The novel complex allele [A238V;F508del] of the CFTR gene: clinical phenotype and possible implications for cystic fibrosis etiological therapies.

PubMed

Diana, Anna; Polizzi, Angela Maria; Santostasi, Teresa; Ratclif, Luigi; Pantaleo, Maria Giuseppina; Leonetti, Giuseppina; Iusco, Danila Rosa; Gallo, Crescenzio; Conese, Massimo; Manca, Antonio

2016-06-01

Few mutations in cis have been annotated for F508del homozygous patients. Southern Italy patients who at a first analysis appeared homozygous for the F508del mutation (n=63) or compound heterozygous for the F508del and another mutation in the cystic fibrosis transmembrane conductance regulator gene (n=155) were searched for the A238V mutation in exon 6. The allelic frequency of the complex allele [A238V;F508del] was 0.04. When the whole data set was used (comprised also of 56 F508del/F508del and 34 F508del/other mutation controls), no differences reached the statistical significance in the clinical parameters, except chloride concentrations which were lower in [A238V;F508del]/other mutation compared with F508del/other mutation (P=0.03). The two study groups presented less complications than the control groups. Within the minimal data set (34 F508del/F508del, 27 F508del/other mutation, 4 [A238V;F508del]/F508del cases and 5 [A238V;F508del]/other mutation cases); that is, presenting all the variables in each patient, forced expiratory volume in 1 s and forced vital capacity presented a trend to lower levels in the study groups in comparison with the F508del/F508del group, and C-reactive protein approximated statistically significant higher levels in the [A238V;F508del]/other mutation as compared with F508del/F508del patients (P=0.09). The analysis of statistical dependence among the variables showed a significant anticorrelation between chloride and body mass index in the [A238V;F508del]/other mutation group. In conclusion, the complex allele [A238V;F508del] seems to be associated with less general complications than in the control groups, on the other hand possibly giving a worse pulmonary phenotype and higher systemic/local inflammatory response. These findings have implications for the correct recruitment and clinical response of F508del patients in the clinical trials testing the new etiological drugs for cystic fibrosis.

Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene

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

Kerem, B.; Zielenski, J.; Markiewicz, D.

1990-11-01

Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found in the second NBF, but none was found in the R-domain. Seven of the mutations were of the missense type affecting some of the highly conserved amino acid residues in the first NBF; 3 were nonsense mutations; 2 would probablymore » affect mRNA splicing; 2 corresponded to small deletions, including another 3-base-pair deletion different from the major mutation ({delta}F508), which could account for 70% of the CF chromosomes in the population. Nine of these mutations accounted for 12 of the 31 non-{delta}F508 CF chromosomes in the Canadian families. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but it also suggests that DNA-based genetic screening for CF carrier status will not be straightforward.« less

Mutation analysis of the cystic fibrosis transmembrane regulator gene in native American populations of the southwest

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

Grebe, T.A.; Doane, W.W.; Norman, R.A.

1992-10-01

The authors report DNA and clinical analysis of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene - namely, [Delta]F508, G542X, G551D, R553X, N1303K, and W1282X - was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM29/PstI were performed as well. Of the 12 affected individuals studied, no [Delta]F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype,more » except for the one carrying the G542X mutation, who has the haplotye AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. The DNA data have serious implications for risk assessment of CF carrier status for these people. 14 refs., 3 tabs.« less

Cystic fibrosis transmembrane conductance regulator gene mutations: do they play a role in the aetiology of allergic bronchopulmonary aspergillosis?

PubMed

Eaton, T E; Weiner Miller, P; Garrett, J E; Cutting, G R

2002-05-01

Previous work suggests that cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations may be implicated in the aetiology of allergic bronchopulmonary aspergilosis (ABPA). To compare the frequency of CF gene mutations in asthmatics with ABPA of varying severity with asthmatics who were skin prick test (SPT)-positive to Aspergillus fumigatus (Af) without evidence of ABPA and asthmatics SPT-negative to Af. Thirty-one Caucasian patients with ABPA were identified, together with asthmatics SPT positive to Af without evidence of ABPA (n = 23) and SPT negative to Af (n = 28). Genomic DNA was tested for 16 CF mutations accounting for approximately 85% of CF alleles in Caucasian New Zealanders. Four (12.9%) ABPA patients were found to be carriers of a CF mutation (DeltaF508 n = 3, R117H n = 1), one (4.3%) asthmatic SPT positive to Af without ABPA (DeltaF508), and one (3.6%) asthmatic SPT negative to Af (R117H). All patients with a CF mutation had normal sweat chloride (< 40 mM). There was no significant difference between the frequency of CF mutations in the ABPA patients and asthmatics without ABPA. However, the frequency of CF mutations in the ABPA patients was significantly different (P = 0.0125) to the expected carrier rate in the general population. These results lend further support to a possible link between CF mutations and ABPA.

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.

Mutation detection by mismatch binding protein, MutS, in amplified DNA: Application to the cystic fibrosis gene

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

Lishanski, A.; Ostrander, E.A.; Rine, J.

1994-03-29

An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene. Genomic DNA samples were amplified with 5{prime}-end-labeled primers that bracket the site of the {Delta}F508 3-bp deletion in exon 10 of the CFTR gene. The renatured PCR products from homozygotes produced homoduplexes; the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS proteinmore » bound more strongly to heteroduplexes that correspond to heterozygous carriers of {Delta}F508 and contain a CTT or a GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS protein to detect heteroduplexes in PCR-amplified DNA extended to fragments {approximately} 500 bp long. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.« less

A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability.

PubMed

Wellhauser, Leigh; Kim Chiaw, Patrick; Pasyk, Stan; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E

2009-06-01

The deletion of Phe-508 (DeltaPhe508) constitutes the most prevalent of a number of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that cause cystic fibrosis (CF). This mutation leads to CFTR misfolding and retention in the endoplasmic reticulum, as well as impaired channel activity. The biosynthetic defect can be partially overcome by small-molecule "correctors"; once at the cell surface, small-molecule "potentiators" enhance the channel activity of DeltaPhe508-CFTR. Certain compounds, such as VRT-532, exhibit both corrector and potentiator functions. In the current studies, we confirmed that the inherent chloride channel activity of DeltaPhe508-CFTR (after biosynthetic rescue) is potentiated in studies of intact cells and membrane vesicles. It is noteworthy that we showed that the ATPase activity of the purified and reconstituted mutant protein is directly modulated by binding of VRT-532 [4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol] ATP turnover by reconstituted DeltaPhe508-CFTR is decreased by VRT-532 treatment, an effect that may account for the increase in channel open time induced by this compound. To determine whether the modification of DeltaPhe508-CFTR function caused by direct VRT-532 binding is associated with structural changes, we evaluated the effect of VRT-532 binding on the protease susceptibility of the major mutant. We found that binding of VRT-532 to DeltaPhe508-CFTR led to a minor but significant decrease in the trypsin susceptibility of the full-length mutant protein and a fragment encompassing the second half of the protein. These findings suggest that direct binding of this small molecule induces and/or stabilizes a structure that promotes the channel open state and may underlie its efficacy as a corrector of DeltaPhe508-CFTR.

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

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

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.

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.

Genetic analysis of hispanic individuals with cystic fibrosis

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

Grebe, T.A.; Doane, W.W.; Norman, R.A.

1994-03-01

The authors have performed molecular genetic analysis of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, oly 46% (59/129) carry [Delta]F508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC[yields]T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductancemore » regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of [Delta]508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analysis demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling. 22 refs., 2 tabs.« less

Highly preferential association of NonF508del CF mutations with the M470 allele.

PubMed

Ciminelli, B M; Bonizzato, A; Bombieri, C; Pompei, F; Gabaldo, M; Ciccacci, C; Begnini, A; Holubova, A; Zorzi, P; Piskackova, T; Macek, M; Castellani, C; Modiano, G; Pignatti, P F

2007-01-01

On the basis of previous findings on random individuals, we hypothesized a preferential association of CF causing mutations with the M allele of the M470V polymorphic site of the CFTR gene. We have determined the M/V-CF mutation haplotype in a series of 201 North East Italian and 73 Czech CF patients who were not F508del homozygotes, as F508del was already known to be fully associated with the M allele. Out of 358 not F508del CF genes, 84 carried the V allele and 274 the less common M allele. In the N-E Italian population, MM subjects have a risk of carrying a CF causing mutation 6.9x greater than VV subjects when F508del is excluded and 15.4x when F508del is included. In the Czech population a similar, although less pronounced, association is observed. Besides the possible biological significance of this association, the possibility of exploiting it for a pilot screening program has been explored in a local North East Italian population for which CF patients were characterized for their CF mutation. General M470V genotyping followed by common CF mutation screening limited to couples in which each partner carries at least one M allele would need testing only 39% of the couples, which contribute 89% of the total risk, with a cost benefit.

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

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.

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.

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.

Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis.

PubMed

Esposito, Speranza; Tosco, Antonella; Villella, Valeria R; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

2016-12-01

Cystic fibrosis (CF) is a lethal monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that entails the (diagnostic) increase in sweat electrolyte concentrations, progressive lung disease with chronic inflammation and recurrent bacterial infections, pancreatic insufficiency, and male infertility. Therapies aimed at restoring the CFTR defect have emerged. Thus, a small molecule which facilitates chloride channel opening, the potentiator Ivacaftor, has been approved for the treatment of CF patients bearing a particular class of rare CFTR mutations. However, small molecules that directly target the most common misfolded CFTR mutant, F508del, and improve its intracellular trafficking in vitro, have been less effective than expected when tested in CF patients, even in combination with Ivacaftor. Thus, new strategies are required to circumvent the F508del-CFTR defect. Airway and intestinal epithelial cells from CF patients bearing the F508del-CFTR mutation exhibit an impressive derangement of cellular proteostasis, with oxidative stress, overactivation of the tissue transglutaminase (TG2), and disabled autophagy. Proteostasis regulators such as cysteamine can rescue and stabilize a functional F508del-CFTR protein through suppressing TG2 activation and restoring autophagy in vivo in F508del-CFTR homozygous mice, in vitro in CF patient-derived cell lines, ex vivo in freshly collected primary patient's nasal cells, as well as in a pilot clinical trial involving homozygous F508del-CFTR patients. Here, we discuss how the therapeutic normalization of defective proteostasis can be harnessed for the treatment of CF patients with the F508del-CFTR mutation.

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.

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.

PubMed

Chang, Xiu-Bao; Mengos, April; Hou, Yue-Xian; Cui, Liying; Jensen, Timothy J; Aleksandrov, Andrei; Riordan, John R; Gentzsch, Martina

2008-09-01

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis

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

Trapnell, B.C.; Chinshyan Chu; Paakko, P.K.

1991-08-01

The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called {Delta}Phe{sup 508}, a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR protein. In the context that the lung is the major site of morbidity and mortality in CF, the authors evaluated airway epithelial cells for CFTR mRNA transcripts in normal individuals, normal-{Delta}Phe{sup 508} heterozygotes, and {Delta}Phe{sup 508} homozygotes to determine if the normal and {Delta}Phe{sup 508} CFTR alleles are expressed in the respiratory epithelium, to what extent they aremore » expressed, and whether there are relative differences in the expression of the normal and abnormal alleles at the mRNA level. Respiratory tract epithelial cells recovered by fiberoptic bronchoscopy with a cytology brush demonstrated CFTR mRNA transcripts with sequences appropriately reflecting the normal and {Delta}Phe{sup 508} CFTR alleles of the various study groups. CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells.« less

Analysis of some polymorphic markers of the CFTR gene in cystic fibrosis patients and healthy donors from the Moscow region

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

Amosenko, F.A.; Sazonova, M.A.; Kapranov, N.I.

1995-04-01

Allelic frequencies of three polymorphic markers in the CFTR gene were estimated on chromosomes derived from cystic fibrosis (CF) patients and healthy donors from Moscow and the Moscow region. These polymorphic markers are tetranucleotide tandem repeats GATT in intron 6B, M470V in exon 10, and T854T in exon 14 (fragment A). Frequencies at allele 1 of the M470V marker, along with allele 2 of GATT and T854T, are two times higher for CF patients without {Delta}F508 mutation than for healthy donors, and there is linkage disequilibrium of these alleles of the polymorphic markers analyzed with the CF gene. Allele 1more » of M470V and T854T markers, as well as allele 2 of the GATT marker (six repeats), are absolutely linked to mutation F508 of the CFTR gene. Using the polymorphic markers studied, family analysis of CF was carried out in two families. 10 refs., 1 fig., 1 tab.« less

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

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.

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.

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

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.

