenes-infected control mice. Additionally, the percentage of neutrophils was also significantly increased after Thd treatment inmore » L. monocytogenes-infected mice. Further studies using antibodies to deplete corresponding cells indicated that thalidomide-mediated increase in primary host resistance (both the moribundity and colony counts in the liver and spleen) to L. monocytogenes infection was due to its effect on neutrophils but not CD8{sup +} T cells or NK cells. Finally, Thd exposure also increased host resistance to secondary host resistance to L. monocytogenes infection, and depletion of neutrophils abolished the protective effect. In conclusion, thalidomide enhanced host resistance to both primary and secondary L. monocytogenes infections by a neutrophil-related mechanism in female B6C3F1 mice.« less

Angiopoietin-1 deficiency increases tumor metastasis in mice.

PubMed

Michael, Iacovos P; Orebrand, Martina; Lima, Marta; Pereira, Beatriz; Volpert, Olga; Quaggin, Susan E; Jeansson, Marie

2017-08-11

Angipoietin-1 activation of the tyrosine kinase receptor Tek expressed mainly on endothelial cells leads to survival and stabilization of endothelial cells. Studies have shown that Angiopoietin-1 counteracts permeability induced by a number of stimuli. Here, we test the hypothesis that loss of Angiopoietin-1/Tek signaling in the vasculature would increase metastasis. Angiopoietin-1 was deleted in mice just before birth using floxed Angiopoietin-1 and Tek mice crossed to doxycycline-inducible bitransgenic ROSA-rtTA/tetO-Cre mice. By crossing Angiopoietin-1 knockout mice to the MMTV-PyMT autochthonous mouse breast cancer model, we investigated primary tumor growth and metastasis to the lung. Furthermore, we utilized B16F10 melanoma cells subcutaneous and experimental lung metastasis models in Angiopoietin-1 and Tek knockout mice. We found that primary tumor growth in MMTV-PyMT mice was unaffected, while metastasis to the lung was significantly increased in Angiopoietin-1 knockout MMTV-PyMT mice. In addition, angiopoietin-1 deficient mice exhibited a significant increase in lung metastasis of B16F10 melanoma cells, compared to wild type mice 3 weeks after injection. Additional experiments showed that this was likely an early event due to increased attachment or extravasation of tumor cells, since seeding of tumor cells was significantly increased 4 and 24 h post tail vein injection. Finally, using inducible Tek knockout mice, we showed a significant increase in tumor cell seeding to the lung, suggesting that Angiopoietin-1/Tek signaling is important for vascular integrity to limit metastasis. This study show that loss of the Angiopoietin-1/Tek vascular growth factor system leads to increased metastasis without affecting primary tumor growth.

In Vivo Interplay between p27Kip1, GATA3, ATOH1, and POU4F3 Converts Non-sensory Cells to Hair Cells in Adult Mice.

PubMed

Walters, Bradley J; Coak, Emily; Dearman, Jennifer; Bailey, Grace; Yamashita, Tetsuji; Kuo, Bryan; Zuo, Jian

2017-04-11

Hearing loss is widespread and persistent because mature mammalian auditory hair cells (HCs) are nonregenerative. In mice, the ability to regenerate HCs from surrounding supporting cells (SCs) declines abruptly after postnatal maturation. We find that combining p27 Kip1 deletion with ectopic ATOH1 expression surmounts this age-related decline, leading to conversion of SCs to HCs in mature mouse cochleae and after noise damage. p27 Kip1 deletion, independent of canonical effects on Rb-family proteins, upregulated GATA3, a co-factor for ATOH1 that is lost from SCs with age. Co-activation of GATA3 or POU4F3 and ATOH1 promoted conversion of SCs to HCs in adult mice. Activation of POU4F3 alone also converted mature SCs to HCs in vivo. These data illuminate a genetic pathway that initiates auditory HC regeneration and suggest p27 Kip1 , GATA3, and POU4F3 as additional therapeutic targets for ATOH1-mediated HC regeneration. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR

PubMed Central

Tosco, A; De Gregorio, F; Esposito, S; De Stefano, D; Sana, I; Ferrari, E; Sepe, A; Salvadori, L; Buonpensiero, P; Di Pasqua, A; Grassia, R; Leone, C A; Guido, S; De Rosa, G; Lusa, S; Bona, G; Stoll, G; Maiuri, M C; Mehta, A; Kroemer, G; Maiuri, L; Raia, V

2016-01-01

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts ‘on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment. PMID:27035618

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

PubMed Central

Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

2015-01-01

Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53−/− mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy. PMID:25656653

NTP Toxicology and Carcinogenesis Studies of Molybdenum Trioxide (CAS No. 1313-27-5) in F344 Rats and B6C3F1 Mice (Inhalation Studies).

PubMed

1997-04-01

Molybdenum is an essential element for the function of nitrogenase in plants and as a cofactor for enzymes including xanthine oxidoreductase, aldehyde oxidase, and sulfide oxidase in animals. Molybdenum trioxide is used primarily as an additive to steel and corrosion-resistant alloys. It is also used as a chemical intermediate for molybdenum products; an industrial catalyst; a pigment; a crop nutrient; components of glass, ceramics, and enamels; a flame retardant for polyester and polyvinyl chloride resins; and a reagent in chemical analyses. Molybdenum trioxide was nominated by the NCI for toxicity and carcinogenicity studies as a representative inorganic molybdenum compound. The production of molybdenum trioxide is the largest of all the molybdenum compounds examined. Male and female F344/N rats and B6C3F1 mice were exposed to molybdenum trioxide (approximately 99% pure) by inhalation for 14 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and cultured Chinese hamster ovary cells. 14-DAY STUDY IN RATS: Groups of five male and five female F344/N rats were exposed to 0, 3, 10, 30, 100, or 300 mg molybdenum trioxide/m(3). Rats were exposed for 6 hours per day, 5 days per week, for a total of 10 exposure days during a 14-day period. All rats survived to the end of the study. The final mean body weights of male rats exposed to 100 mg/m(3) and male and female rats exposed to 300 mg/m(3) were significantly lower than those of the control groups. Male rats exposed to 300 mg/m(3) lost weight during the study. There were no clinical findings related to exposure to molybdenum trioxide. No chemical-related lesions were observed. 14-DAY STUDY IN MICE: Groups of five male and five female B6C3F1 mice were exposed to 0, 3, 10, 30, 100, or 300 mg molybdenum trioxide/m(3). Mice were exposed 6 hours per day, 5 days per week, for a total of 10 exposure days during a 14-day period. All mice survived to the end of the study. Final mean

Efficacy and safety of lumacaftor/ivacaftor combination therapy in patients with cystic fibrosis homozygous for Phe508del CFTR by pulmonary function subgroup: a pooled analysis.

PubMed

Elborn, J Stuart; Ramsey, Bonnie W; Boyle, Michael P; Konstan, Michael W; Huang, Xiaohong; Marigowda, Gautham; Waltz, David; Wainwright, Claire E

2016-08-01

Lumacaftor/ivacaftor combination therapy has shown clinical benefits in patients with cystic fibrosis homozygous for the Phe508del CFTR mutation; however, pretreatment lung function is a confounding factor that potentially affects the efficacy and safety of this therapy. We aimed to assess the efficacy and safety of lumacaftor/ivacaftor therapy in these patients, defined by specific categories of lung function. Both trials (TRAFFIC and TRANSPORT) included in this pooled analysis were multinational, randomised, double-blind, placebo-controlled, parallel-group, phase 3 studies. Eligible patients from 187 participating centres in North America, Australia, and the European Union (both trials) were aged 12 years or older with a confirmed diagnosis of cystic fibrosis, homozygous for the Phe508del CFTR mutation, and with a percent predicted FEV1 (ppFEV1) of 40-90 at the time of screening. Patients were randomly assigned with an interactive web response system (1:1:1) to receive placebo, lumacaftor (600 mg once daily) plus ivacaftor (250 mg every 12 h), or lumacaftor (400 mg every 12 h) plus ivacaftor (250 mg every 12 h) for 24 weeks. Prespecified subgroup analyses of pooled efficacy and safety data by lung function, as measured by ppFEV1, were done for patients with baseline ppFEV1 (<40 and ≥40) and screening ppFEV1 (<70 and ≥70). The primary endpoint was the absolute change from baseline in ppFEV1 at week 24 analysed in all randomised patients who received at least one dose of study drug. Both trials are registered with ClinicalTrials.gov (TRAFFIC: NCT01807923; TRANSPORT: NCT01807949). Both trials were done between April, 2013, and April, 2014. Of the 1108 patients included in the efficacy analysis, 81 patients had a ppFEV1 that decreased to lower than 40 between screening and baseline and 1016 had a ppFEV1 of 40 or higher at baseline. At screening, 730 had a ppFEV1 of less than 70, and 342 had a ppFEV1 of 70 or higher. Improvements in the absolute change from

Comparison of the Effects of Hexavalent Chromium in the Alimentary Canal of F344 Rats and B6C3F1 Mice Following Exposure in Drinking Water: Implications for Carcinogenic Modes of Action

PubMed Central

Thompson, Chad M.; Proctor, Deborah M.; Suh, Mina; Haws, Laurie C.; Hébert, Charles D.; Mann, Jill F.; Shertzer, Howard G.; Hixon, J. Gregory; Harris, Mark A.