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

Mechanistic Approaches to Improve Correction of the Most Common Disease-Causing Mutation in Cystic Fibrosis

PubMed Central

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

2016-01-01

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to deletion of the phenylalanine at position 508 (ΔF508) in the CFTR protein and causes multiple folding and functional defects. Contrary to large-scale efforts by industry and academia, no significant therapeutic benefit has been achieved with a single “corrector”. Therefore, investigations concentrate on drug combinations. Orkambi (Vertex Pharmaceuticals), the first FDA-approved drug for treatment of cystic fibrosis (CF) caused by this mutation, is a combination of a corrector (VX-809) that facilitates ΔF508 CFTR biogenesis and a potentiator (VX-770), which improves its function. Yet, clinical trials utilizing this combination showed only modest therapeutic benefit. The low efficacy Orkambi has been attributed to VX-770-mediated destabilization of VX-809-rescued ΔF508 CFTR. Here we report that the negative effects of VX-770 can be reversed by increasing the half-life of the endoplasmic reticulum (ER) form (band B) of ΔF508 CFTR with another corrector (Corr-4a.) Although Corr-4a alone has only minimal effects on ΔF508 CFTR rescue, it increases the half-life of ΔF508 CFTR band B when it is present during half-life measurements. Our data shows that stabilization of band B ΔF508 CFTR with Corr-4a and simultaneous rescue with VX-809, leads to a >2-fold increase in cAMP-activated, CFTRinh-172-inhibited currents compared to VX-809 alone, or VX-809+VX-770. The negative effects of VX-770 and the Corr-4a protection are specific to the native I507-ATT ΔF508 CFTR without affecting the inherently more stable, synonymous variant I507-ATC ΔF508 CFTR. Our studies emphasize that stabilization of ΔF508 CFTR band B in the ER might improve its functional rescue by Orkambi. PMID:27214033

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.

Preimplantation genetic diagnosis for cystic fibrosis: a case report.

PubMed

Biazotti, Maria Cristina Santoro; Pinto Junior, Walter; Albuquerque, Maria Cecília Romano Maciel de; Fujihara, Litsuko Shimabukuro; Suganuma, Cláudia Haru; Reigota, Renata Bednar; Bertuzzo, Carmen Sílvia

2015-01-01

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby.

Preimplantation genetic diagnosis for cystic fibrosis: a case report

PubMed Central

Biazotti, Maria Cristina Santoro; Pinto, Walter; de Albuquerque, Maria Cecília Romano Maciel; Fujihara, Litsuko Shimabukuro; Suganuma, Cláudia Haru; Reigota, Renata Bednar; Bertuzzo, Carmen Sílvia

2015-01-01

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby. PMID:25993078

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 sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. PMID:25763566

[Pseudo-Bartter syndrome as manifestation of cystic fibrosis with DF508 mutation].

PubMed

Galaviz-Ballesteros, María de Jesús; Acosta-Rodríguez-Bueno, Carlos Patricio; Consuelo-Sánchez, Alejandra; Franco-Álvarez, Isidro; Olalla-Mora, Odilo Iván; Vázquez-Frias, Rodrigo

Pseudo Bartter syndrome (PBS) is defined as hypokalaemic hypochloraemic metabolic alkalosis in the absence of renal tubular pathology. Children with cystic fibrosis (CF) are at risk of developing electrolyte abnormalities and even PBS may occur. 5 months old female infant with a history of two events of dehydration with vomit, refusal to eat, chronic cough, polyuria, malnutrition, metabolic alkalosis, hypokalemia, hyponatremia, hypochloremia and acute renal failure. Chronic cough study was performed, discarding pulmonary tuberculosis, gastroesophageal reflux disease and impaired swallowing. PBS was diagnosed due to hypokalaemic hypochloraemic metabolic alkalosis in the absence of renal tubular pathology. CF was corroborated by electrolytes in sweat and through molecular analysis of the delta F508 mutation. This is one of the few reported cases linking PBS and this mutation. In patients with hyponatremic dehydration episodes with hypokalaemic hypochloraemic metabolic alkalosis, PBS should be considered as differential diagnosis. CF could be presented as PBS, mainly in patients younger than 2 years. Copyright © 2016 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

PubMed

Sangiuolo, Federica; Scaldaferri, Maria Lucia; Filareto, Antonio; Spitalieri, Paola; Guerra, Lorenzo; Favia, Maria; Caroppo, Rosa; Mango, Ruggiero; Bruscia, Emanuela; Gruenert, Dieter C; Casavola, Valeria; De Felici, Massimo; Novelli, Giuseppe

2008-01-01

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.

Modeling cystic fibrosis disease progression in patients with the rare CFTR mutation P67L.

PubMed

MacKenzie, Isobel E R; Paquette, Valerie; Gosse, Frances; George, Sheenagh; Chappe, Frederic; Chappe, Valerie

2017-05-01

The progression of cystic fibrosis (CF) in patients with the rare mutation P67L was examined to determine if it induced a milder form of CF compared to the common severe ΔF508 mutation. Parameters of lung function, level of bacterial infection, nutritional status and hospitalization were used to represent CF progression. Age at diagnosis and pancreatic status were used to assess CF presentation. Analysis of data from the CF Canada Registry collected over a 15-year period included 266 ΔF508/ΔF508 homozygote patients from CF clinics in Atlantic Canada and 26 compound heterozygote patients with the rare P67L mutation from clinics across Canada. Late age at diagnosis, high incidence of pancreatic sufficiency, maintained Body Mass Index (BMI) with age, delayed life-threatening bacterial infection, and fewer days in hospital were observed for P67L heterozygote patients included in this study. Although the decline of lung function did not differ from ΔF508 homozygotes, the fact that a greater proportion of P67L heterozygotes live to an older age suggests that lung function is not the primary factor determining CF progression for P67L heterozygote patients. The P67L mutation is associated with a mild disease, even when combined with the severe ΔF508 mutation. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809

PubMed Central

Van Goor, Fredrick; Hadida, Sabine; Grootenhuis, Peter D. J.; Burton, Bill; Stack, Jeffrey H.; Straley, Kimberly S.; Decker, Caroline J.; Miller, Mark; McCartney, Jason; Olson, Eric R.; Wine, Jeffrey J.; Frizzell, Ray A.; Ashlock, Melissa; Negulescu, Paul A.

2011-01-01

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that impair the function of CFTR, an epithelial chloride channel required for proper function of the lung, pancreas, and other organs. Most patients with CF carry the F508del CFTR mutation, which causes defective CFTR protein folding and processing in the endoplasmic reticulum, resulting in minimal amounts of CFTR at the cell surface. One strategy to treat these patients is to correct the processing of F508del-CFTR with small molecules. Here we describe the in vitro pharmacology of VX-809, a CFTR corrector that was advanced into clinical development for the treatment of CF. In cultured human bronchial epithelial cells isolated from patients with CF homozygous for F508del, VX-809 improved F508del-CFTR processing in the endoplasmic reticulum and enhanced chloride secretion to approximately 14% of non-CF human bronchial epithelial cells (EC50, 81 ± 19 nM), a level associated with mild CF in patients with less disruptive CFTR mutations. F508del-CFTR corrected by VX-809 exhibited biochemical and functional characteristics similar to normal CFTR, including biochemical susceptibility to proteolysis, residence time in the plasma membrane, and single-channel open probability. VX-809 was more efficacious and selective for CFTR than previously reported CFTR correctors. VX-809 represents a class of CFTR corrector that specifically addresses the underlying processing defect in F508del-CFTR. PMID:21976485

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation.

PubMed

De Stefano, Daniela; Villella, Valeria R; Esposito, Speranza; Tosco, Antonella; Sepe, Angela; De Gregorio, Fabiola; Salvadori, Laura; Grassia, Rosa; Leone, Carlo A; De Rosa, Giuseppe; Maiuri, Maria C; Pettoello-Mantovani, Massimo; Guido, Stefano; Bossi, Anna; Zolin, Anna; Venerando, Andrea; Pinna, Lorenzo A; Mehta, Anil; Bona, Gianni; Kroemer, Guido; Maiuri, Luigi; Raia, Valeria

2014-01-01

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Combined effects of VX-770 and VX-809 on several functional abnormalities of F508del-CFTR channels.

PubMed

Kopeikin, Z; Yuksek, Z; Yang, H-Y; Bompadre, S G

2014-09-01

The most common cystic fibrosis-associated mutation, the deletion of phenylalanine 508 (F508del), results in channels with poor membrane expression and impaired function. VX-770, a clinically approved drug for treatment of CF patients carrying the G551D mutation, and VX-809, a corrector shown in vitro to increase membrane expression of mutant channels, are currently undergoing clinical trials, but functional data at the molecular level is still lacking. The effect of VX-770 and VX-809 on the multiple functional defects of F508del-CFTR was assessed via excised inside-out patch-clamp experiments. VX-770 completely restores the low opening-rate of F508del-CFTR, with smaller open-time increase, in temperature-corrected and VX-809-treated channels. The shorter locked-open time of hydrolysis-deficient F508del-CFTR is also prolonged by VX-770. VX-809 does not improve channel function by itself as previously reported. The results from these studies can be interpreted as an equilibrium shift toward the open-channel conformation of F508del-CFTR channels. Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Pharmacological treatment of the ion transport defect in cystic fibrosis.

PubMed

Roomans, G M

2001-01-01

Cystic fibrosis (CF) is a lethal monogenetic disease characterised by impaired water and ion transport over epithelia. The lung pathology is fatal and causes death in 95% of CF patients. The genetic basis of the disease is a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most common mutation, DeltaF508, results in a protein that cannot properly be folded in the endoplasmic reticulum, is destroyed and hence does not reach the apical cell membrane. This paper will discuss those pharmacological approaches that are directed at correcting the defect in ion transport. At present, no clinically effective drug is available, although research has defined areas in which progress might be made. These are the following: (1) the drug 4-phenylbutyrate (4PBA) increases the expression of DeltaF508-CFTR in the cell membrane, probably by breaking the association between DeltaF508-CFTR and a chaperone; (2) a number of xanthines, in particular 8-cyclopentyl-1, 3-dipropylxanthine (CPX), are effective in activating CFTR, presumably by direct binding and also possibly by correcting the trafficking defect; (3) the isoflavone genistein can activate both wild-type and mutant CFTR, probably through direct binding to the channel; (4) purinergic agonists (ATP and UTP) can stimulate chloride secretion via a Ca(2+)-dependent chloride channel and in this way compensate for the defect in CFTR, but stable analogues will be required before this type of treatment has clinical significance; (5) treatment with inhaled amiloride may correct the excessive absorption of Na(+) ions and water by airway epithelial cells that appears connected to the defect in CFTR; although clinical tests have not been very successful so far, amiloride analogues with a longer half-life may give better results. The role of CFTR in bicarbonate secretion has not yet been established with certainty, but correction of the defect in bicarbonate secretion may be important in clinical treatment of the disease. Currently, major efforts are directed at developing a pharmacological treatment of the ion transport defect in CF, but much basic research remains to be done, in particular, with regard to the mechanism by which defective CFTR is removed in the endoplasmic reticulum by the ubiquitin-proteasome pathway, which is a central pathway in protein production and of significance for several other diseases apart from CF.

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

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.

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

PubMed Central

Stefano, Daniela De; Villella, Valeria R; Esposito, Speranza; Tosco, Antonella; Sepe, Angela; Gregorio, Fabiola De; Salvadori, Laura; Grassia, Rosa; Leone, Carlo A; Rosa, Giuseppe De; Maiuri, Maria C; Pettoello-Mantovani, Massimo; Guido, Stefano; Bossi, Anna; Zolin, Anna; Venerando, Andrea; Pinna, Lorenzo A; Mehta, Anil; Bona, Gianni; Kroemer, Guido; Maiuri, Luigi; Raia, Valeria

2014-01-01

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in CftrF508del homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation. PMID:25350163

Screening for F508del as a first step in the molecular diagnosis of cystic fibrosis.

PubMed

Marson, Fernando Augusto de Lima; Bertuzzo, Carmen Silvia; Ribeiro, Maria Ângela Gonçalves de Oliveira; Ribeiro, Antônio Fernando; Ribeiro, José Dirceu

2013-01-01

To determine the relevance of screening for the F508del mutation of the cystic fibrosis transmembrane conductance regulator gene as a first step in the genetic diagnosis of cystic fibrosis (CF) by associating the genotype with various clinical variables. We evaluated 180 CF patients regarding the F508del mutation. The clinical data were obtained from the medical records of the patients and from interviews with their parents or legal guardians. Of the 180 patients studied, 65 (36.1%) did not carry the F508del mutation (group 0 [G0]), 67 (37.2%) were F508del heterozygous (G1), and 48 (26.7%) were F508del homozygous (G2). All three groups showed associations with the clinical variables. Homozygosis was associated with younger patients, younger age at CF diagnosis, and younger age at the first isolation of Pseudomonas aeruginosa (PA), as well as with higher prevalence of pancreatic insufficiency (PI) and non-mucoid PA (NMPA) colonization. In comparison with G1+G2 patients, G0 patients were older; first experienced clinical symptoms, digestive disease, and pulmonary disease at an older age; were older at CF diagnosis and at first PA isolation; and had a lower prevalence of PI and meconium ileus, as well as of colonization by NMPA, mucoid PA, and Burkholderia cepacia. In G1 patients, values were intermediate for age at CF diagnosis; age at first PA isolation, first pulmonary symptoms, and first clinical manifestations; MPA colonization; and OR for PI. The identification of F508del in 63.9% of the patients studied showed that this can be a useful tool as a first step in the genetic diagnosis of CF. The F508del genotype was associated with clinical severity of the disease, especially with the variables related to CF onset.

Identification of eight mutations and three sequence variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene

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

Ghanem, N.; Costes, B.; Girodon, E.

1994-05-15

To determine cystic fibrosis (CF) defects in a sample of 224 non-[Delta]F508 CF chromosomes, the authors used denaturing gradient gel multiplex analysis of CF transmembrane conductance regulator gene segments, a strategy based on blind exhaustive analysis rather than a search for known mutations. This process allowed detection of 11 novel variations comprising two nonsense mutations (Q890X and W1204X), a splice defect (405 + 4 A [yields] G), a frameshift (3293delA), four presumed missense mutations (S912L, H949Y, L1065P, Q1071P), and three sequence polymorphisms (R31C or 223 C/T, 3471 T/C, and T1220I or 3791 C/T). The authors describe these variations, together withmore » the associated phenotype when defects on both CF chromosomes were identified. 8 refs., 1 fig., 1 tab.« less

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.

Cystic fibrosis transmembrane regulator haplotypes in households of patients with cystic fibrosis.

PubMed

Furgeri, Daniela Tenório; Marson, Fernando Augusto Lima; Correia, Cyntia Arivabeni Araújo; Ribeiro, José Dirceu; Bertuzzo, Carmen Sílvia

2018-01-30

Nearly 2000 mutations in the cystic fibrosis transmembrane regulator (CFTR) gene have been reported. The F508del mutation occurs in approximately 50-65% of patients with cystic fibrosis (CF). However, molecular diagnosis is not always possible. Therefore, silent polymorphisms can be used to label the mutant allele in households of patients with CF. To verify the haplotypes of four polymorphisms at the CFTR locus in households of patients with CF for pre-fertilization, pre-implantation, and prenatal indirect mutation diagnosis to provide better genetic counseling for families and patients with CF and to associate the genotypes/haplotypes with the F508del mutation screening. GATT polymorphism analysis was performed using direct polymerase chain reaction amplification, and the MP6-D9, TUB09 and TUB18 polymorphism analyses were performed using restriction fragment length polymorphism. Nine haplotypes were found in 37 CFTR alleles, and of those, 24 were linked with the F508del mutation and 13 with other CFTR mutations. The 6 (GATT), C (MP6-D9), G (TUB09), and C (TUB18) haplotypes showed the highest prevalence (48%) of the mutant CFTR allele and were linked to the F508del mutation (64%). In 43% of households analyzed, at least one informative polymorphism can be used for the indirect diagnostic test. CFTR polymorphisms are genetic markers that are useful for identifying the mutant CFTR alleles in households of patients with CF when it is not possible to establish the complete CFTR genotype. Moreover, the polymorphisms can be used for indirect CFTR mutation identification in cases of pre-fertilization, pre-implantation and prenatal analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolic interactions between cysteamine and epigallocatechin gallate.

PubMed

Izzo, Valentina; Pietrocola, Federico; Sica, Valentina; Durand, Sylvère; Lachkar, Sylvie; Enot, David; Bravo-San Pedro, José Manuel; Chery, Alexis; Esposito, Speranza; Raia, Valeria; Maiuri, Luigi; Maiuri, Maria Chiara; Kroemer, Guido

2017-02-01

Phase II clinical trials indicate that the combination of cysteamine plus epigallocatechin gallate (EGCG) is effective against cystic fibrosis in patients bearing the most frequent etiological mutation (CFTRΔF508). Here, we investigated the interaction between both agents on cultured respiratory epithelia cells from normal and CFTRΔF508-mutated donors. We observed that the combination of both agents affected metabolic circuits (and in particular the tricarboxylic acid cycle) in a unique way and that cysteamine plus EGCG reduced cytoplasmic protein acetylation more than each of the 2 components alone. In a cell-free system, protein cross-linking activity of EGCG was suppressed by cysteamine. Finally, EGCG was able to enhance the conversion of cysteamine into taurine in metabolic flux experiments. Altogether, these results indicate that multiple pharmacological interactions occur between cysteamine and EGCG, suggesting that they contribute to the unique synergy of both agents in restoring the function of mutated CFTRΔF508.

Metabolic interactions between cysteamine and epigallocatechin gallate

PubMed Central

Izzo, Valentina; Pietrocola, Federico; Sica, Valentina; Durand, Sylvère; Lachkar, Sylvie; Enot, David; Bravo-San Pedro, José Manuel; Chery, Alexis; Esposito, Speranza; Raia, Valeria; Maiuri, Luigi; Maiuri, Maria Chiara; Kroemer, Guido

2017-01-01

ABSTRACT Phase II clinical trials indicate that the combination of cysteamine plus epigallocatechin gallate (EGCG) is effective against cystic fibrosis in patients bearing the most frequent etiological mutation (CFTRΔF508). Here, we investigated the interaction between both agents on cultured respiratory epithelia cells from normal and CFTRΔF508-mutated donors. We observed that the combination of both agents affected metabolic circuits (and in particular the tricarboxylic acid cycle) in a unique way and that cysteamine plus EGCG reduced cytoplasmic protein acetylation more than each of the 2 components alone. In a cell-free system, protein cross-linking activity of EGCG was suppressed by cysteamine. Finally, EGCG was able to enhance the conversion of cysteamine into taurine in metabolic flux experiments. Altogether, these results indicate that multiple pharmacological interactions occur between cysteamine and EGCG, suggesting that they contribute to the unique synergy of both agents in restoring the function of mutated CFTRΔF508. PMID:28059601

Cystic fibrosis mutations in North American populations of French ancestry: Analysis of Quebec French-Canadian and Louisiana Acadian families

PubMed Central

Rozen, Rima; Schwartz, Robert H.; Hilman, Bettina C.; Stanislovitis, Pat; Horn, Glenn T.; Klinger, Katherine; Daigneault, Jocelyne; De Braekeleer, Marc; Kerem, Bat-sheva; Tsui, Lap-Chee; Fujiwara, T. Mary; Morgan, Kenneth

1990-01-01

A 3-bp deletion (ΔF508) in the cystic fibrosis (CF) gene is the mutation on the majority of CF chromosomes. We studied 112 CF families from North American populations of French ancestry: French-Canadian families referred from hospitals in three cities in Quebec and from the Saguenay-Lac St. Jean region of northeastern Quebec and Acadian families living in Louisiana. ΔF508 was present on 71%, 55%, and 70% of the CF chromosomes from the major-urban Quebec, Saguenay-Lac St. Jean, and Louisiana Acadian families, respectively. A weighted estimate of the proportion of ΔF508 in the French-Canadian patient population of Quebec was 70%. We found that 95% of the CF chromosomes with ΔF508 had D7S23 haplotype B, the most frequent haplotype on CF chromosomes. In the Saguenay-Lac St. Jean families, 86% of the CF chromosomes without ΔF508 had the B haplotype, compared with 31% for the major-urban Quebec and Louisiana Acadian families. The incidence of CF in the Saguenay-Lac St. Jean population was 1/895 live-born infants. PMID:2220803

An unexpected effect of TNF-α on F508del-CFTR maturation and function

PubMed Central

Bitam, Sara; Urbach, Valérie; Sermet-Gaudelus, Isabelle; Hinzpeter, Alexandre; Edelman, Aleksander

2015-01-01

Cystic fibrosis (CF) is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene ( CFTR), which encodes a cAMP-dependent Cl - channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT) CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml) of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE) leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC) signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular. PMID:26594334

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.

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.

Identification of eight novel mutations in a collaborative analysis of a part of the second transmembrane domain of the CFTR gene

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

Mercier, B.; Audrezet, M.P.; Guillermit, H.

Cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible, when mutated, for cystic fibrosis (CF), spans over 230 kb on the long arm of chromosome 7 and is composed of 27 exons. The most common mutation responsible for CF worldwide is the deletion of a phenylalanine amino acid at codon 508 in the first nucleotide-binding fold and accounts for approximately 70% of CF chromosomes studied. More than 250 other mutations have been reported through the CF Genetic Analysis Consortium. The majority of the mutations previously described lie in the two nucleotide-binding folds. To explore exhaustively other regions of the gene,more » particularly exons coding for transmembrane domains, the authors have initiated a collaborative study between different laboratories to screen 369 non-[Delta]F508 CF chromosomes of seven ethnic European populations (Belgian, French, Breton, Irish, Italian, Yugoslavian, Russian). Among these chromosomes carrying an unidentified mutation, 63 were from Brittany, 50 of various French origin, 45 of Irish origin, 56 of Italian origin, 41 of Belgian origin, 2 of Turkish origin, 38 of Yugoslavian origin, 22 of Russian origin, and 52 of Bulgarian origin. Diagnostic criteria for CF included at least one positive sweat test and pulmonary disease with or without pancreatic disease. Using a denaturing gradient gel electrophoresis (DGGE) assay, they have identified eight novel mutations in exon 17b coding for part of the second transmembrane domain of the CFTR and they describe them in this report. 8 refs., 1 fig., 1 tab.« less

∆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.

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

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

Pseudomonas aeruginosa Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells

PubMed Central

Stanton, Bruce A.; Coutermarsh, Bonita; Barnaby, Roxanna; Hogan, Deborah

2015-01-01

Background P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE) cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second) by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770. Methods and Results F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del) were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR. Conclusion The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials. PMID:26018799

Pseudomonas aeruginosa Reduces VX-809 Stimulated F508del-CFTR Chloride Secretion by Airway Epithelial Cells.

PubMed

Stanton, Bruce A; Coutermarsh, Bonita; Barnaby, Roxanna; Hogan, Deborah

2015-01-01

P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE) cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second) by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770. F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del) were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR. The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials.

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 American Society for Pharmacology and Experimental Therapeutics.

Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease

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

Shoshani, T.; Bashan, N.; Seret, H.

1992-01-01

Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation ({Delta}F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cysticmore » fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57(60%) are of Ashkenazi origin. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease.« less

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.

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.

Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening.

PubMed

Perone, C; Medeiros, G S; del Castillo, D M; de Aguiar, M J B; Januário, J N

2010-02-01

The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.

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.

Cystic fibrosis transmembrane conductance regulator modulators: precision medicine in cystic fibrosis.

PubMed

Burgener, Elizabeth B; Moss, Richard B

2018-06-01

The aim of this study was to describe the newest development in cystic fibrosis (CF) care, CF transmembrane conductance regulator (CFTR) modulator therapies. Phase II results showing CFTR modulator triple therapies are more effective than current CFTR modulators. CFTR modulator therapy targets the protein defective in CF and boosts its function, but the drug must match mutation pathobiology. Ivacaftor, a CFTR potentiator, was the first modulator approved in 2012, with impressive improvement in lung function and other measures of disease in patients with gating and other residual function mutations (∼10% of CF patients). In 2015, the combination of lumacaftor, a CFTR corrector, and ivacaftor was approved for patients homozygous for the F508del mutation (∼40-50% of the CF population) with positive but less impressive clinical response and 10-20% incidence of intolerance. A next-generation CFTR corrector, tezacaftor, with ivacaftor equally effective and better tolerated than lumacaftor, has also received US Food and Drug Administration approval. Novel CFTR correctors, entering Phase 3 trials in triple modulator combination with tezacaftor-ivacaftor, appear substantially more effective for patients who are homozygous for the F508del mutation and can provide benefit for patients with a single F508del mutation. This offers promise of effective CFTR modulator therapy for nearly 90% of CF patients.

Comparative processing and function of human and ferret cystic fibrosis transmembrane conductance regulator.

PubMed

Fisher, John T; Liu, Xiaoming; Yan, Ziying; Luo, Meihui; Zhang, Yulong; Zhou, Weihong; Lee, Ben J; Song, Yi; Guo, Chenhong; Wang, Yujiong; Lukacs, Gergely L; Engelhardt, John F

2012-06-22

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR(ΔF508/ΔF508) animal model may be useful.

Comparative Processing and Function of Human and Ferret Cystic Fibrosis Transmembrane Conductance Regulator*

PubMed Central

Fisher, John T.; Liu, Xiaoming; Yan, Ziying; Luo, Meihui; Zhang, Yulong; Zhou, Weihong; Lee, Ben J.; Song, Yi; Guo, Chenhong; Wang, Yujiong; Lukacs, Gergely L.; Engelhardt, John F.

2012-01-01

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTRΔF508/ΔF508 animal model may be useful. PMID:22570484

Increasing the Endoplasmic Reticulum Pool of the F508del Allele of the Cystic Fibrosis Transmembrane Conductance Regulator Leads to Greater Folding Correction by Small Molecule Therapeutics.

PubMed

Chung, W Joon; Goeckeler-Fried, Jennifer L; Havasi, Viktoria; Chiang, Annette; Rowe, Steven M; Plyler, Zackery E; Hong, Jeong S; Mazur, Marina; Piazza, Gary A; Keeton, Adam B; White, E Lucile; Rasmussen, Lynn; Weissman, Allan M; Denny, R Aldrin; Brodsky, Jeffrey L; Sorscher, Eric J

2016-01-01

Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ~2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ~10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41-a compound that inhibits the E1 ubiquitin activating enzyme-was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR.

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

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.

A chemical corrector modifies the channel function of F508del-CFTR.