2012-01-01

Exposure to high concentrations of hexavalent chromium (Cr[VI]) in drinking water is reported to induce oral mucosa tumors in F344 rats and intestinal tumors in B6C3F1 mice. To investigate the modes of action underlying these tumors, 90-day drinking water studies (with interim necropsy at day 8) were conducted with concentrations of 0.1–182 mg/l Cr(VI), administered as 0.3–520 mg/l sodium dichromate dihydrate. Blood and tissue samples were analyzed for chromium content, oxidative stress, iron levels, and gross and microscopic lesions. Results for the F344 rats are described herein and compared with results from B6C3F1 mice published previously. After 90 days of exposure, total chromium concentrations in the rat and mouse oral mucosae were comparable, yet significant dose-dependent decreases in the reduced-to-oxidized glutathione ratio (GSH/GSSG) were observed only in rats. In the duodenum, changes in GSH/GSSG were only observed in mice. Levels of 8-hydroxydeoxyguanosine were not increased in the oral or duodenal mucosae of either species. Glutathione levels were increased in the duodenum but decreased in the jejunum of both species, indicating potential differential responses in the intestinal segments. Histiocytic infiltration was observed in the duodenum of both species, yet duodenal cytokines were repressed in mice but increased in rats. Serum and bone marrow iron levels were more decreased in rats than mice. Collectively, these data suggest that Cr(VI)-induced carcinogenesis in the rodent alimentary canal involves oxidative stress; however, differences in histopathology, cytokines, and iron status suggest potential contributions from other factors as well. PMID:22011396

Comparison of the effects of hexavalent chromium in the alimentary canal of F344 rats and B6C3F1 mice following exposure in drinking water: implications for carcinogenic modes of action.

PubMed

Thompson, Chad M; Proctor, Deborah M; Suh, Mina; Haws, Laurie C; Hébert, Charles D; Mann, Jill F; Shertzer, Howard G; Hixon, J Gregory; Harris, Mark A

2012-01-01

Exposure to high concentrations of hexavalent chromium (Cr[VI]) in drinking water is reported to induce oral mucosa tumors in F344 rats and intestinal tumors in B6C3F1 mice. To investigate the modes of action underlying these tumors, 90-day drinking water studies (with interim necropsy at day 8) were conducted with concentrations of 0.1-182 mg/l Cr(VI), administered as 0.3-520 mg/l sodium dichromate dihydrate. Blood and tissue samples were analyzed for chromium content, oxidative stress, iron levels, and gross and microscopic lesions. Results for the F344 rats are described herein and compared with results from B6C3F1 mice published previously. After 90 days of exposure, total chromium concentrations in the rat and mouse oral mucosae were comparable, yet significant dose-dependent decreases in the reduced-to-oxidized glutathione ratio (GSH/GSSG) were observed only in rats. In the duodenum, changes in GSH/GSSG were only observed in mice. Levels of 8-hydroxydeoxyguanosine were not increased in the oral or duodenal mucosae of either species. Glutathione levels were increased in the duodenum but decreased in the jejunum of both species, indicating potential differential responses in the intestinal segments. Histiocytic infiltration was observed in the duodenum of both species, yet duodenal cytokines were repressed in mice but increased in rats. Serum and bone marrow iron levels were more decreased in rats than mice. Collectively, these data suggest that Cr(VI)-induced carcinogenesis in the rodent alimentary canal involves oxidative stress; however, differences in histopathology, cytokines, and iron status suggest potential contributions from other factors as well.

Genetic basis of HDL variation in 129/SvImJ and C57BL/6J mice: importance of testing candidate genes in targeted mutant mice*s⃞

PubMed Central

Su, Zhiguang; Wang, Xiaosong; Tsaih, Shirng-Wern; Zhang, Aihong; Cox, Allison; Sheehan, Susan; Paigen, Beverly

2009-01-01

To evaluate the effect of genetic background on high-density lipoprotein cholesterol (HDL) levels in Soat1−/− mice, we backcrossed sterol O-acyltransferase 1 (Soat1)−/− mice, originally reported to have elevated HDL levels, to C57BL/6 mice and constructed a congenic strain with only a small region (3.3Mb) of 129 alleles, specifically excluding the nearby apolipoprotein A-II (Apoa2) gene from 129. HDL levels in these Soat1−/− mice were no different from C57BL/6, indicating that the passenger gene Apoa2 caused the previously reported elevation of HDL in these Soat1−/− mice. Because many knockouts are made in strain 129 and then subsequently backcrossed into C57BL/6, it is important to identify quantitative trait loci (QTL) that differ between 129 and C57BL/6 so that one can guard against effects ascribed to a knockout but really caused by a passenger gene from 129. To provide such data, we generated 528 F2 progeny from an intercross of 129S1/SvImJ and C57BL/6 and measured HDL concentrations in F2 animals first fed chow and then atherogenic diet. A genome wide scan using 508 single-nucleotide polymorphisms (SNPs) identified 19 QTL, 2 of which were male specific and 2 were female specific. Using comparative genomics and haplotype analysis, we narrowed QTL on chromosomes 3, 5, 8, 17, and 18 to 0.5, 6.3, 2.6, 1.1, and 0.6 Mb, respectively. These data will serve as a reference for any effort to test the impact of candidate genes on HDL using a knockout strategy. PMID:18772481

33 CFR 165.508 - Security Zone; Georgetown Channel, Potomac River, Washington, DC.

Code of Federal Regulations, 2010 CFR

2010-07-01

..., Potomac River, Washington, DC. 165.508 Section 165.508 Navigation and Navigable Waters COAST GUARD... § 165.508 Security Zone; Georgetown Channel, Potomac River, Washington, DC. (a) Definitions. (1) The... zone: All waters of the Georgetown Channel of the Potomac River, from the surface to the bottom, 75...

33 CFR 165.508 - Security Zone; Georgetown Channel, Potomac River, Washington, DC.

Code of Federal Regulations, 2014 CFR

2014-07-01

..., Potomac River, Washington, DC. 165.508 Section 165.508 Navigation and Navigable Waters COAST GUARD... § 165.508 Security Zone; Georgetown Channel, Potomac River, Washington, DC. (a) Definitions. (1) The... zone: All waters of the Georgetown Channel of the Potomac River, from the surface to the bottom, 75...

33 CFR 165.508 - Security Zone; Georgetown Channel, Potomac River, Washington, DC.

Code of Federal Regulations, 2012 CFR

2012-07-01

..., Potomac River, Washington, DC. 165.508 Section 165.508 Navigation and Navigable Waters COAST GUARD... § 165.508 Security Zone; Georgetown Channel, Potomac River, Washington, DC. (a) Definitions. (1) The... zone: All waters of the Georgetown Channel of the Potomac River, from the surface to the bottom, 75...

33 CFR 165.508 - Security Zone; Georgetown Channel, Potomac River, Washington, DC.

Code of Federal Regulations, 2013 CFR

2013-07-01

..., Potomac River, Washington, DC. 165.508 Section 165.508 Navigation and Navigable Waters COAST GUARD... § 165.508 Security Zone; Georgetown Channel, Potomac River, Washington, DC. (a) Definitions. (1) The... zone: All waters of the Georgetown Channel of the Potomac River, from the surface to the bottom, 75...

33 CFR 165.508 - Security Zone; Georgetown Channel, Potomac River, Washington, DC.

Code of Federal Regulations, 2011 CFR

2011-07-01

..., Potomac River, Washington, DC. 165.508 Section 165.508 Navigation and Navigable Waters COAST GUARD... § 165.508 Security Zone; Georgetown Channel, Potomac River, Washington, DC. (a) Definitions. (1) The... zone: All waters of the Georgetown Channel of the Potomac River, from the surface to the bottom, 75...