PubMed

Kim Chiaw, Patrick; Wellhauser, Leigh; Huan, Ling Jun; Ramjeesingh, Mohabir; Bear, Christine E

2010-09-01

The deletion of Phe-508 (F508del) constitutes the most prevalent cystic fibrosis-causing mutation. This mutation leads to cystic fibrosis transmembrane conductance regulator (CFTR) misfolding and retention in the endoplasmic reticulum and altered channel activity in mammalian cells. This folding defect can however be partially overcome by growing cells expressing this mutant protein at low (27 degrees C) temperature. Chemical "correctors" have been identified that are also effective in rescuing the biosynthetic defect in F508del-CFTR, thereby permitting its functional expression at the cell surface. The mechanism of action of chemical correctors remains unclear, but it has been suggested that certain correctors [including 4-cyclohexyloxy-2-(1-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-yl]-ethyl)-quinazoline (VRT-325)] may act to promote trafficking by interacting directly with the mutant protein. To test this hypothesis, we assessed the effect of VRT-325 addition on the channel activity of F508del-CFTR after its surface expression had been "rescued" by low temperature. It is noteworthy that short-term pretreatment with VRT-325 [but not with an inactive analog, 4-hydroxy-2-(1-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-yl]-ethyl)-quinazoline (VRT-186)], caused a modest but significant inhibition of cAMP-mediated halide flux. Furthermore, VRT-325 decreased the apparent ATP affinity of purified and reconstituted F508del-CFTR in our ATPase activity assay, an effect that may account for the decrease in channel activity by temperature-rescued F508del-CFTR. These findings suggest that biosynthetic rescue mediated by VRT-325 may be conferred (at least in part) by direct modification of the structure of the mutant protein, leading to a decrease in its ATP-dependent conformational dynamics. Therefore, the challenge for therapy discovery will be the design of small molecules that bind to promote biosynthetic maturation of the major mutant without compromising its activity in vivo.

Effect of the F508del genotype on outcomes of endoscopic sinus surgery in children with cystic fibrosis.

PubMed

Do, Bao Anh Julie; Lands, Larry C; Saint-Martin, Christine; Mascarella, Marco A; Manoukian, John J; Daniel, Sam J; Nguyen, Lily H P

2014-07-01

Numerous authors have sought to describe genotype-phenotype correlations in cystic fibrosis (CF), notably to pancreatic insufficiency and lung disease. However, few studies have focused on the association between the F508del genotype and response to sinus surgery. The objective of this study is to assess the effect of the F508del genotype on sinonasal disease severity and outcomes following functional endoscopic sinus surgery (FESS) in a pediatric population. A retrospective chart review of 153 children with CF seen at a tertiary care pediatric hospital from 1995 to 2008 was performed. Patients were classified into one of three groups according to F508del genotype, either as homozygous, heterozygous or not carrying a F508del mutation. The sinonasal disease phenotype of the three groups was compared based on clinical and radiological findings, extent of endoscopic sinus surgery and rate of revision surgery. The relationship between the F508del genotype and pancreatic insufficiency was confirmed (p<0.05). There was no association between the F508del genotype and increased need for FESS (p=0.75). Moreover, no association was established between F508del homozygosity and presence of nasal polyps, Lund-Mackay score, extent of surgery or length of postoperative hospitalization. The rates of revision surgery did not differ significantly among the three genotypes analyzed (p=0.59). There is no clear association between the F508del genotype and an increased need for FESS, extent of surgery, or revision surgery. Given the phenotypic variability of sinonasal disease in patients with CF, a prospective study is needed to better understand outcomes following FESS and the contribution of gene modifiers to this effect. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).

PubMed

Sargent, R Geoffrey; Suzuki, Shingo; Gruenert, Dieter C

2014-01-01

Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.

Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

PubMed

Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

2013-12-13

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

PubMed Central

Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

2013-01-01

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation.

PubMed

Rubenstein, R C; Lyons, B M

2001-07-01

Intracellular trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) is repaired by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA downregulates protein and mRNA expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family) by approximately 40-50% and decreases formation of a HSC70-DeltaF508 CFTR complex that may be important in the intracellular degradation of DeltaF508 CFTR. We examined the potential mechanisms by which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells, 1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70 promoter or of a human HSC70 promoter fragment in luciferase reporter assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays. In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA degradation by approximately 40%. The initial rate of 35S-HSC70 protein synthesis in 4PBA-treated IB3-1 cells was reduced by approximately 40%, consistent with the steady-state mRNA level, whereas its rate of degradation was unaltered by 4PBA. 4PBA also reduced the steady-state accumulation of (35)S-HSC70 by approximately 40%. These data suggest that 4PBA decreases the expression of HSC70 mRNA and protein by inducing cellular adaptations that result in the decreased stability of HSC70 mRNA.

The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9.

PubMed

Bertrand, Carol A; Mitra, Shalini; Mishra, Sanjay K; Wang, Xiaohui; Zhao, Yu; Pilewski, Joseph M; Madden, Dean R; Frizzell, Raymond A

2017-06-01

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9. Copyright © 2017 the American Physiological Society.

[Hyperechogenic fetal bowel as a marker of fetal cystic fibrosis].

PubMed

Sukupová, M; Dhaifalah, I; Adamík, Z; Havalová, J

2015-01-01

Hyperechogenic bowel (HB) occurs in 0.1 to 1.8% of normal pregnancies. In most cases it has no consequence for the foetus, but can be associated with cystic fibrosis (CF), chromosomal defects, genetic syndromes, viral infections, gastrointestinal pathology, missed gravidity, IUGR and preterm labour. Assessment the risk of the foetus having CF or other abnormalities when HB was detected during ultrasound screening in the second trimester of pregnancy in our centre. Retrospective study. Department of Obstetrics and Gynecology, Centre of Fetal Medicine and Genetics, KNTB a.s. Zlín. Retrospective analysis of 149 cases of HB between 17 to 22 weeks of pregnancy detected from January 2008 to April 2012. HB was evaluated according to its degree of echogenicity (Slotnik/Abuhamed classification), presence or absence of other ultrasound markers and the result of first trimester combined screening result. When stage II or III HB and/or borderline risk in first trimester screening, and presence of other ultrasound markers was detected, amniocentesis (AMC) was performed to investigate the karyotype, mutations in the CFTR gene and presence of viral infections (cytomegalovirus and parvovirus B19). If stage I or II HB and/or negative I. trimester screening and no other ultrasound markers, viral infections and mutations in the CFTR gene were investigated form maternal blood. If positive, paternal blood sampling testing for mutation in the CFTR gene was performed. If a mutation was detected in both parents, AMC was performed. Mutations of the CFTR gene was investigated with a commercial panel of 33 to 50 most common mutations. Postnatally the outcome of neonatal screening for CF(IRT) and any newborns with congenital malformations were ascertained. HB was seen in 149 foetuses, AMC was performed in 94 (63%), and blood sampling in 55 (37%). Two mutations in the CFTR gene associated with a severe form of CF (deltaF508/3849 KBC +10 T) were found in one foetus from the AMC group with stage III HB. The parents decided to terminate the pregnancy. The incidence of HB in our group was 0.7%. In 4 foetuses (2.7%) with stage II HB heterozygous deltaF508 mutation was found, in the rest no mutations were detected. Parents of heterozygous carriers underwent genetic consultation. Postnatal CF screening (IRT level from a heel prick sample) was negative; therefore no further molecular genetic analysis was performed. Infection was detected in three foetuses; one case was managed with intrauterine transfusion and in the other two cases parents decided for termination. Four cases (2.7%) were terminated because of severe congenital anomalies. Minor congenital abnormalities were detected in seven (4.7%) cases. Intrauterine death was detected in three (2%) pregnancies. Based on our results, HB can be considered as a significant marker for the risk of CF, especially in HB stages II and III. It also demonstrates the importance of this marker for the risk of other foetal abnormalities.

Predictors of deterioration of lung function in Polish children with cystic fibrosis.

PubMed

Olszowiec-Chlebna, Małgorzata; Koniarek-Maniecka, Agnieszka; Stelmach, Włodzimierz; Smejda, Katarzyna; Jerzyńska, Joanna; Majak, Paweł; Białas, Monika; Stelmach, Iwona

2016-04-01

Severity of lung disease varies in patients with the same CFTR genotype. It suggests that other factors affect the severity of cystic fibrosis (CF). The aim of the study was to identify risk factors that determine lung function decline in Polish cystic fibrosis children. The follow-up time was no less than 5 years of respiratory status observation based on the forced expiratory volume in 1 s value (FEV1). The socio-economic data, perinatal interview, presence of meconium ileus (MI), time of CF diagnosis, initiation of tobramycin inhalation solution (TIS), pancreatic function, sensitization to Aspergillus fumigatus, presence of impaired glucose tolerance (IGT) or diabetes mellitus, chronic bacterial colonization and number of exacerbations and hospitalizations were assessed. The mean age of 61 included children was 13.3 ±7.6 years. Delta F508 homozygosity was detected in 45.9%, 44.3% were delta F508 heterozygous, and 9.8% had other genotypes. FEV1 decline was observed among 20% of patients; the rest of the patients presented stable values of FEV1 during at least 5 years of observation. The most significant predictors related to the decline of FEV1 were presentation of MI (p = 0.0344), IGT (p = 0.0227), number of exacerbations (p = 0.0288), and early Pseudomonas aeruginosa (PA) chronic colonization (p = 0.0165) followed by late TIS initiation after the first detection of PA (p=0.0071). Neither time of diagnosis nor type of CFTR mutation was statistically significant as a predictor of lung deterioration. The presence of MI, IGT, chronic PA colonization, and number of exacerbations are risk factors for lung function deterioration.

[Analysis of energy expenditure in adults with cystic fibrosis: comparison of indirect calorimetry and prediction equations].

PubMed

Fuster, Casilda Olveira; Fuster, Gabriel Olveira; Galindo, Antonio Dorado; Galo, Alicia Padilla; Verdugo, Julio Merino; Lozano, Francisco Miralles

2007-07-01

Undernutrition, which implies an imbalance between energy intake and energy requirements, is common in patients with cystic fibrosis. The aim of this study was to compare resting energy expenditure determined by indirect calorimetry with that obtained with commonly used predictive equations in adults with cystic fibrosis and to assess the influence of clinical variables on the values obtained. We studied 21 patients with clinically stable cystic fibrosis, obtaining data on anthropometric variables, hand grip dynamometry, electrical bioimpedance, and resting energy expenditure by indirect calorimetry. We used the intraclass correlation coefficient (ICC) and the Bland-Altman method to assess agreement between the values obtained for resting energy expenditure measured by indirect calorimetry and those obtained with the World Health Organization (WHO) and Harris-Benedict prediction equations. The prediction equations underestimated resting energy expenditure in more than 90% of cases. The agreement between the value obtained by indirect calorimetry and that calculated with the prediction equations was poor (ICC for comparisons with the WHO and Harris-Benedict equations, 0.47 and 0.41, respectively). Bland-Altman analysis revealed a variable bias between the results of indirect calorimetry and those obtained with prediction equations, irrespective of the resting energy expenditure. The difference between the values measured by indirect calorimetry and those obtained with the WHO equation was significantly larger in patients homozygous for the DeltaF508 mutation and in those with exocrine pancreatic insufficiency. The WHO and Harris-Benedict prediction equations underestimate resting energy expenditure in adults with cystic fibrosis. There is poor agreement between the values for resting energy expenditure determined by indirect calorimetry and those estimated with prediction equations. Underestimation was greater in patients with exocrine pancreatic insufficiency and patients who were homozygous for DeltaF508.

Rescue of murine F508del CFTR activity in native intestine by low temperature and proteasome inhibitors.

PubMed

Wilke, Martina; Bot, Alice; Jorna, Huub; Scholte, Bob J; de Jonge, Hugo R

2012-01-01

Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.

Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds

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

Doerk, T.; Wulbrand, U.; Tuemmler, B.

1993-03-01

Single cases of the four novel splice site mutations 1525[minus]1 G [r arrow] A (intron 9), 3601[minus]2 A [r arrow] G (intron 18), 3850[minus]3 T [r arrow] G (intron 19), and 4374+1 G [r arrow] T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and Germany descent. The nucleotide substitutions at the +1, [minus]1, and [minus]2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850[minus]3 T-to-G change was discovered in a very mildly affected 33-year-old [Delta]F508 compoundmore » heterozygote, suggesting that the T-to-G transversion at the less conserved [minus]3 position of the acceptor splice site may retain some wildtype function. 13 refs., 1 fig., 2 tabs.« less

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

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

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

Binding screen for cystic fibrosis transmembrane conductance regulator correctors finds new chemical matter and yields insights into cystic fibrosis therapeutic strategy.