NTP Carcinogenesis Bioassay of Diallyl Phthalate (CAS No. 131-17-9) in B6C3F1 Mice (Gavage Study).

PubMed

1983-04-01

Diallyl phthalate is a widely used crosslinking agent for unsaturated polyesters. Diallyl phthalate or diallyl phthalate polyester blends are used primarily as plasticizers and carriers for adding catalysts and pigments to polyesters and in molding, electrical parts, laminating compounds, and impregnation of metal castings. Rubber compounds, epoxy formulations, and polyurethane foams may also contain diallyl phthalate. Annual production of diallyl phthalate in the United States exceeds 5,000 pounds; precise figures are not available. A NTP Carcinogenesis bioassay of diallyl phthalate (99% pure) was conducted by administering 0 (vehicle control), 150, or 300 mg/kg diallyl phthalate in corn oil by gavage, 5 days per week for 103 weeks, to groups of 50 male and 50 female B6C3F1 mice. Survival rates and mean body weights of dosed mice were not different from those of the controls, and pathological lesions unrelated to proliferative changes were not observed. Therefore, a maximally tolerated dose for the purposes of carcinogenicity testing may not have been achieved. The incidences of lymphoma and either lymphoma or leukemia in dosed male mice were no significantly greater than those in the controls according to pairwise comparisons (P=0.051 to P=0.096), but the trend tests were statistically significant by either life table or incidental tumor analysis (P=0.031 to P=0.045). The incidence of lymphomas in the high-dose male mice was 12/50 (24%) in comparison with 6/50 (12%) in the controls. Recent historical incidences at the performing laboratory and in the NTP Bioassay Program were 18/120 (15%) and 71/661 (11%), respectively. Since the incidence of high-dose male mice with leukemia was not significantly greater than that of concurrent or historical controls at the performing laboratory by pairwise comparisons, this marginal increase was considered only to be equivocally related to diallyl phthalate administration. Increased incidences of squamous cell papillomas

DIFFERING HEPATOTOXICITY AND LETHALITY AFTER SUBACUTE TRICHLOROETHYLENE EXPOSURE IN AQUEOUS OR CORN OIL GAVAGE VEHICLES IN B6C3F1 MICE

EPA Science Inventory

Subacute toxicity of trichloroethylene (TCE) was evaluated in male and female B6C3F1 mice using corn oil or aqueous gavage vehicles. Mice received oral doses of TCE five times a week for 4 weeks at 600, 1200 and 2400 mg/kg/day for males and 450, 900 and 1800 mg/kg/day for females...

Toxicity of inhaled methyl isocyanate in F344/N rats and B6C3F1 mice. I. Acute exposure and recovery studies.

PubMed Central

Bucher, J R; Gupta, B N; Adkins, B; Thompson, M; Jameson, C W; Thigpen, J E; Schwetz, B A

1987-01-01

Male and female F344/N rats and B6C3F1 mice were exposed to lethal and sublethal concentrations of methyl isocyanate by inhalation. Mortality, clinical signs, body and organ weights, and changes in clinical pathology and hematology were monitored immediately after 2-hr exposures and during the ensuing 3 months. Additional studies investigated the possible involvement of cyanide in the toxicity of methyl isocyanate. During exposures, signs of restlessness, lacrimation, and a reddish discharge from the nose and mouth were evident in rats and mice. Following exposures, rats and mice were dyspneic and weak. Deaths of rats and mice exposed to lethal concentrations (20 to 30 ppm) began within 15-18 hr, with males more prone to early death than females. A second wave of deaths occurred after 8 to 10 days, affecting primarily female rats and mice exposed to 20 to 30 ppm of methyl isocyanate, and male and female rats exposed to 10 ppm. Most deaths occurred during the first month following the exposures and were preceded by periods of severe respiratory distress. Body weights decreased in proportion to dose early, but then weight gain resumed in survivors at control rates. The only organ with a consistent, dose-related weight change was the lung, which was heavier throughout the studies in animals exposed to high concentrations of methyl isocyanate. No significant clinical pathology, or hematologic changes were observed in exposed rats. Blood and brain cholinesterase were not inhibited. Studies attempting to measure cyanide in the blood of methyl isocyanate-exposed rats, and attempting to affect lethality with a cyanide antidote (sodium nitrite and sodium thiosulfate) gave negative results.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3622444

Hematopoiesis and aging. V. A decline in hematocrit occurs in all aging female B6D2F1 mice

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

Boggs, D.R.; Patrene, K.

Longitudinal studies of hematocrits were done in aging B6D2F1 female mice at 54, 64, 91, 105 and 115 weeks of age. A modest decline in hematocrit was observed in 41/42 mice; we have previously shown that the decreased hematocrit of aged as compared to young mice is due to an expansion of plasma volume. Mice which died spontaneously after 91 weeks had lower hematocrits at 91 weeks and 105 weeks than did those which survived to 115 weeks. At each time interval, a sub-group of mice was killed and uptake of /sup 59/Fe into blood, foreleg, spleen and liver wasmore » studied and total nucleated cells per humerus was determined. The results were generally compatible with the thesis that aging mice maintain normal rates of erythropoiesis under basal conditions. Thus, it would appear that a decrease in hematocrit can be considered an expected part of the aging process in this mouse.« less

Quantitative anti-F1 and anti-V IgG ELISAs as serological correlates of protection against plague in female Swiss Webster mice.

PubMed

Little, S F; Webster, W M; Wilhelm, H; Fisher, D; Norris, S L W; Powell, B S; Enama, J; Adamovicz, J J

2010-01-22

A recombinant fusion protein composed of Yersinia pestis fraction 1 capsule (F1) and virulence-associated V antigen (V) (F1-V) has been developed as the next-generation vaccine against plague. In this study, female Swiss Webster mice received a single intramuscular vaccination with one of eight doses of the F1-V vaccine and exposed 4 weeks later to either Y. pestis CO92 or C12 organisms by the subcutaneous or aerosol routes of infection. Quantitative anti-F1 and anti-V immunoglobulin G (IgG) ELISAs were used to examine the relationship between survival outcome and antibody titers to F1 and V. Results suggested that each 1log(10) increase in week 4 quantitative anti-F1 and anti-V IgG ELISA titers were associated with a 1.7-fold (p=0.0051) and 2.5-fold (p=0.0054) increase in odds of survival, respectively, against either bubonic or pneumonic plague and may serve as serological correlates of protection.

12 CFR 508.7 - Conduct of hearings.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Conduct of hearings. 508.7 Section 508.7 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.7 Conduct of hearings. (a) Hearings provided by this...

NTP Toxicology and Carcinogenesis Studies of EMODIN (CAS NO. 518-82-1) Feed Studies in F344/N Rats and B6C3F1 Mice.

PubMed

2001-06-01

Emodin is a naturally occurring anthraquinone present in the roots and bark of numerous plants of the genus Rhamnus. Extracts from the roots, bark, and/or dried leaves of buckthorn, senna, cascara, aloe, frangula, and rhubarb have been used as laxatives since ancient times and currently are widely used in the preparation of herbal laxative preparations. Anthraquinone glycosides are poorly absorbed from the gastrointestinal tract but are cleaved by gut bacteria to produce aglycones (such as emodin) that are more readily absorbed and are responsible for the purgative properties of these preparations. There is extensive exposure to emodin and other anthraquinones resulting from the use of herb-based stimulant laxatives. Reports that 1,8-dihydroxyanthraquinone, a commonly used laxative ingredient, caused tumors in the gastrointestinal tract of rats raised the possibility of an association between colorectal cancer and the use of laxatives containing anthraquinones. Because emodin is a hydroxyanthraquinone structurally similar to 1,8-dihydroxyanthraquinone, is present in herbal laxatives, and was reported to be mutagenic in bacteria, it was considered a potential carcinogen and was selected for in-depth evaluation. Male and female F344/N rats and B6C3F1 mice were exposed to emodin (at least 94% pure) in feed for 16 days, 14 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 16-DAY STUDY IN RATS: Groups of five male and five female rats were fed diets containing 0, 600, 2,000, 5,500, 17,000, or 50,000 ppm emodin (equivalent to average daily doses of approximately 50, 170, 480, 1,400, or 3,700 mg emodin/kg body weight to males and 50, 160, 460, 1,250, or 2,000 mg/kg to females) for 15 (males) or 16 (females) days. Three female rats died before the end of the study. Mean body weights of males and females exposed to 5,500 ppm

Basal and induced granulopoiesis in outbred, F1 hybrid and inbred mice: can inbreeding depression influence the experimental practice?

PubMed

Hofer, Michal; Pospísil, Milan; Dusek, Ladislav; Holá, Jirina; Hoferová, Zuzana; Weiterová, Lenka

2010-08-01

In this study we examined differences in selected indices of granulopoiesis in outbred, F(1) hybrid and inbred mouse strains. Specifically, serum granulocyte colony-stimulating factor (G-CSF) levels, numbers of marrow granulocyte-macrophage progenitor cells and morphologically recognizable proliferative marrow granulocytic precursor cells were evaluated. These parameters were determined in untreated controls, and in mice exposed either to a non-specific stimulus (injection of saline) or to a granulopoiesis-enhancing stimulus (administration of a cyclooxygenase-2 inhibitor, meloxicam). Lower levels of G-CSF were detectable in the outbred ICR mice, which also demonstrated an enhanced response to both types of the stimuli. Considering the fact that outbred mice are closer to natural mammalian populations, including human ones, the possibility of using outbred mice, instead of the often used inbred strains, for experiments evaluating the effects of pharmacological interventions on hematopoiesis should be investigated.

12 CFR 508.10 - Burden of persuasion.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 12 Banks and Banking 5 2011-01-01 2011-01-01 false Burden of persuasion. 508.10 Section 508.10 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.10 Burden of persuasion. The petitioner has the burden of...

12 CFR 508.10 - Burden of persuasion.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 12 Banks and Banking 6 2014-01-01 2012-01-01 true Burden of persuasion. 508.10 Section 508.10 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.10 Burden of persuasion. The petitioner has the burden of...

12 CFR 508.10 - Burden of persuasion.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 12 Banks and Banking 6 2013-01-01 2012-01-01 true Burden of persuasion. 508.10 Section 508.10 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.10 Burden of persuasion. The petitioner has the burden of...

12 CFR 508.10 - Burden of persuasion.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 12 Banks and Banking 6 2012-01-01 2012-01-01 false Burden of persuasion. 508.10 Section 508.10 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.10 Burden of persuasion. The petitioner has the burden of...

12 CFR 508.5 - Petition for hearing.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Petition for hearing. 508.5 Section 508.5 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.5 Petition for hearing. (a) To obtain a hearing, the...

12 CFR 508.6 - Initiation of hearing.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Initiation of hearing. 508.6 Section 508.6 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.6 Initiation of hearing. (a) Within 10 days of the filing...

12 CFR 508.10 - Burden of persuasion.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Burden of persuasion. 508.10 Section 508.10 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.10 Burden of persuasion. The petitioner has the burden of...

12 CFR 508.9 - Rules of evidence.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Rules of evidence. 508.9 Section 508.9 Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY REMOVALS, SUSPENSIONS, AND PROHIBITIONS WHERE A CRIME IS CHARGED OR PROVEN § 508.9 Rules of evidence. (a) Formal rules of evidence shall...

ETC 408/508: Technical Editing

ERIC Educational Resources Information Center

Charlton, Michael

2013-01-01

ETC 408/508: Technical Editing is a cross-listed undergraduate and graduate course at Missouri Western State University, an open admissions public university with approximately 6,000 students. 508 is an elective course for students in the Master of Applied Arts in Written Communication degree and highly recommended for those in the Technical…

Synergistic protection of mice against plague with monoclonal antibodies specific for the F1 and V antigens of Yersinia pestis.

PubMed

Hill, Jim; Copse, Catherine; Leary, Sophie; Stagg, Anthony J; Williamson, E Diane; Titball, Richard W

2003-04-01

Monoclonal antibodies specific for Yersinia pestis V antigen and F1 antigen, administered singly or in combination, protected mice in models of bubonic and pneumonic plague. Antibodies showed synergy when administered prophylactically and as a therapy 48 h postinfection. Monoclonal antibodies therefore have potential as a treatment for plague.

Adrenergic pathway activation enhances brown adipose tissue metabolism: A [18F]FDG PET/CT study in mice

PubMed Central

Mirbolooki, M. Reza; Upadhyay, Sanjeev Kumar; Constantinescu, Cristian C.; Pan, Min-Liang; Mukherjee, Jogeshwar

2013-01-01

Objective Pharmacologic approaches to study brown adipocyte activation in vivo with a potential of being translational to humans are desired. The aim of this study was to examine pre- and postsynaptic targeting of adrenergic system for enhancing brown adipose tissue (BAT) metabolism quantifiable by [18F]fluoro-2-deoxyglucose ([18F]FDG) positron emission tomography (PET)/ computed tomography (CT) in mice. Methods A β3-adrenoreceptor selective agonist (CL 316243), an adenylyl cyclase enzyme activator (forskolin) and a potent blocker of presynaptic norepinephrine transporter (atomoxetine) were injected through the tail vein of Swiss Webster mice 30 minutes before intravenous (iv) administration of [18F]FDG. The mice were placed on the PET/CT bed for 30 min PET acquisition followed by 10 min CT acquisition for attenuation correction and anatomical delineation of PET images. Results Activated interscapular (IBAT), cervical, periaortic and intercostal BAT were observed in 3-dimentional analysis of [18F]FDG PET images. CL 316243 increased the total [18F]FDG standard uptake value (SUV) of IBAT 5-fold greater compared to that in placebo-treated mice. It also increased the [18F]FDG SUV of white adipose tissue (2.4-fold), and muscle (2.7-fold), as compared to the control. There was no significant difference in heart, brain, spleen and liver uptakes between groups. Forskolin increased [18F]FDG SUV of IBAT 1.9-fold greater than that in placebo-treated mice. It also increased the [18F]FDG SUV of white adipose tissue (2.2-fold) and heart (5.4-fold) compared to control. There was no significant difference in muscle, brain, spleen, and liver uptakes between groups. Atomoxetine increased [18F]FDG SUV of IBAT 1.7-fold greater than that in placebo-treated mice. There were no significant differences in all other organs compared to placebo-treated mice except liver (1.6 fold increase). A positive correlation between SUV levels of IBAT and CT hounsfiled unit (HU) (R2=0.55, p<0.001) and

Immune-Enhancing Effect of Nanometric Lactobacillus plantarum nF1 (nLp-nF1) in a Mouse Model of Cyclophosphamide-Induced Immunosuppression.

PubMed

Choi, Dae-Woon; Jung, Sun Young; Kang, Jisu; Nam, Young-Do; Lim, Seong-Il; Kim, Ki Tae; Shin, Hee Soon

2018-02-28

Nanometric Lactobacillus plantarum nF1 (nLp-nF1) is a biogenics consisting of dead L. plantarum cells pretreated with heat and a nanodispersion process. In this study, we investigated the immune-enhancing effects of nLp-nF1 in vivo and in vitro. To evaluate the immunostimulatory effects of nLp-nF1, mice immunosuppressed by cyclophosphamide (CPP) treatment were administered with nLp-nF1. As expected, CPP restricted the immune response of mice, whereas oral administration of nLp-nF1 significantly increased the total IgG in the serum, and cytokine production (interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α)) in bone marrow cells. Furthermore, nLp-nF1 enhanced the production of splenic cytokines such as IL-12, TNF-α, and interferon gamma (IFN-γ). In vitro, nLp-nF1 stimulated the immune response by enhancing the production of cytokines such as IL-12, TNF-α, and IFN-γ. Moreover, nLp-nF1 given a food additive enhanced the immune responses when combined with various food materials in vitro. These results suggest that nLp-nF1 could be used to strengthen the immune system and recover normal immunity in people with a weak immune system, such as children, the elderly, and patients.