PubMed

Hall, Justin D; Wang, Hong; Byrnes, Laura J; Shanker, Suman; Wang, Kelong; Efremov, Ivan V; Chong, P Andrew; Forman-Kay, Julie D; Aulabaugh, Ann E

2016-02-01

The most common mutation in cystic fibrosis (CF) patients is deletion of F508 (ΔF508) in the first nucleotide binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR). ΔF508 causes a decrease in the trafficking of CFTR to the cell surface and reduces the thermal stability of isolated NBD1; it is well established that both of these effects can be rescued by additional revertant mutations in NBD1. The current paradigm in CF small molecule drug discovery is that, like revertant mutations, a path may exist to ΔF508 CFTR correction through a small molecule chaperone binding to NBD1. We, therefore, set out to find small molecule binders of NBD1 and test whether it is possible to develop these molecules into potent binders that increase CFTR trafficking in CF-patient-derived human bronchial epithelial cells. Several fragments were identified that bind NBD1 at either the CFFT-001 site or the BIA site. However, repeated attempts to improve the affinity of these fragments resulted in only modest gains. Although these results cannot prove that there is no possibility of finding a high-affinity small molecule binder of NBD1, they are discouraging and lead us to hypothesize that the nature of these two binding sites, and isolated NBD1 itself, may not contain the features needed to build high-affinity interactions. Future work in this area may, therefore, require constructs including other domains of CFTR in addition to NBD1, if high-affinity small molecule binding is to be achieved. © 2016 The Protein Society.

A new mutation in the CFTR gene, composed of two adjacent DNA alterations, is a common cause of cystic fibrosis among Georgian Jews

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

Shoshani, T.; Berkun, Y.; Yahav, Y.

Five Jewish cystic fibrosis (CF) patients from four unrelated families, all of whom emigrated from what was Soviet Georgia were studied. The parents in two of the families are first-degree relatives. The clinical phenotype of the patients seems to be associated with a severe disease, as reflected by early age of diagnosis (before the age of 1 year), high sweat chloride level (105-140 meq/liter), and pancreatic insufficiency. The pulmonary function and nutritional status of these patients are normal. These patients were tested for [Delta]F508 by analysis of heteroduplex DNA (4). None of the CF chromosomes was found to carry themore » [Delta]F508 mutation. Subsequently, PCR-amplified genomic DNA samples from two of these patients were subjected to direct sequencing (5) of regions containing exons 7, 9-12, an 19-21 of the CF gene using the oligonucleotides previously described (3, 6). In exon 7, two DNA alterations 3 bp apart were identified in both patients. The first alteration in a C [yields] A transversion at nucleotide position 1207, changing the glutamine codon to lysine (Q359K). The second DNA alteration is a C [yields] A transversion at nucleotide position 1211 changing the threonine codon to lysine (T360K). The two DNA alterations cause nonconservative amino acid substitutions, changing each of the two uncharged polar amino acids (glutamine and threonine) to a basic amino acid, lysine. The Q359K substitution destroys an Rsal recognition site and can be detected by PCR amplification of exon 7 using 7i-5 and 7i-3 oligonucleotides (6), followed by Rsal digestion and electrophoresis on 10% polyacrylamide gels. Two Rsal sites are found in a normal amplified DNA fragment, resulting in three restriction fragments of 292, 68, and 50 bp. Digestion of the PCR fragment of an individual homozygous for this substitution resulted in only two fragments of 342 and 68 bp. 6 refs., 3 figs.« less

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.

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

The 3849 + 10 kB C-->T mutation in a 21-year-old patient with cystic fibrosis.

PubMed

Kaplan, D M; Niv, A; Aviram, M; Parvari, R; Leiberman, A; Fliss, D M

1996-12-01

Cystic fibrosis (CF) is the most common lethal inherited disease in the white population. It is characterized by exocrine gland epithelia dysfunction, which leads to pulmonary and pancreatic insufficiency. Since the cloning of the CF gene in 1989 and the identification of the most common CF mutation (delta F508), more than 400 different mutations have been described. These mutations appear to contribute to the heterogeneity of the CF phenotype and several reports have speculated on the relationship between the most common CF mutations and the patient's clinical status. We report the case of a 21-year-old woman with longstanding chronic pansinusitis, nasal polyposis, chronic cough and severe nasal crusting. During a period of five years she had been followed by her otolaryngologist and pediatric pulmonologist. Sweat tests performed at the age of 17 and 18 were within normal limits and she underwent repeated conventional sinonasal procedures, with no improvement in her clinical status. On her present admission, sweat tests showed a 70 meq/l chloride concentration. The diagnosis of CF was then confirmed by DNA analysis and the patient was found to carry the 3849 + 10 kB C-->T mutation. The early detection of this newly recognized form of CF in adults as well as in children presenting with sinonasal symptoms is critical for life expectancy and quality.

Genetic and clinical features of false-negative infants in a neonatal screening programme for cystic fibrosis.

PubMed

Padoan, R; Genoni, S; Moretti, E; Seia, M; Giunta, A; Corbetta, C

2002-01-01

A study was performed on the delayed diagnosis of cystic fibrosis (CF) in infants who had false-negative results in a neonatal screening programme. The genetic and clinical features of false-negative infants in this screening programme were assessed together with the efficiency of the screening procedure in the Lombardia region. In total, 774,687 newborns were screened using a two-step immunoreactive trypsinogen (IRT) (in the years 1990-1992), IRT/IRT + delF508 (1993-1998) or IRT/IRT + polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) protocol (1998-1999). Out of 196 CF children born in the 10 y period 15 were false negative on screening (7.6%) and molecular analysis showed a high variability in the genotypes. The cystic fibrosis transmembrane regulator (CFTR) gene mutations identified were delF508, D1152H, R1066C, R334W, G542X, N1303K, F1052V, A120T, 3849 + 10kbC --> T, 2789 + 5G --> A, 5T-12TG and the novel mutation D110E. In three patients no mutation was identified after denaturing gradient gel electrophoresis of the majority of CFTR gene exons. The clinical phenotypes of CF children diagnosed by their symptoms at different ages were very mild. None of them presented with a severe lung disease. The majority of them did not seem to have been damaged by the delayed diagnosis. The combination of IRT assay plus genotype analysis (1998-1999) appears to be a more reliable method of detecting CF than IRT measurement alone or combined with only the delF508 mutation.

Impact of Gene Patents and Licensing Practices on Access to Genetic Testing for Cystic Fibrosis

PubMed Central

Chandrasekharan, Subhashini; Heaney, Christopher; James, Tamara; Conover, Chris; Cook-Deegan, Robert

2010-01-01

Cystic fibrosis (CF) is one of the most commonly tested autosomal recessive disorders in the US. Clinical CF is associated with mutations in the CFTR gene, of which the most common mutation among Caucasians, ΔF508, was identified in 1989. The University of Michigan, Johns Hopkins University, and the Hospital for Sick Children, where much of the initial research occurred, hold key patents for CF genetic sequences, mutations and methods for detecting them. Several patents including the one that covers detection of the ΔF508 mutation are jointly held by the University of Michigan and the Hospital for Sick Children in Toronto, with Michigan administering patent licensing in the US. The University of Michigan broadly licenses the ΔF508 patent for genetic testing with over 60 providers of genetic testing to date. Genetic testing is now used in newborn screening, diagnosis, and reproductive decisions. Interviews with key researchers and intellectual property managers, a survey of laboratories’ prices for CF genetic testing, a review of literature on CF tests’ cost effectiveness, and a review of the developing market for CF testing provide no evidence that patents have significantly hindered access to genetic tests for CF or prevented financially cost-effective screening. Current licensing practices for cystic fibrosis (CF) genetic testing appear to facilitate both academic research and commercial testing. More than one thousand different CFTR mutations have been identified, and research continues to determine their clinical significance. Patents have been nonexclusively licensed for diagnostic use, and have been variably licensed for gene transfer and other therapeutic applications. The Cystic Fibrosis Foundation has been engaged in licensing decisions, making CF a model of collaborative and cooperative patenting and licensing practice. PMID:20393308

A novel MPL point mutation resulting in thrombopoietin-independent activation.

PubMed

Abe, M; Suzuki, K; Inagaki, O; Sassa, S; Shikama, H

2002-08-01

Thrombopoietin (TPO) and its receptor (MPL) are important regulators of megakaryopoiesis. MPL belongs to a cytokine receptor superfamily. To date, all constitutively active MPL mutants have been artificially constructed with amino acid substitutions in the transmembrane domain or extracellular domain of the protein, and they activate signal transduction pathways in Ba/F3 cells that can also be activated by the normal MPL. In this paper, we report a novel spontaneously occurring mutation of MPL, with an amino acid substitution of Trp(508) to Ser(508) in the intracellular domain of MPL, that induces the factor-independent growth of Ba/F3 cells. Examination of intracellular signaling pathways demonstrated that the mutant MPL protein constitutively activates three distinct signaling pathways, SHC-Ras-Raf-MAPK/JNK, JAK-STAT, and PI3K-Akt-Bad.

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.

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

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Enhances Activation of p38 and ERK MAPKs, Increasing Interleukin-6 Synthesis in Airway Epithelial Cells Exposed to Pseudomonas aeruginosa*

PubMed Central

Bérubé, Julie; Roussel, Lucie; Nattagh, Leila; Rousseau, Simon

2010-01-01

In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRΔF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRΔF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRΔF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRΔF508 mutation in airway epithelial cells contributes to increase inflammation of the airways. PMID:20460375

Ivacaftor and sinonasal pathology in a cystic fibrosis patient with genotype deltaF508/S1215N.

PubMed

Vreede, C L; Berkhout, M C; Sprij, A J; Fokkens, W J; Heijerman, H G M

2015-05-01

In patients with Cystic Fibrosis and a type III mutation, ivacaftor (Kalydeco(®), Vertex) can increase the opening time of the CFTR channel and improve chloride transport. Research showed significant improvement of lung function and increase in weight following ivacaftor use. However, ivacaftor showed to have adverse events on the sinonasal system as well, such as upper respiratory tract infections, nasal congestion and headaches. This case report showed a positive effect of ivacaftor on the sinonasal pathology in a 17 year old patient with CF. After 5 months of ivacaftor use, the CT-sinus showed complete resolution of the opacification of the paranasal sinuses and a decrease in symptoms of sinonasal disease. This positive effect of ivacaftor on sinonasal pathology seems promising, therefore more research is needed to evaluate the effect of ivacaftor on the upper airways in CF. Copyright © 2014. Published by Elsevier B.V.

Mutations of the cystic fibrosis gene, but not cationic trypsinogen gene, are associated with recurrent or chronic idiopathic pancreatitis.

PubMed

Ockenga, J; Stuhrmann, M; Ballmann, M; Teich, N; Keim, V; Dörk, T; Manns, M P

2000-08-01

We investigated whether mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and cationic trypsinogen gene are associated with recurrent acute, or chronic idiopathic pancreatitis. Twenty patients with idiopathic pancreatitis (11 women, nine men; mean age, 30 yr) were studied for the presence of a CFTR mutation by screening the genomic DNA for more than 30 mutations and variants in the CFTR gene. Selected mutations of the cationic trypsinogen gene were screened by Afl III restriction digestion or by a mutation-specific polymerase chain reaction (PCR). In each patient exons 1, 2, and 3 of the cationic trypsinogen gene were sequenced. Patients with a CFTR mutation underwent evaluation of further functional electrophysiological test (intestinal current measurement). No mutation of the cationic trypsinogen gene was detected. A CFTR mutation was detected in 6/20 (30.0%) patients. Three patients (15.0%) had a cystic fibrosis (CF) mutation on one chromosome (deltaF508, I336K, Y1092X), which is known to cause phenotypical severe cystic fibrosis. One patient was heterozygous for the 5T allele. In addition, two possibly predisposing CFTR variants (R75Q, 1716G-->A) were detected on four patients, one of these being a compound heterozygous for the missense mutation I336K and R75Q. No other family member (maternal I336K; paternal R75Q; sister I1336K) developed pancreatitis. An intestinal current measurement in rectum samples of patients with a CFTR mutation revealed no CF-typical constellations. CFTR mutations are associated with recurrent acute, or chronic idiopathic pancreatitis, whereas mutations of the cationic trypsinogen mutation do not appear to be a frequent pathogenetic factor.

Spectrum of CFTR gene mutations in Ecuadorian cystic fibrosis patients: the second report of the p.H609R mutation.

PubMed

Ortiz, Sofía C; Aguirre, Santiago J; Flores, Sofía; Maldonado, Claudio; Mejía, Juan; Salinas, Lilian

2017-11-01

High heterogeneity in the CFTR gene mutations disturbs the molecular diagnosis of cystic fibrosis (CF). In order to improve the diagnosis of CF in our country, the present study aims to define a panel of common CFTR gene mutations by sequencing 27 exons of the gene in Ecuadorian Cystic Fibrosis patients. Forty-eight Ecuadorian individuals with suspected/confirmed CF diagnosis were included. Twenty-seven exons of CFTR gene were sequenced to find sequence variations. Prevalence of pathogenic variations were determined and compared with other countries' data. We found 70 sequence variations. Eight of these are CF-causing mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. Also this study is the second report of p.H609R in Ecuadorian population. Mutation prevalence differences between Ecuadorian population and other Latin America countries were found. The panel of mutations suggested as an initial screening for the Ecuadorian population with cystic fibrosis should contain the mutations: p.F508del, p.G85E, p.G330E, p.A455E, p.G970S, W1098X, R1162X, and N1303K. © 2017 NETLAB Laboratorios Especializados. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770

PubMed Central

Van Goor, Fredrick; Hadida, Sabine; Grootenhuis, Peter D. J.; Burton, Bill; Cao, Dong; Neuberger, Tim; Turnbull, Amanda; Singh, Ashvani; Joubran, John; Hazlewood, Anna; Zhou, Jinglan; McCartney, Jason; Arumugam, Vijayalaksmi; Decker, Caroline; Yang, Jennifer; Young, Chris; Olson, Eric R.; Wine, Jeffery J.; Frizzell, Raymond A.; Ashlock, Melissa; Negulescu, Paul

2009-01-01

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (Po) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl− secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by ≈10-fold, to ≈50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na+ and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway. PMID:19846789

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.