Development of Yersinia pestis F1 antigen-loaded microspheres vaccine against plague

PubMed Central

Huang, Shih-shiung; Li, I-Hsun; Hong, Po-da; Yeh, Ming-kung

2014-01-01

Yersinia pestis F1 antigen-loaded poly(DL-lactide-co-glycolide)/polyethylene glycol (PEG) (PLGA/PEG) microspheres were produced using a water-in-oil-in-water emulsion/solvent extraction technique and assayed for their percent yield, entrapment efficiency, surface morphology, particle size, zeta potential, in vitro release properties, and in vivo animal protect efficacy. The Y. pestis F1 antigen-loaded microspheres (mean particle size 3.8 μm) exhibited a high loading capacity (4.5% w/w), yield (85.2%), and entrapment efficiency (38.1%), and presented a controlled in vitro release profile with a low initial burst (18.5%), then continued to release Y. pestis F1 antigen over 70 days. The distribution (%) of Y. pestis F1 on the microspheres surface, outer layer, and core was 3.1%, 28.9%, and 60.7%, respectively. A steady release rate was noticed to be 0.55 μg Y. pestis F1 antigen/mg microspheres/day of Y. pestis F1 antigen release maintained for 42 days. The cumulative release amount at the 1st, 28th, and 42nd days was 8.2, 26.7, and 31.0 μg Y. pestis F1 antigen/mg microspheres, respectively. The 100 times median lethal dose 50% (LD50) of Y. pestis Yokohama-R strain by intraperitoneal injection challenge in mice test, in which mice received one dose of 40 μg F1 antigen content of PLGA/PEG microspheres, F1 antigen in Al(OH)3, and in comparison with F1 antigen in Al(OH)3 vaccine in two doses, was evaluated after given by subcutaneous immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with one dose of F1 antigen-loaded PLGA/PEG microspheres, and two doses of F1 antigen in Al(OH)3 vaccine (100%). In vivo vaccination studies also demonstrated that F1 vaccines microspheres had a protective ability; its steady-state IgG immune protection in mice plasma dramatic increased from 2 weeks (18,764±3,124) to 7 weeks (126,468±19,176) after vaccination. These findings strongly suggest that F1-antigen loaded

Creep of A508/533 Pressure Vessel Steel

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

Richard Wright

2014-08-01

ABSTRACT Evaluation of potential Reactor Pressure Vessel (RPV) steels has been carried out as part of the pre-conceptual Very High Temperature Reactor (VHTR) design studies. These design studies have generally focused on American Society of Mechanical Engineers (ASME) Code status of the steels, temperature limits, and allowable stresses. Initially, three candidate materials were identified by this process: conventional light water reactor (LWR) RPV steels A508 and A533, 2¼Cr-1Mo in the annealed condition, and Grade 91 steel. The low strength of 2¼Cr-1Mo at elevated temperature has eliminated this steel from serious consideration as the VHTR RPV candidate material. Discussions with themore » very few vendors that can potentially produce large forgings for nuclear pressure vessels indicate a strong preference for conventional LWR steels. This preference is based in part on extensive experience with forging these steels for nuclear components. It is also based on the inability to cast large ingots of the Grade 91 steel due to segregation during ingot solidification, thus restricting the possible mass of forging components and increasing the amount of welding required for completion of the RPV. Grade 91 steel is also prone to weld cracking and must be post-weld heat treated to ensure adequate high-temperature strength. There are also questions about the ability to produce, and very importantly, verify the through thickness properties of thick sections of Grade 91 material. The availability of large components, ease of fabrication, and nuclear service experience with the A508 and A533 steels strongly favor their use in the RPV for the VHTR. Lowering the gas outlet temperature for the VHTR to 750°C from 950 to 1000°C, proposed in early concept studies, further strengthens the justification for this material selection. This steel is allowed in the ASME Boiler and Pressure Vessel Code for nuclear service up to 371°C (700°F); certain excursions above that

The effect of study type on body weight and tumor incidence in B6C3F1 mice fed the NTP-2000 diet.

PubMed

Marino, Dale J

2012-07-01

The B6C3F1 mouse is the standard mouse strain used in National Toxicology Program (NTP) carcinogenesis studies. Over time, increased liver tumorigenesis that was correlated with elevated body weights was noted in males and females. NTP therefore replaced the NIH-07 diet with the NTP-2000 diet and returned to group housing of females as lower body weights were noted in group housed mice. However, recent studies reported study-type differences in body weights at 3 months using the NTP-2000 diet with higher weights evident in drinking water and inhalation studies compared to feed studies. Therefore, body weight and tumor incidence data were collected for untreated control mice from all 2-year NTP feed (12), drinking water (8), water gavage (6) and inhalation (10) studies that used the NTP-2000 diet in order to assess the impact of study type on body weights and tumor incidences. Results show statistically significant elevated body weights and liver tumor incidences in males and females from drinking water, water gavage and inhalation studies compared to results from feed studies. Thus, the elevated body weights and liver tumorigenesis noted in mice using the NIH-07 diet were also evident using the NTP-2000 diet, which was introduced to address body weight elevations. Given the study-type dependent effects noted, these results emphasize the importance of carefully selecting historical control data for B6C3F1 mice. Moreover, because of the association between body weight and liver tumorigenesis, these results may have implications regarding dose-level selection for carcinogenicity studies involving B6C3F1 mice based on the maximum tolerated dose.

Perk Ablation Ameliorates Myelination in S63del-Charcot–Marie–Tooth 1B Neuropathy

PubMed Central

Musner, Nicolò; Sidoli, Mariapaola; Zambroni, Desireè; Del Carro, Ubaldo; Ungaro, Daniela; D’Antonio, Maurizio; Feltri, Maria L.

2016-01-01

In peripheral nerves, P0 glycoprotein accounts for more than 20% of myelin protein content. P0 is synthesized by Schwann cells, processed in the endoplasmic reticulum (ER) and enters the secretory pathway. However, the mutant P0 with S63 deleted (P0S63del) accumulates in the ER lumen and induces a demyelinating neuropathy in Charcot–Marie–Tooth disease type 1B (CMT1B)–S63del mice. Accumulation of P0S63del in the ER triggers a persistent unfolded protein response. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER stress sensor that phosphorylates eukaryotic initiation factor 2 alpha (eIF2alpha) in order to attenuate protein synthesis. We have shown that increasing phosphophorylated-eIF2alpha (P-eIF2alpha) is a potent therapeutic strategy, improving myelination and motor function in S63del mice. Here, we explore the converse experiment: Perk haploinsufficiency reduces P-eIF2alpha in S63del nerves as expected, but surprisingly, ameliorates, rather than worsens S63del neuropathy. Motor performance and myelin abnormalities improved in S63del//Perk+/− compared with S63del mice. These data suggest that mechanisms other than protein translation might be involved in CMT1B/S63del neuropathy. In addition, Perk deficiency in other cells may contribute to demyelination in a non–Schwann-cell autonomous manner. PMID:27095827

NTP Toxicology and Carcinogenesis Studies of Cobalt Sulfate Heptahydrate (CAS No. 10026-24-1) in F344/N Rats and B6C3F1 Mice (Inhalation Studies).

PubMed

1998-08-01

Cobalt sulfate is used in the electroplating and electro chemical industries. It is also used as a coloring agent for ceramics and as a drying agent in inks, paints, varnishes, and linoleum. Cobalt sulfate may be added to animal feed as a mineral supplement and has been used as a top dressing on pasture lands. Cobalt sulfate was nominated by the National Cancer Institute for study based on a lack of information on the toxicity of soluble salts. Male and female F344/N rats and B6C3F1 mice were exposed to cobalt sulfate heptahydrate (approximately 99% pure) by inhalation for 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium. The results of prechronic inhalation toxicity studies were reported previously (Bucher et al., 1990; NTP, 1991). 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed to aerosols containing 0, 0.3, 1.0, or 3.0 mg/m3 cobalt sulfate heptahydrate 6 hours per day, 5 days per week, for 105 weeks. Survival and Body Weights Survival of exposed males and females was similar to that of the chamber controls. Mean body weights of exposed male and female rats were similar to those of the chamber controls throughout the study. Pathology Findings The incidences and severities of proteinosis, alveolar epithelial metaplasia, granulomatous alveolar inflammation, and interstitial fibrosis were markedly greater in all exposed groups of male and female rats than in the chamber controls. The incidences of alveolar epithelial hyperplasia in all groups of exposed males and in females exposed to 3.0 mg/m3 were significantly greater than those in the chamber control groups, as were the incidences of squamous metaplasia in 1.0 mg/m3 females and atypical alveolar epithelial hyperplasia in 3.0 mg/m3 females. In 3.0 mg/m3 males, the combined incidence of alveolar/ bronchiolar neoplasms (adenoma and/or carcinoma) was significantly greater than in the chamber controls. In female rats exposed to 1.0 or 3.0 mg/m3, the incidences of

IGF-1 deficiency impairs cerebral myogenic autoregulation in hypertensive mice.

PubMed

Toth, Peter; Tucsek, Zsuzsanna; Tarantini, Stefano; Sosnowska, Danuta; Gautam, Tripti; Mitschelen, Matthew; Koller, Akos; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan

2014-12-01

Aging impairs autoregulatory protection in the brain, exacerbating hypertension-induced cerebromicrovascular injury, neuroinflammation, and development of vascular cognitive impairment. Despite the importance of the age-related decline in circulating insulin-like growth factor-1 (IGF-1) levels in cerebrovascular aging, the effects of IGF-1 deficiency on functional adaptation of cerebral arteries to high blood pressure remain elusive. To determine whether IGF-1 deficiency impairs autoregulatory protection, hypertension was induced in control and IGF-1-deficient mice (Igf1(f/f)+TBG-iCre-AAV8) by chronic infusion of angiotensin-II. In hypertensive control mice, cerebral blood flow (CBF) autoregulation was extended to higher pressure values and the pressure-induced tone of middle cerebral arteries (MCAs) was increased. In hypertensive IGF-1-deficient mice, autoregulation was markedly disrupted, and MCAs did not show adaptive increases in myogenic tone. In control mice, the mechanism of adaptation to hypertension involved upregulation of TRPC channels in MCAs and this mechanism was impaired in hypertensive IGF-1-deficient mice. Likely downstream consequences of cerebrovascular autoregulatory dysfunction in hypertensive IGF-1-deficient mice included exacerbated disruption of the blood-brain barrier and neuroinflammation (microglia activation and upregulation of proinflammatory cytokines and chemokines), which were associated with impaired hippocampal cognitive function. Collectively, IGF-1 deficiency impairs autoregulatory protection in the brain of hypertensive mice, potentially exacerbating cerebromicrovascular injury and neuroinflammation mimicking the aging phenotype.

Effects of oral Lactobacillus administration on antioxidant activities and CD4+CD25+forkhead box P3 (FoxP3)+ T cells in NZB/W F1 mice.

PubMed

Tzang, Bor-Show; Liu, Chung-Hsien; Hsu, Kuo-Ching; Chen, Yi-Hsing; Huang, Chih-Yang; Hsu, Tsai-Ching

2017-09-01

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterised by a dysregulation of the immune system, which causes inflammation responses, excessive oxidative stress and a reduction in the number of cluster of differentiation (CD)4+CD25+forkhead box P3 (FoxP3)+ T cells. Supplementation with certain Lactobacillus strains has been suggested to be beneficial in the comprehensive treatment of SLE. However, little is known about the effect and mechanism of certain Lactobacillus strains on SLE. To investigate the effects of Lactobacillus on SLE, NZB/W F1 mice were orally gavaged with Lactobacillus paracasei GMNL-32 (GMNL-32), Lactobacillus reuteri GMNL-89 (GMNL-89) and L. reuteri GMNL-263 (GMNL-263). Supplementation with GMNL-32, GMNL-89 and GMNL-263 significantly increased antioxidant activity, reduced IL-6 and TNF-α levels and significantly decreased the toll-like receptors/myeloid differentiation primary response gene 88 signalling in NZB/W F1 mice. Notably, supplementation with GMNL-263, but not GMNL-32 and GMNL-89, in NZB/W F1 mice significantly increased the differentiation of CD4+CD25+FoxP3+ T cells. These findings reveal beneficial effects of GMNL-32, GMNL-89 and GMNL-263 on NZB/W F1 mice and suggest that these specific Lactobacillus strains can be used as part of a comprehensive treatment of SLE patients.

Atypical E2F transcriptional repressor DEL1 acts at the intersection of plant growth and immunity by controlling the hormone salicylic acid.

PubMed

Chandran, Divya; Rickert, Joshua; Huang, Yingxiang; Steinwand, Michael A; Marr, Sharon K; Wildermuth, Mary C

2014-04-09

In plants, the activation of immunity is often inversely correlated with growth. Mechanisms that control plant growth in the context of pathogen challenge and immunity are unclear. Investigating Arabidopsis infection with the powdery mildew fungus, we find that the Arabidopsis atypical E2F DEL1, a transcriptional repressor known to promote cell proliferation, represses accumulation of the hormone salicylic acid (SA), an established regulator of plant immunity. DEL1-deficient plants are more resistant to pathogens and slightly smaller than wild-type. The resistance and size phenotypes of DEL1-deficient plants are due to the induction of SA and activation of immunity in the absence of pathogen challenge. Moreover, Enhanced Disease Susceptibility 5 (EDS5), a SA transporter required for elevated SA and immunity, is a direct repressed target of DEL1. Together, these findings indicate that DEL1 control of SA levels contributes to regulating the balance between growth and immunity in developing leaves. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of bone marrow-derived CD11b(+)F4/80 (+) immature dendritic cells on the balance between pro-inflammatory and anti-inflammatory cytokines in DBA/1 mice with collagen-induced arthritis.

PubMed

Fu, Jingjing; Zhang, Lingling; Song, Shanshan; Sheng, Kangliang; Li, Ying; Li, Peipei; Song, Shasha; Wang, Qingtong; Chu, Jianhong; Wei, Wei

2014-05-01

To explore the effect of bone marrow-derived CD11b(+)F4/80(+) immature dendritic cells (BM CD11b(+)F4/80(+)iDC) on the balance between pro-inflammatory and anti-inflammatory cytokines in DBA/1 mice with collagen-induced arthritis (CIA). BM CD11b(+)F4/80(+)iDC were induced with rmGM-CSF and rmIL-4, and were identified by the expressions of toll-like receptor 2 (TLR-2), indoleamine 2,3-deoxygenase (IDO), interleukin (IL)-10, transforming growth factor (TGF)-β1 and mixed leukocyte reaction (MLR). CIA was established in DBA/1 mice by immunization with type II collagen. CIA mice were injected intravenously with BM CD11b(+)F4/80(+)iDC three times after immunization. The effect of BM CD11b(+)F4/80(+)iDC on CIA was evaluated by the arthritis index, joint histopathology, body weight, thymus index, thymocytes proliferation, IL-1β, tumor necrosis factor (TNF)-α, IL-17, IL-10 and TGF-β1 levels. BM CD11b(+)F4/80(+)iDC induced with rmGM-CSF and rmIL-4 expressed high levels of TLR-2, IDO, IL-10 and TGF-β1. Infusion of BM CD11b(+)F4/80(+)iDC in CIA mice significantly reduced the arthritis index and pathological scores of joints, recovered the weight, decreased the thymus index and inhibited thymocyte proliferation. Levels of IL-1β, TNF-α and IL-17 were decreased in BM CD11b(+)F4/80(+)iDC-treated mice. BM CD11b(+)F4/80(+)iDC can be induced successfully with rmGM-CSF and rmIL-4. BM CD11b(+)F4/80(+)iDC treatment can ameliorate the development and severity of CIA by regulating the balance between pro-inflammatory cytokines and anti-inflammatory cytokines.

Sensory irritation structure-activity study of inhaled aldehydes in B6C3F1 and Swiss-Webster mice

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

Steinhagen, W.H.; Barrow, C.S.

1984-03-15

The sensory irritation potential of a series of saturated and unsaturated aliphatic and cyclic aldehydes was investigated in B6C3F1 and Swiss-Webster mice. With the reflex decrease in respiratory rate as the endpoint response, alpha, beta-unsaturated aliphatic aldehydes yielded RD/sub 50/ values (concentration which elicits a 50% decrease in respiratory rate) ranging from 1 to 5 ppm while saturated aliphatic aldehydes with two or more carbons produced RD/sub 50/ values from 750 to 4200 ppm. Cyclic aldehydes produced intermediate RD/sub 50/ values which ranged from 60 to 400 ppm. No statistically significant differences were found between concentration-response curves of B6C3F1 andmore » Swiss-Webster mice. Saturated aliphatic aldehydes with two or more carbons were nearly 1000 times less potent than formaldehyde. Although the mechanisms responsible for stimulation of trigeminal nerve endings by airborne chemicals are poorly understood, several hypotheses may help to explain the differences seen in this study. For example, the sensory irritation potency of the saturated aliphatic aldehydes diminished with their reported dehydration constants which may determine the degree to which these aldehydes crosslink with receptor proteins. The sensory irritation potency of acrolein and crotonaldehyde was probably due to 1,2 or 1,4 addition reactions. Additionally, molecular conformation and a recently published physical mechanism may contribute to sensory irritation responses, particularly for the less reactive aldehydes. Tentative threshold limit values (TLVs), based upon prevention of sensory irritation, were extrapolated from the RD/sub 50/ values of Swiss-Webster mice. With the exception of crotonaldehyde, good agreement was found with currently published TLVs.« less

18 CFR 50.8 - Acceptance/rejection of applications.

Code of Federal Regulations, 2010 CFR

2010-04-01

... applications. 50.8 Section 50.8 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY REGULATIONS UNDER THE FEDERAL POWER ACT APPLICATIONS FOR PERMITS TO SITE INTERSTATE ELECTRIC TRANSMISSION FACILITIES § 50.8 Acceptance/rejection of applications. (a) Applications will be...