Orkambi® and amplifier co-therapy improves function from a rare CFTR mutation in gene-edited cells and patient tissue.

PubMed

Molinski, Steven V; Ahmadi, Saumel; Ip, Wan; Ouyang, Hong; Villella, Adriana; Miller, John P; Lee, Po-Shun; Kulleperuma, Kethika; Du, Kai; Di Paola, Michelle; Eckford, Paul Dw; Laselva, Onofrio; Huan, Ling Jun; Wellhauser, Leigh; Li, Ellen; Ray, Peter N; Pomès, Régis; Moraes, Theo J; Gonska, Tanja; Ratjen, Felix; Bear, Christine E

2017-09-01

The combination therapy of lumacaftor and ivacaftor (Orkambi ® ) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi ® could treat patients with rarer mutations of similar "theratype"; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function-similar to ΔF508-CFTR, are unlikely to yield a robust Orkambi ® response. While  in silico  and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi ® response. Importantly, this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi ® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach, including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

The Frequency of CCR5del32 Mutation in Populations of Russians, Tatars and Bashkirs of Chelyabinsk Region, Russia.

PubMed

Govorovskaya, Irina; Khromova, Elena; Suslova, Tatiana; Alexeev, Leonid; Kofiadi, Ilya

2016-12-01

The distribution of genetic variants associated with natural resistance to viral infections can vary among human ethnic groups due to evolutionary factors, defining the different epidemiologic background of world populations. The polymorphisms, defining the natural resistance to HIV-infection and the rate of progression up to AIDS, are very important since epidemic is still on rise. We have studied the distribution of allele and genotype frequencies of CCR5delta32 mutation in major populations inhabiting Chelyabinsk region of the Russian Federation. Genetic survey included the population of 509 potential blood marrow donors: Russians (N = 300), Bashkirs (N = 118) and Tatars (N = 91). The genotyping assay was performed using real-time polymerase chain reaction (real-time PCR). The genotypes were defined by melting curve analysis. The CCR5delta32 allele and CCR5delta32/delta32 genotype are presented in population of Russians in Chelyabinsk region with the frequencies of F x  = 10.83% and P x  = 1.67, for the CCR5delta32 allele and its homozygosity, respectively. In populations of Bashkirs and Tatars CCR5delta32 allele and CCR5delta32/delta32 genotype are presented at lower frequencies of F x  = 6.36%/P x  = 0.85 and F x  = 7.14%/P x  = 1.10, respectively. These data are consistent with the theory of northern origin of the CCR5delta32 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.

The CF Canada-Sick Kids Program in individual CF therapy: A resource for the advancement of personalized medicine in CF.

PubMed

Eckford, Paul D W; McCormack, Jacqueline; Munsie, Lise; He, Gengming; Stanojevic, Sanja; Pereira, Sergio L; Ho, Karen; Avolio, Julie; Bartlett, Claire; Yang, Jin Ye; Wong, Amy P; Wellhauser, Leigh; Huan, Ling Jun; Jiang, Jia Xin; Ouyang, Hong; Du, Kai; Klingel, Michelle; Kyriakopoulou, Lianna; Gonska, Tanja; Moraes, Theo J; Strug, Lisa J; Rossant, Janet; Ratjen, Felix; Bear, Christine E

2018-04-20

Therapies targeting certain CFTR mutants have been approved, yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal, the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a "first of its kind", comprehensive resource containing patient-specific cell cultures and data from 100 CF individuals that will enable modeling of therapeutic responses. The CFIT program is generating: 1) nasal cells from drug naïve patients suitable for culture and the study of drug responses in vitro, 2) matched gene expression data obtained by sequencing the RNA from the primary nasal tissue, 3) whole genome sequencing of blood derived DNA from each of the 100 participants, 4) induced pluripotent stem cells (iPSCs) generated from each participant's blood sample, 5) CRISPR-edited isogenic control iPSC lines and 6) prospective clinical data from patients treated with CF modulators. To date, we have recruited 57 of 100 individuals to CFIT, most of whom are homozygous for F508del (to assess in-vitro: in-vivo correlations with respect to ORKAMBI response) or heterozygous for F508del and a minimal function mutation. In addition, several donors are homozygous for rare nonsense and missense mutations. Nasal epithelial cell cultures and matched iPSC lines are available for many of these donors. This accessible resource will enable development of tools that predict individual outcomes to current and emerging modulators targeting F508del-CFTR and facilitate therapy discovery for rare CF causing mutations. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

PubMed Central

Grove, Diane E.; Houck, Scott A.

2011-01-01

Cystic fibrosis (CF) is a lethal recessive genetic disease caused by mutations in the CFTR gene. The gene product is a PKA-regulated anion channel that is important for fluid and electrolyte transport in the epithelia of lung, gut, and ducts of the pancreas and sweat glands. The most common CFTR mutation, ΔF508, causes a severe, but correctable, folding defect and gating abnormality, resulting in negligible CFTR function and disease. There are also a large number of rare CF-related mutations where disease is caused by CFTR misfolding. Yet the extent to which defective biogenesis of these CFTR mutants can be corrected is not clear. CFTRV232D is one such mutant that exhibits defective folding and trafficking. CFTRΔF508 misfolding is difficult to correct, but defective biogenesis of CFTRV232D is corrected to near wild-type levels by small-molecule folding correctors in development as CF therapeutics. To determine if CFTRV232D protein is competent as a Cl− channel, we utilized single-channel recordings from transfected human embryonic kidney (HEK-293) cells. After PKA stimulation, CFTRV232D channels were detected in patches with a unitary Cl− conductance indistinguishable from that of CFTR. Yet the frequency of detecting CFTRV232D channels was reduced to ∼20% of patches compared with 60% for CFTR. The folding corrector Corr-4a increased the CFTRV232D channel detection rate and activity to levels similar to CFTR. CFTRV232D-corrected channels were inhibited with CFTRinh-172 and stimulated fourfold by the CFTR channel potentiator VRT-532. These data suggest that CF patients with rare mutations that cause CFTR misfolding, such as CFTRV232D, may benefit from treatment with folding correctors and channel potentiators in development to restore CFTRΔF508 function. PMID:21642448

A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype

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

Chillon, M.; Casals, T.; Gimenez, J.

1995-03-01

mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6bA{yields}G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA{r_arrow}G-mRNA was 5-10-fold less abundant than {triangle}F508 mRNA. Mutations 1811+1.6kbA{yields}G was found in 21 Spanish and 1 German CF chromosome(s), making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype {triangle}F508/1811+1.6kbA{yields}G have only 1%-3% of normal CFTRmore » mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. 30 refs., 3 figs., 2 tabs.« less

Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients

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

Mercier, B.; Verlingue, C.; Audrezet, M.P.

1995-01-01

Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of sterility in men. Although the genetic basis of this condition is still unclear, it has been shown recently that some of these patients carry mutations in their cystic fibrosis transmembrane conductance regulator (CFTR) genes. To extend this observation, we have analyzed the entire coding sequence of the CFTR gene in a cohort of 67 men with CBAVD, who are otherwise healthy. We have identified four novel missense mutations (A800G, G149R, R258G, and E193K). We have shown that 42% of subjects were carriers of one CFTR allele andmore » that 24% are compound heterozygous for CFTR alleles. Thus, we have been unable to identify 76% of these patients as carrying two CFTR mutations. Furthermore, we have described the segregation of CFTR haplotypes in the family of one CBAVD male; in this family are two male siblings, with identical CFTR loci but displaying different phenotypes, one of them being fertile and the other sterile. The data presented in this family, indicating a discordance between the CBAVD phenotype and a marked carrier ({delta}F508) chromosome, support the involvement of another gene(s), in the etiology of CBAVD. 35 refs., 2 figs., 1 tab.« less

F508del-CFTR rescue: a matter of cell stress response.

PubMed

Nieddu, Erika; Pollarolo, Benedetta; Merello, Luisa; Schenone, Silvia; Mazzei, Mauro

2013-01-01

Cystic fibrosis (CF) is a common inherited fatal disease affecting 70,000 people worldwide, with a median predicted age of survival of approximately 38 years. The deletion of Phenylalanine in position 508 of the Cystic Fibrosis Transmembrane conductance Regulator (F508del-CFTR) is the most common mutation in CF patients: the deleted protein, not properly folded, is degraded. To date no commercial drugs are available. Low temperature, some osmolytes and conditions able to induce heat shock protein 70 (Hsp70) expression and heat shock cognate 70 (Hsc70) inhibition result in F508del-CFTR rescue, hence restoring its physiological function: this review sheds light on the correlation between these several evidences. Interestingly, all these approaches have a role in the cell stress response (CSR), a set of cell reactions to stress. In addition, unpredictably, F508del-CFTR rescue has to be considered in the frame of CSR: entities that induce - or are induced during - the CSR are, in general, also able to correct trafficking defect of CFTR. Specifically, the low temperature induces, by definition, a CSR; osmolytes, such as glycerol and trimethylamine N-oxide (TMAO), are products of the CSR; pharmacological correctors, such as Matrine and 4-phenylbutirric acid (4PBA), down-regulate the constitutive Hsc70 in favor of an up-regulation of the inducible chaperone Hsp70, another component of the CSR. The identification of a common mechanism of action for different types of correctors could drive the discovery of new active molecules in CF, overcoming methods clinically inapplicable, such as the low temperature.

Prevalence of meconium ileus marks the severity of mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene.

PubMed

Dupuis, Annie; Keenan, Katherine; Ooi, Chee Y; Dorfman, Ruslan; Sontag, Marci K; Naehrlich, Lutz; Castellani, Carlo; Strug, Lisa J; Rommens, Johanna M; Gonska, Tanja

2016-04-01

Meconium ileus (MI) is a perinatal complication in cystic fibrosis (CF), which is only minimally influenced by environmental factors. We derived and examined MI prevalence (MIP) scores to assess CFTR phenotype-phenotype correlation for severe mutations. MIP scores were established using a Canadian CF population (n = 2,492) as estimates of the proportion of patients with MI among all patients carrying the same CFTR mutation, focusing on patients with p.F508del as the second allele. Comparisons were made to the registries from the US CF Foundation (n = 43,432), Italy (Veneto/Trentino/Alto Adige regions) (n = 1,788), and Germany (n = 3,596). The prevalence of MI varied among the different registries (13-21%). MI was predominantly prevalent in patients with pancreatic insufficiency carrying "severe" CFTR mutations. In this severe spectrum MIP scores further distinguished between mutation types, for example, G542X (0.31) with a high, F508del (0.22) with a moderate, and G551D (0.08) with a low MIP score. Higher MIP scores were associated with more severe clinical phenotypes, such as a lower forced expiratory volume in 1 second (P = 0.01) and body mass index z score (P = 0.04). MIP scores can be used to rank CFTR mutations according to their clinical severity and provide a means to expand delineation of CF phenotypes.Genet Med 18 4, 333-340.

Novel methods to enhance single strand conformation polymorphism (SSCP) senstivity and efficiency: Application to mutation detection in cystic fibrosis (CF)

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

Hagstrom, D.J.; Snow, K.; Yuan, Z.

1994-09-01

For single gene defects in which there are a variety of mutations with significant frequencies, it is a challenge to find an efficient and sensitive method for mutation detection. For example, although 70% to 75% of CF chromosomes in a North American Caucasian population have the mutation {delta}F508, more than 400 mutations (mostly single base pair substitutions) are represented on the remaining chromosomes. SSCP analysis is a relatively straightforward procedure and therefore suitable for routine use in a clinical laboratory. However, previous reports have demonstrated suboptimal sensitivity rates in screening for mutations. We have developed a novel set of conditionsmore » which greatly enhances sensitivity and efficiency of SSCP. Our protocol incorporates multiplex PCR, stepping of wattages during electrophoresis and increased salt concentration at the anode relative to the gel. To screen for mutations in the CFTR gene, three multiplex PCR reactions are performed using identical thermocycler parameters. Sizes of PCR products range from 441 bp to 196 bp: size differences of > 30 bp are necessary to ensure separation during electrophoresis. All PCR products are separated by electrophoresis at room temperature on a single gel containing 8% (37.5:1) polyacrylamide, 5% glycerol and 1x TBE. Using an anode buffer with increased salt (2x TBE) sharpens smaller sized bands, and stepping watts from 5W to 20W during electrophoresis enhances sensitivity. Positive controls were used to demonstrate that mutations could be detected. Other mutations or polymorphisms were verified by cycle sequencing of PCR products or by alternative PCR-based assays for the more common mutations. Thus, using 3 PCR reactions per patient and one gel condition, we are able to achieve a CF mutation detection rate of approximately 90% in a North American Caucasian population.« less

Preconceptional identification of cystic fibrosis carriers in the Sardinian population: A pilot screening program.