NTP Toxicology and Carcinogenesis Studies of l-Epinephrine Hydrochloride (CAS No. 55-31-2) in F344/N Rats and B6C3F1 Mice (Inhalation Studies).

PubMed

1990-03-01

l-Epinephrine, an endogenous neurotransmitter hormone, is widely used for the treatment of allergic and respiratory disorders. NTP Toxicology and Carcinogenesis studies of epinephrine hydrochloride were conducted by exposing groups of F344/N rats and B6C3F1 mice of each sex to an aerosol containing epinephrine hydrochloride for 14 days, 13 weeks, 15 months, or 2 years. During the 14-day and 13-week studies, control animals were exposed to dilute aerosols of hydrochloric acid (pH 2.8), whereas during the 15-month and 2-year studies, controls were exposed to aerosols of water. Genetic toxicology studies of epinephrine were conducted in Salmonella typhimurium and Chinese hamster ovary (CHO) cells. Fourteen-Day Studies: Rats and mice were exposed to 0 or 12.5-200 mg/m3 epinephrine hydrochloride. Deaths occurred in male rats exposed to 12.5 mg/m3 or more and in females exposed to 25 mg/m3 or more. Deaths of mice occurred at concentrations of 50 mg/m3 or higher. Compound-related clinical signs included an increased respiratory rate in all groups of epinephrine-exposed rats and mice. At higher concentrations (100 and 200 mg/m3), excessive lacrimation and dyspnea in rats and exaggerated visual and auditory reflexes in mice were observed. Thirteen-Week Studies: Rats and mice were exposed to 0 or 2.5-40 mg/m3 epinephrine hydrochloride. Deaths in rats and mice were not concentration related. Final mean body weights of chemically exposed and hydrochloric acid aerosol control rats and mice were generally similar. Increased respiratory rates were noted in rats and mice exposed to 40 mg/m3. Heart and adrenal gland weights of rats and mice and liver weights of mice exposed to 40 mg/m3 were greater than those of aerosol controls. Squamous metaplasia occurred in the respiratory epithelium of the nasal mucosa of rats and mice exposed to 40 mg/m3. Degenerative lesions of the laryngeal muscle were seen in male and female rats exposed to 20 or 40 mg/m3. Inflammation in the glandular

Prolonged action potential duration in cardiac ablation of PDK1 mice.

PubMed

Han, Zhonglin; Jiang, Yu; Yang, Zhongzhou; Cao, Kejiang; Wang, Dao W

2015-01-01

The involvement of the AGC protein kinase family in regulating arrhythmia has drawn considerable attention, but the underlying mechanisms are still not clear. The aim of this study is to explore the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1), one of upstream protein kinases of the AGC protein kinase family, in the pathogenesis of dysregulated electrophysiological basis. PDK1(F/F) αMHC-Cre mice and PDK1(F/F) mice were divided into experiment group and control group. Using patch clamping technology, we explored action potential duration in both groups, and investigated the functions of transient outward potassium channel and L-type Ca(2+) channel to explain the abnormal action potential duration. Significant prolongation action potential duration was found in mice with PDK1 deletion. Further, the peak current of transient outward potassium current and L-type Ca(2+) current were decreased by 84% and 49% respectively. In addition, dysregulation of channel kinetics lead to action potential duration prolongation further. In conclusion, we have demonstrated that PDK1 participates in action potential prolongation in cardiac ablation of PDK1 mice. This effect is likely to be mediated largely through downregulation of transient outward potassium current. These findings indicate the modulation of the PDK1 pathway could provide a new mechanism for abnormal electrophysiological basis.

Chlorogenic Acid Activates CFTR-Mediated Cl- Secretion in Mice and Humans: Therapeutic Implications for Chronic Rhinosinusitis

PubMed Central

Illing, Elisa; Cho, Do-Yeon; Zhang, Shaoyan; Skinner, Daniel F.; Dunlap, Quinn A.; Sorscher, Eric J.; Woodworth, Bradford A.

2016-01-01

Objectives Salubrious effects of the green coffee bean are purportedly secondary to high concentrations of chlorogenic acid. Chlorogenic acid has a molecular structure similar to bioflavonoids that activate transepithelial Cl- transport in sinonasal epithelia. In contrast to flavonoids, the drug is freely soluble in water. The objective of this study is to evaluate the Cl- secretory capability of chlorogenic acid and its potential as a therapeutic activator of mucus clearance in sinus disease. Study Design Basic research Setting Laboratory Subjects and Methods Chlorogenic acid was tested on primary murine nasal septal epithelial(MNSE)[CFTR+/+ and transgenic CFTR-/-] and human sinonasal epithelial(HSNE)[CFTR+/+ and F508del/F508del] cultures under pharmacologic conditions in Ussing chambers to evaluate effects on transepithelial Cl- transport. Cellular cAMP, phosphorylation of the CFTR regulatory domain(R-D), and CFTR mRNA transcription were also measured. Results Chlorogenic acid stimulated transepithelial Cl- secretion [(change in short-circuit current(ΔISC=μA/cm2)] in MNSE(13.1+/-0.9 vs. 0.1+/-0.1, p<0.05) and HSNE(34.3+/-0.9 vs. 0.0+/-0.1, p<0.05). The drug had a long duration until peak effect at 15-30 minutes after application. Significant inhibition with INH-172, as well as absent stimulation in cultures lacking functional CFTR, suggests effects are dependent on CFTR-mediated pathways. However, the absence of elevated cellular cAMP and phosphorylation the CFTR R-D indicates chlorogenic acid does not work through a PKA-dependent mechanism. Conclusion Chlorogenic acid is a water soluble agent that promotes CFTR-mediated Cl- transport in mouse and human sinonasal epithelium. Translating activators of mucociliary transport to clinical use provides a new therapeutic approach to sinus disease. Further in vivo evaluation is planned. PMID:26019132

Chlorogenic Acid Activates CFTR-Mediated Cl- Secretion in Mice and Humans: Therapeutic Implications for Chronic Rhinosinusitis.

PubMed

Illing, Elisa A; Cho, Do-Yeon; Zhang, Shaoyan; Skinner, Daniel F; Dunlap, Quinn A; Sorscher, Eric J; Woodworth, Bradford A

2015-08-01

Salubrious effects of the green coffee bean are purportedly secondary to high concentrations of chlorogenic acid. Chlorogenic acid has a molecular structure similar to bioflavonoids that activate transepithelial Cl(-) transport in sinonasal epithelia. In contrast to flavonoids, the drug is freely soluble in water. The objective of this study is to evaluate the Cl(-) secretory capability of chlorogenic acid and its potential as a therapeutic activator of mucus clearance in sinus disease. Basic research. Laboratory. Chlorogenic acid was tested on primary murine nasal septal epithelial (MNSE) (CFTR(+/+) and transgenic CFTR(-/-)) and human sinonasal epithelial (HSNE) (CFTR(+/+) and F508del/F508del) cultures under pharmacologic conditions in Ussing chambers to evaluate effects on transepithelial Cl(-) transport. Cellular cyclic adenosine monophosphate (cAMP), phosphorylation of the CFTR regulatory domain (R-D), and CFTR mRNA transcription were also measured. Chlorogenic acid stimulated transepithelial Cl(-) secretion (change in short-circuit current [ΔISC = µA/cm(2)]) in MNSE (13.1 ± 0.9 vs 0.1 ± 0.1; P < .05) and HSNE (34.3 ± 0.9 vs 0.0 ± 0.1; P < .05). The drug had a long duration until peak effect at 15 to 30 minutes after application. Significant inhibition with INH-172 as well as absent stimulation in cultures lacking functional CFTR suggest effects are dependent on CFTR-mediated pathways. However, the absence of elevated cellular cAMP and phosphorylation the CFTR R-D indicates chlorogenic acid does not work through a PKA-dependent mechanism. Chlorogenic acid is a water-soluble agent that promotes CFTR-mediated Cl(-) transport in mouse and human sinonasal epithelium. Translating activators of mucociliary transport to clinical use provides a new therapeutic approach to sinus disease. Further in vivo evaluation is planned. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

Amniotic fluid digestive enzyme analysis is useful for identifying CFTR gene mutations of unclear significance.

PubMed

Oca, Florine; Dreux, Sophie; Gérard, Bénédicte; Simon-Bouy, Brigitte; de Becdelièvre, Alix; Ferec, Claude; Girodon, Emmanuelle; Muller, Françoise

2009-12-01

The large number of CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations and the existence of variants of unclear significance complicate the prenatal diagnosis of cystic fibrosis (CF). The aim of this study was to determine whether the pattern of amniotic fluid digestive enzymes (AF-DEs) could be correlated with the severity of CFTR mutations. The AF-DE pattern (gamma-glutamyltranspeptidase, aminopeptidase M, and the intestinal isoform of alkaline phosphatase) was retrospectively analyzed in 43 AF samples. All fetuses presented 2 CFTR mutations, which were classified according to the severity of the disease: CF/CF (n = 38); CF/CFTR-related disorders (n = 1); and CF/unknown variant (n = 4). The relationships between clinical CF status, CFTR mutations, and AF-DE pattern were studied. Of 38 severely affected CF fetuses, an "obstructive" AF-DE pattern was observed in 15 of 15 samples collected before 22 weeks, irrespective of the CFTR mutation (diagnostic sensitivity, 100%; diagnostic specificity, 99.8%). In the 23 fetuses evaluated after 22 weeks, the AF-DE pattern was abnormal in 7 cases and noncontributive in 16 (diagnostic sensitivity, 30.4%; diagnostic specificity, 99.8%). Of the 5 questionable cases (F508del/N1224K, F508del/L73F, 3849+10kbC>T/G1127E, F508del/S1235R, F508del/G622D), all were CF symptom free at 2-4 years of follow-up. The AF-DE pattern (<22 weeks) was typical in 3 cases but abnormal in the last 2 cases. AF-DE analysis is of value for prenatal CF diagnosis in classic forms of CF and could be helpful in nonclassic CF.

NTP toxicology and carcinogenesis studies of chromium picolinate monohydrate (CAS No. 27882-76-4) in F344/N rats and B6C3F1 mice (feed studies).

PubMed

2010-06-01

Chromium picolinate monohydrate is the commercially available form of chromium picolinate. Chromium picolinate is one of a number of compounds that contain chromium in the trivalent state (Cr III), which is the predominant form of chromium in nature. Humans ingest Cr III in food and dietary supplements. The major uses of Cr III in the chemical and manufacturing industries include production of chromium pigments and leather tanning. Chromium picolinate was nominated by the National Cancer Institute and a private individual for testing based on the potential for widespread consumer exposure from use as a dietary supplement. Male and female F344/N rats and B6C3F1 mice were exposed to chromium picolinate monohydrate (95% to 96% pure) in feed for 3 months or 2 years. Genetic toxicology studies with chromium picolinate monohydrate were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes. Genetic toxicology studies with chromium picolinate were conducted in S. typhimurium and rat bone marrow cells. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets containing 0, 80, 240, 2,000, 10,000, or 50,000 ppm chromium picolinate monohydrate (equivalent to average daily doses of approximately 7, 20, 160, 800, and 4,240 mg chromium picolinate monohydrate/kg body weight to males and 6, 20, 160, 780, and 4,250 mg/kg to females) for 14 weeks. All rats survived to the end of the study. Mean body weights and feed consumption of all exposed groups of males and females were similar to those of the control groups throughout the study. No exposure-related lesions occurred in males or females. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were fed diets containing 0, 80, 240, 2,000, 10,000, or 50,000 ppm chromium picolinate monohydrate (equivalent to average daily doses of approximately 17, 50, 450, 2,300, and 11,900 mg chromium picolinate monohydrate/kg body weight to males and 14, 40, 370, 1,775, and 9,140 mg/kg to females) for 14

Evaluation of the potential of triethanolamine to alter hepatic choline levels in female B6C3F1 mice.

PubMed

Stott, W T; Radtke, B J; Linscombe, V A; Mar, M-H; Zeisel, S H

2004-06-01

Triethanolamine (TEA), a widely used nongenotoxic alcohol-amine, has recently been reported to cause an increased incidence of liver tumors in female B6C3F1 mice, but not in males nor in Fischer 344 rats. Choline deficiency induces liver cancer in rodents, and TEA could compete with choline uptake into tissues. The potential of TEA to cause choline deficiency in the liver of these mice as a mode of tumorigenesis was investigated. Groups of female B6C3F1 mice were administered 0 (vehicle) or a maximum tolerated dosage (MTD) of 1000 mg/kg/day TEA (Trial I) and 0, 10, 100, 300, or 1000 mg/kg/day TEA (Trial II) in acetone vehicle via skin painting 5 days/week for 3 weeks. Female CDF(R) rats were also administered 0 or an MTD dosage of 250 mg/kg/day TEA (Trial II) in a similar manner. No clinical signs of toxicity were noted, and upon sacrifice, levels of hepatic choline, its primary storage form, phosphocholine (PCho), and its primary oxidation product, betaine, were determined. A statistically significant decrease in PCho and betaine, was observed at the high dosage (26-42%) relative to controls and a dose-related, albeit variable, decrease was noted in PCho levels. Choline levels were also decreased 13-35% at the high dose level in mice. No changes in levels of choline or metabolites were noted in treated rats. A subsequent evaluation of the potential of TEA to inhibit the uptake of (3)H-choline by cultured Chinese Hamster Ovary Cells revealed a dose-related effect upon uptake. It was concluded that TEA may cause liver tumors in mice via a choline-depletion mode of action and that this effect is likely caused by the inhibition of choline uptake by cells.

Evaluation of the Potential of Triethanolamine to Alter Hepatic Choline Levels in Female B6C3F1 Mice

PubMed Central

Stott, W. T.; Radtke, B. J.; Linscombe, V. A.; Mar, M-H; Zeisel, S. H.

2006-01-01

Triethanolamine (TEA), a widely used nongenotoxic alcoholamine, has recently been reported to cause an increased incidence of liver tumors in female B6C3F1 mice, but not in males nor in Fischer 344 rats. Choline deficiency induces liver cancer in rodents, and TEA could compete with choline uptake into tissues. The potential of TEA to cause choline deficiency in the liver of these mice as a mode of tumorigenesis was investigated. Groups of female B6C3F1 mice were administered 0 (vehicle) or a maximum tolerated dosage (MTD) of 1000 mg/kg/day TEA (Trial I) and 0, 10, 100, 300, or 1000 mg/kg/day TEA (Trial II) in acetone vehicle via skin painting 5 days/week for 3 weeks. Female CDF® rats were also administered 0 or an MTD dosage of 250 mg/kg/day TEA (Trial II) in a similar manner. No clinical signs of toxicity were noted, and upon sacrifice, levels of hepatic choline, its primary storage form, phosphocholine (PCho), and its primary oxidation product, betaine, were determined. A statistically significant decrease in PCho and betaine, was observed at the high dosage (26–42%) relative to controls and a dose-related, albeit variable, decrease was noted in PCho levels. Choline levels were also decreased 13–35% at the high dose level in mice. No changes in levels of choline or metabolites were noted in treated rats. A subsequent evaluation of the potential of TEA to inhibit the uptake of 3H-choline by cultured Chinese Hamster Ovary Cells revealed a dose-related effect upon uptake. It was concluded that TEA may cause liver tumors in mice via a choline-depletion mode of action and that this effect is likely caused by the inhibition of choline uptake by cells. PMID:15056812

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

PubMed

Giannattasio, S; Bobba, A; Jurgelevicius, V; Vacca, R A; Lattanzio, P; Merafina, R S; Utkus, A; Kucinskas, V; Marra, E

2006-01-01

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.

46 CFR 185.508 - Wearing of life jackets.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Wearing of life jackets. 185.508 Section 185.508... TONS) OPERATIONS Preparations for Emergencies § 185.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including...

46 CFR 185.508 - Wearing of life jackets.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Wearing of life jackets. 185.508 Section 185.508... TONS) OPERATIONS Preparations for Emergencies § 185.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including...

46 CFR 185.508 - Wearing of life jackets.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 7 2012-10-01 2012-10-01 false Wearing of life jackets. 185.508 Section 185.508... TONS) OPERATIONS Preparations for Emergencies § 185.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including...

46 CFR 185.508 - Wearing of life jackets.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 7 2014-10-01 2014-10-01 false Wearing of life jackets. 185.508 Section 185.508... TONS) OPERATIONS Preparations for Emergencies § 185.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including...

46 CFR 185.508 - Wearing of life jackets.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false Wearing of life jackets. 185.508 Section 185.508... TONS) OPERATIONS Preparations for Emergencies § 185.508 Wearing of life jackets. (a) The master of a vessel shall require passengers to don life jackets when possible hazardous conditions exist, including...

COMPARATIVE METABOLISM OF ARSENIC IN MICE AFTER A SINGLE OR REPEATED ORAL ADMINISTRATION OF ARSENATE

EPA Science Inventory