PubMed

Coiana, Alessandra; Faa', Valeria; Carta, Daniela; Puddu, Rosalba; Cao, Antonio; Rosatelli, Maria Cristina

2011-05-01

In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable population for a preconceptional screening. Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia. We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%), followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele. In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the preconceptional screening program and the high participation rate of the Sardinian population (99%). Copyright © 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Short communication: novel truncating mutations in the CFTR gene causing a severe form of cystic fibrosis in Italian patients.

PubMed

Lenarduzzi, S; Morgutti, M; Crovella, S; Coiana, A; Rosatelli, M C

2014-11-14

Cystic fibrosis (CF) is a common recessive genetic disease caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. More than 1800 different mutations have been described to date. Here, we report 3 novel mutations in CFTR in 3 Italian CF patients. To detect and identify 36 frequent mutations in Caucasians, we used the INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn Update kits (Innogenetics; Ghent, Belgium). Our first analysis did not reveal both of the responsible mutations; thus, direct sequencing of the CFTR gene coding region was performed. The 3 patients were compound heterozygous. In one allele, the F508del (c.1521_1523delCTT, p.PHE508del) mutation in exon 11 was observed in each case. For the second allele, in patient No.1, direct sequencing revealed an 11-base pair deletion (GAGGCGATACT) in exon 14 (c.2236_2246del; pGlu746Alafs*29). In patient No. 2, direct sequencing revealed a nonsense mutation at nucleotide 3892 (c.3892G>T) in exon 24. In patient No. 3, direct sequencing revealed a deletion of cytosine in exon 27 (c.4296delC; p.Asn1432Lysfs*16). These 3 novel mutations indicate the production of a truncated protein, which consequently results in a non-functional polypeptide.

Is acute recurrent pancreatitis in children a precursor of chronic pancreatitis? A long-term follow-up study of 93 cases.

PubMed

Poddar, Ujjal; Yachha, Surender K; Borkar, Vibhor; Srivastava, Anshu

2017-07-01

In view of paucity of literature we analyzed our experience of acute recurrent pancreatitis (ARP) to study clinical profile and long-term outcome. Over 13 years, 93 consecutive children (≤18 years) diagnosed to have ARP were included in this study. Magnetic resonance cholangiopancreatography was done at baseline and on follow-up. Common mutations for serine-protease-inhibitor (SPINK1 N34S), protease inhibitor (PRSS1 R122S) and cystic fibrosis transmembrane conductance regulator (CFTR deltaF508, 5T) were studied in 22 idiopathic cases. The median age of the children with ARP was 13 (10-14.5) years, 53 were males. Etiology included biliary in 14 (15%), pancreas divisum in 6 (7%), others in 3 (3.5%) and idiopathic in the remaining 70 (75%). SPINK1 mutation was found in 10/22 (45%) cases. Over a median follow-up of 25.5 (8.25-48) months, 37 (42%) of 88 (5 lost to follow-up) developed chronic pancreatitis (CP). On multivariate analysis idiopathic etiology (p<0.03), presence of SPINK1 mutation (p=0.01), longer follow-up (p<0.001) were associated with progression to CP. Biliopancreatic structural/obstructive causes should always be looked for. It seems ARP is a precursor of CP and progression is associated with idiopathic etiology and presence of genetic mutations. Hence, patients with ARP should be kept on regular follow-up to detect CP. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Simple image-based no-wash method for quantitative detection of surface expressed CFTR

PubMed Central

Larsen, Mads Breum; Hu, Jennifer; Frizzell, Raymond A.; Watkins, Simon C.

2016-01-01

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR’s anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins. PMID:26361332

Molecular analysis of patients with {Beta}-glucuronidase deficiency presenting as hydrops fetalis or as early mucopolysaccharidosis VII

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

Vervoort, R.; Liebaers, I.; Lissens, W.

1996-03-01

Although not all mucopolysaccharidosis type VII (MPS VII) neonates present with hydrops fetalis or with related symptoms, hydrops fetalis is a common form of presentation of this mucopolysaccharidosis. We used reverse-transcription-PCR-SSCP and direct sequencing to screen for mutations in the human {beta}-glucuronidase cDNA of 17 MPS VII patients with severe presentation of the disease. Mutations resulting in an unstable mRNA were detected in genomic DNA with direct sequencing of the PCR-amplified {beta}-glucuronidase exons. We found extensive genetic heterogeneity in MPS VII alleles: in addition to 6 of 12 previously reported mutations (L176F, R216W, R357X, R382C, W507X, and W627C), we detectedmore » 14 undescribed mutations in the {beta}-glucuronidase coding region that produce MPS VII alleles (G136R, E150K, S312X, Y320S, Y320C, H351Y, R382H, R374C, R435P, R477W, G572D, Y508C, K606N, and 1900{Delta}GA). The mutations in hydropic fetuses were widely scattered in the {beta}-glucuronidase gene. Analysis of three polymorphic sites of the mutant alleles (1766T/C, 1972C/T, and a new 1091+27C/G polymorphism) allowed exclusion of identity by descent for some recurrent mutations. Three of four mutations introducing a premature translation stop codon were found to affect mRNA abundance and/or structure. Expression studies provided evidence for the causal relationship between each of the mutations found in MPS VII alleles and the enzyme deficiency, in that all mutations identified exhibited markedly reduced enzyme activity expressed in COS7 cells following transfection with the mutant cDNA. 52 refs., 4 figs., 5 tabs.« less

Molecular analysis of patients with beta-glucuronidase deficiency presenting as hydrops fetalis or as early mucopolysaccharidosis VII.

PubMed Central

Vervoort, R.; Islam, M. R.; Sly, W. S.; Zabot, M. T.; Kleijer, W. J.; Chabas, A.; Fensom, A.; Young, E. P.; Liebaers, I.; Lissens, W.

1996-01-01

Although not all mucopolysaccharidosis type VII (MPS VII) neonates present with hydrops fetalis or with related symptoms, hydrops fetalis is a common form of presentation of this mucopolysaccharidosis. We used reverse-transcription-PCR-SSCP and direct sequencing to screen for mutations in the human beta-glucuronidase cDNA of 17 MPS VII patients with severe presentation of the disease. Mutations resulting in an unstable mRNA were detected in genomic DNA with direct sequencing of the PCR-amplified beta-glucuronidase exons. We found extensive genetic heterogeneity in MPS VII alleles: in addition to 6 or 12 previously reported mutations (L176F, R216W, R357X, R382C, W507X, and W627C), we detected 14 undescribed mutations in the beta-glucuronidase coding region that produce MPS VII alleles (G136R, E150K, S312X, Y320S, Y320C, H351Y, R382H, R374C, R435P, R477W, G572D, Y508C, K606N and 1900 delta GA). The mutations in hydropic fetuses were widely scattered in the beta-glucuronidase gene. Analysis of three polymorphic sites of the mutant alleles (1766T/C, 1972C/T and a new 1091+27C/G polymorphism) allowed exclusion of identity by descent for some recurrent mutations. Three of four mutations introducing a premature translation stop codon were found to affect mRNA abundance and/or structure. Expression studies provided evidence for the causal relationship between each of the mutations found in MPS VII alleles and the enzyme deficiency, in that all mutations identified exhibited markedly reduced enzyme activity expressed in COS7 cells following transfection with the mutant cDNA. Images Figure 2 Figure 3A Figure 3BC Figure 4 PMID:8644704

[Clinical significance of JAK2、CALR and MPL gene mutations in 1 648 Philadelphia chromosome negative myeloproliferative neoplasms patients from a single center].

PubMed

Li, M Y; Chao, H Y; Sun, A N; Qiu, H Y; Jin, Z M; Tang, X W; Han, Y; Fu, C C; Chen, S N; Wu, D P

2017-04-14

Objective: To explore the prevalences of JAK2, CALR and MPL gene mutations and the mutation types in patients with Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) , and to compare their clinical characteristics of different mutation types with each other and mutation negative group. Methods: The mutations of JAK2 V617F, JAK2 gene at exon 12, CALR gene at exon 9 and MPL gene at exon 10 in 1 648 Ph negative MPNs patients were detected by direct sequencing. Results: ① The JAK2V617F mutation was found in 471 (92.7%) of 508 PV patients, 819 (78.1%) of 1 049 ET patients and 74 (81.3%) of 91 PMF patients respectively, with the total mutation rate as 82.8% (1 364/1 648) . The JAK2 exon12 mutation was found in 9 (1.7%) of 508 PV patients, none was found in ET or PMF patients, with the total mutation rate as 0.5% (9/1 648) . The CALR mutation was found in 132 (12.6%) of 1 049 ET patients and 11 (12.1%) of 91 PMF patients respectively, with the total mutation rate as 8.7% (143/1 648) ; the MPL mutation was found in 9 (0.9%) of 1 049 ET patients and 1 (1.1%) of 91 PMF patients respectively, with the total mutation rate as 0.6% (10/1 648) . The co-occurrence of any two types of driver gene mutations was not detected by direct sequencing. ②The median onset age of patients with JAK2V617F[61 (15-95) y] was significant higher than of with JAK2 exon12 mutation[49 (33-62) y] or without mutations[42 (3-78) y] ( P <0.001) , but not for patients with CALR[57 (17-89) y] or MPL mutation[59 (22-71) y] ( P >0.05) . Patients with JAK2V617F had higher white blood cell count and hemoglobin level ( P <0.05) when compared with patients with CALR mutation or without mutations, or only significantly higher white blood cell count when compared with patients with MPL mutation ( P =0.013) . The platelet count of patients with CALR mutation was significantly higher than of with JAK2V617F[966 (400-2 069) ×10(9)/L vs 800 (198-3 730) ×10(9)/L, P <0.001]. ③Karyotype analysis was conducted in 1 160 patients with MPNs, the rates of karyotype abnormality of patients with and without CALR mutation were 9.8% (8/82) and 7.4% (80/1 078) ( P =0.441) respectively; The rates of karyotype abnormality of patients with and without JAK2V617F mutation were 7.7% (75/971) and 6.9% (13/189) ( P =0.688) respectively. The incidence of karyotype abnormality of patients with CALR mutation was higher than of with JAK2V617F[9.8% (8/82) vs 7.7% (75/971) ] without statistically significant difference ( P =0.512) . The karyotype analysis of 7 cases of JAK2 exon12 mutation and 6 ones with MPL gene mutation revealed normal karyotype. Conclusions: Driver gene mutations detection could ensure the diagnosis and prognosis judgment of MPN more reliable, different subtypes of MPNs had different profiles of driver gene mutations, the latter lead to unique clinical phenotype.

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product.

PubMed

Haggie, Peter M; Phuan, Puay-Wah; Tan, Joseph-Anthony; Xu, Haijin; Avramescu, Radu G; Perdomo, Doranda; Zlock, Lorna; Nielson, Dennis W; Finkbeiner, Walter E; Lukacs, Gergely L; Verkman, Alan S

2017-01-20

W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR 1281 , without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR 1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR 1281 , functional screens were done of ∼30,000 synthetic small molecules and drugs/nutraceuticals in CFTR 1281 -transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ∼5-fold greater efficacy than VX-809, some of which were selective for CFTR 1281 , whereas others also corrected ΔF508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR 1281 function ∼8-fold over that of VX-770 alone, normalizing CFTR 1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ΔF508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product*♦

PubMed Central

Haggie, Peter M.; Phuan, Puay-Wah; Tan, Joseph-Anthony; Xu, Haijin; Avramescu, Radu G.; Perdomo, Doranda; Zlock, Lorna; Nielson, Dennis W.; Finkbeiner, Walter E.; Lukacs, Gergely L.; Verkman, Alan S.

2017-01-01

W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR1281, without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR1281, functional screens were done of ∼30,000 synthetic small molecules and drugs/nutraceuticals in CFTR1281-transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ∼5-fold greater efficacy than VX-809, some of which were selective for CFTR1281, whereas others also corrected ΔF508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR1281 function ∼8-fold over that of VX-770 alone, normalizing CFTR1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ΔF508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects. PMID:27895116

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.

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

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

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

Cystic fibrosis identified by neonatal screening: incidence, genotype, and early natural history.

PubMed

Green, M R; Weaver, L T; Heeley, A F; Nicholson, K; Kuzemko, J A; Barton, D E; McMahon, R; Payne, S J; Austin, S; Yates, J R

1993-04-01

The incidence of cystic fibrosis over the last 10 years in East Anglia (a region of the United Kingdom with a population of 2.1 million) has halved. This has happened during the establishment of a neonatal screening programme, which has enabled early diagnosis, genetic counselling, and lately the option of prenatal diagnosis in subsequent pregnancies. One hundred and seven children were born with cystic fibrosis between 1981 and 1990, eight of whom were siblings. The Guthrie blood spots of 82 infants detected by neonatal immunoreactive trypsin screening between 1981 and 1990 were examined for the presence of the most common cystic fibrosis gene mutation (delta F508). It was present in 135 (82%) of the 164 cystic fibrosis genes analysed with 54 (66%) cases being homozygous and 27 (33%) heterozygous. Sixty nine per cent of infants were symptomatic at the time of diagnosis regardless of genotype. No association was found between the early clinical or biochemical features of the disease and homozygosity or heterozygosity for this mutation. Screening for cystic fibrosis using the blood immunoreactive trypsin assay alone remains an effective method of identifying infants with the disease soon after birth, thereby allowing early therapeutic intervention. Genetic counselling and prenatal diagnosis have contributed to a reduction in the number of children born with cystic fibrosis, but may not entirely explain the decreasing incidence of the disease.

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.

Tissue-specific and time-dependent clonal expansion of ENU-induced mutant cells in gpt delta mice.

PubMed

Nakayama, Takafumi; Sawai, Tomoko; Masuda, Ikuko; Kaneko, Shinya; Yamauchi, Kazumi; Blyth, Benjamin J; Shimada, Yoshiya; Tachibana, Akira; Kakinuma, Shizuko

2017-10-01

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N-ethyl-N-nitrosourea (ENU) with the persistence of ENU-induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU-induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592-606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Potentiators (specific therapies for class III and IV mutations) for cystic fibrosis.

PubMed

Patel, Sanjay; Sinha, Ian P; Dwan, Kerry; Echevarria, Carlos; Schechter, Michael; Southern, Kevin W

2015-03-26

Cystic fibrosis is the most common inherited life-shortening illness in Caucasians and caused by a mutation in the gene that codes for the cystic fibrosis transmembrane regulator protein (CFTR), which functions as a salt transporter. This mutation most notably affects the airways of people with cystic fibrosis. Excess salt absorption by defective CFTR dehydrates the airway lining and leads to defective mucociliary clearance. Consequent accumulation of thick, sticky mucus makes the airway prone to chronic infection and progressive inflammation; respiratory failure often ensues. Additionally, abnormalities with CFTR lead to systemic complications like malnutrition, diabetes and subfertility.Since the discovery of the causative gene, our understanding of the structure and function of CFTR and the impact of different mutations has increased and allowed pharmaceutical companies to design new mutation-specific therapies targeting the underlying molecular defect. Therapies targeting mutation classes III and IV (CFTR potentiators) aim to normalise airway surface liquid and help re-establish mucociliary clearance, which then has a beneficial impact on the chronic infection and inflammation that characterizes lung disease in people with cystic fibrosis. These therapies may also affect other mutations. To evaluate the effects of CFTR potentiators on clinically important outcomes in children and adults with cystic fibrosis. We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched the reference lists of relevant articles and reviews. Last search: 05 March 2015.We searched the EU Clinical Trials Register, clinicaltrials.gov (US Clinical Trials Register) and the International Clinical Trials Registry Platform (ICTRP). Last search of clinical trial registries: 06 February 2014. Randomised controlled trials of parallel design comparing CFTR potentiators to placebo in people with cystic fibrosis. In a post hoc change we excluded trials combining CFTR potentiators with other mutation-specific therapies. These will be considered in a separate review. The authors independently extracted data and assessed the risk of bias in included trials; they contacted trial authors for additional data. Meta-analyses were undertaken on outcomes at a number of time points. We included four randomised controlled trials (n = 378), lasting from 28 days to 48 weeks, comparing the potentiator ivacaftor to placebo. Trials differed in terms of design and participant eligibility criteria, which limited the meta-analyses. The phase 2 trial (n = 19) and two phase 3 trials (adult trial (n = 167), paediatric trial (n = 52)), recruited participants with the G551D mutation (class III). The fourth trial (n = 140) enrolled participants homozygous for the ΔF508 mutation (class II).Risks of bias in the trials were moderate. Random sequence generation, allocation concealment and blinding of trial personnel were well-documented. Participant blinding was less clear throughout all trials; in three trials, some participant data were excluded from the analysis. Selective outcome reporting was apparent in three trials. All trials were sponsored by industry and supported by other non-pharmaceutical funding bodies.No trial reported any deaths. Significantly higher quality of life scores in the respiratory domain were reported by the adult phase 3 G551D trial at 24 weeks, mean difference 8.10 (95% confidence interval (CI) 4.77 to 11.43) and 48 weeks, mean difference 8.60 (95% CI 5.27 to 11.93); but not by the paediatric phase 3 G551D trial. The adult phase 3 G551D trial reported improvements in relative change from baseline in forced expiratory volume at one second at 24 weeks, mean difference 16.90% (95% CI 13.60 to 20.20) and 48 weeks, mean difference 16.80% (95% CI 13.50 to 20.10); as did the paediatric G551D trial at 24 weeks, mean difference 17.4% (P < 0.0001)). No improvements in quality of life or lung function were reported in the ΔF508 participants.Combined data from both phase 3 G551D trials demonstrated increased reporting of cough, odds ratio 0.57 (95% CI 0.33 to 1.00) and increased episodes of decreased pulmonary function, odds ratio 0.29 (95% CI 0.10 to 0.82) in the placebo group. The adult phase 3 G551D trial demonstrated increased reporting of dizziness amongst the ivacaftor group, OR 10.55 (95% CI 1.32 to 84.47). No trial showed a difference between treatment arms in the number of participants interrupting or discontinuing the trial drug.In the phase 3 G551D trials, fewer participants assigned to ivacaftor developed serious pulmonary exacerbations. When considering all data for exacerbations, participants taking ivacaftor in the adult phase 3 G551D study developed fewer exacerbations, odds ratio 0.54 (95% CI 0.29 to 1.01). In the other G551D studies and in the ΔF508 study, there was no difference between groups in the number of participants who developed pulmonary exacerbations.Combined data from both phase 3 G551D trials demonstrated significant improvements in absolute change from baseline in forced expiratory volume at one second (% predicted) at 24 weeks, mean difference 10.80% (95% CI 8.91 to 12.69) and 48 weeks, mean difference 10.44% (95% CI 8.56 to 12.32); also in weight at 24 weeks, mean difference 2.37 kg (95% CI 1.68 to 3.06) and 48 weeks, mean difference 2.75 kg (95% CI 1.74 to 3.75). No improvements in these outcomes were reported in the ΔF508 participants.Significant reductions in sweat chloride concentration were reported in both G551D and ΔF508 participants: in combined data from both phase 3 G551D trials at 24 weeks, mean difference -48.98 mmol/L (95% CI -52.07 to -45.89) and 48 weeks, mean difference -49.03 mmol/L (95% CI -52.11 to -45.94); and from the ΔF508 trial at 16 weeks, mean difference -2.90 mmol/L (95% CI -5.60 to -0.20). Both G551D phase 3 trials (n = 219) demonstrated a clinically relevant impact of the potentiator ivacaftor on outcomes at 24 and 48 weeks, providing evidence for the use of this treatment in adults and children (over six years of age) with cystic fibrosis and the G551D mutation (class III). There is no evidence to support the use of ivacaftor in people with the ΔF508 mutation (class II) (n = 140). Trials on ivacaftor in people with different mutations are ongoing.

CFTR rescue with VX-809 and VX-770 favors the repair of primary airway epithelial cell cultures from patients with class II mutations in the presence of Pseudomonas aeruginosa exoproducts.

PubMed

Adam, Damien; Bilodeau, Claudia; Sognigbé, Laura; Maillé, Émilie; Ruffin, Manon; Brochiero, Emmanuelle

2018-04-13

Progressive airway damage due to bacterial infections, especially with Pseudomonas aeruginosa remains the first cause of morbidity and mortality in CF patients. Our previous work revealed a repair delay in CF airway epithelia compared to non-CF. This delay was partially prevented after CFTR correction (with VRT-325) in the absence of infection. Our goals were now to evaluate the effect of the Orkambi combination (CFTR VX-809 corrector + VX-770 potentiator) on the repair of CF primary airway epithelia, in infectious conditions. Primary airway epithelial cell cultures from patients with class II mutations were mechanically injured and wound healing rates and transepithelial resistances were monitored after CFTR rescue, in the absence and presence of P. aeruginosa exoproducts. Our data revealed that combined treatment with VX-809 and VX-770 elicited a greater beneficial impact on airway epithelial repair than VX-809 alone, in the absence of infection. The treatment with Orkambi was effective not only in airway epithelial cell cultures from patients homozygous for the F508del mutation but also from heterozygous patients carrying F508del and another class II mutation (N1303 K, I507del). The stimulatory effect of the Orkambi treatment was prevented by CFTR inhibition with GlyH101. Finally, Orkambi combination elicited a slight but significant improvement in airway epithelial repair and transepithelial resistance, despite the presence of P. aeruginosa exoproducts. Our findings indicate that Orkambi may favor airway epithelial integrity in CF patients with class II mutations. Complementary approaches would however be needed to further improve CFTR rescue and airway epithelial repair. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a

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

Chou, J.Y.; Lei, K.J.; Shelly, L.L.

1994-09-01

Glycogen storage disease (GSD) type la (von Gierke disease) is caused by the deficiency of glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. The disease presents with clinical manifestations of severe hypoglycemia, hepatomegaly, growth retardation, lactic acidemia, hyperlipidemia, and hyperuricemia. We have succeeded in isolating a murine G6Pase cDNA from a normal mouse liver cDNA library by differentially screening method. We then isolated the human G6Pase cDNA and gene. To date, we have characterized the G6Pase genes of twelve GSD type la patients and uncovered a total of six different mutations. The mutations are comprised of R83C (an Arg atmore » codon 83 to a Cys), Q347X (a Gly at codon 347 to a stop codon), 459insTA (a two basepair insertion at nucleotide 459 yielding a truncated G6Pase of 129 residues), R295C (an Arg at codon 295 to a Cys), G222R (a Gly at codon 222 to an Arg) and {delta}F327 (a codon deletion for Phe-327 at nucleotides 1058 to 1060). The relative incidences of these mutations are 37.5% (R83C), 33.3% (Q347X), 16.6% (459insTA), 4.2% (G222R), 4.2% (R295C) and 4.2% ({delta}F327). Site-directed mutagenesis and transient expression assays demonstrated that the R83C, Q347X, R295C, and {delta}F327 mutations abolished whereas the G222R mutation greatly reduced G6Pase activity. We further characterized the structure-function requirements of amino acids 83, 222, and 295 in G6Pase catalysis. The identification of mutations in GSD type la patients has unequivocally established the molecular basis of the type la disorder. Knowledge of the mutations may be applied to prenatal diagnosis and opens the way for developing and evaluating new therapeutic approaches.« less

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.

The cystic fibrosis gene: Medical and social implications for heterozygote detection

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

Wilfond, B.S.; Fost, N.

1990-05-23

The primary goal of mass screening programs for cystic fibrosis carriers should be to allow people to make more informed reproductive decisions. However, previous experience with genetic screening programs, including those for phenylketonuria and sickle cell disease, have revealed complex problems including error, confusion, and stigmatization. These problems could be greater with cystic fibrosis, since more than 8 million Americans may be carriers and entrepreneurial interests can be expected to promote screening in what could become a billion-dollar industry. The present frequency of the detectable mutation ({Delta}F{sub 508}), 75%, will complicate the counseling process. The sensitivity of the test tomore » detect at-risk couples would be 56%. The cost of screening could be as much as $2.2 million for each cystic fibrosis birth avoided. Regardless of improvements in the detection rate, implementation of population screening should be delayed until pilot studies that demonstrate its safety and effectiveness are completed. While studies are in progress, preconception testing should be offered to adult relatives of cystic fibrosis patients as part of a comprehensive program following institutional review board approval for compassionate use.« less

Backbone dynamics and global effects of an activating mutation in minimized Mtu RecA inteins.

PubMed

Du, Zhenming; Liu, Yangzhong; Ban, David; Lopez, Maria M; Belfort, Marlene; Wang, Chunyu

2010-07-23

Inteins mediate protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mycobacterium tuberculosis RecA inteins. However, V67L mutation causes little change in crystal structures. To test whether protein dynamics contribute to activity enhancement in the V67L mutation, we have studied the conformations and dynamics of the minimized and engineered intein DeltaDeltaIhh-V67CM and a single V67L mutant, DeltaDeltaIhh-L67CM, by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N-terminus and C-terminus of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows hydrogen-exchange rates globally, indicating a shift to more stable conformations and reduction in ensemble distribution. Whereas the V67L mutation causes little change for motions on the picosecond-to-nanosecond timescale, motions on the microsecond-to-millisecond timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are residues important for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing the ensemble distribution of the intein structure and by modifying the active sites. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Impact of cystic fibrosis disease on archaea and bacteria composition of gut microbiota.

PubMed

Miragoli, Francesco; Federici, Sara; Ferrari, Susanna; Minuti, Andrea; Rebecchi, Annalisa; Bruzzese, Eugenia; Buccigrossi, Vittoria; Guarino, Alfredo; Callegari, Maria Luisa

2017-02-01

Cystic fibrosis is often associated with intestinal inflammation due to several factors, including altered gut microbiota composition. In this study, we analyzed the fecal microbiota among patients with cystic fibrosis of 10-22 years of age, and compared the findings with age-matched healthy subjects. The participating patients included 14 homozygotes and 14 heterozygotes with the delF508 mutation, and 2 heterozygotes presenting non-delF508 mutations. We used PCR-DGGE and qPCR to analyze the presence of bacteria, archaea and sulfate-reducing bacteria. Overall, our findings confirmed disruption of the cystic fibrosis gut microbiota. Principal component analysis of the qPCR data revealed no differences between homozygotes and heterozygotes, while both groups were distinct from healthy subjects who showed higher biodiversity. Archaea were under the detection limit in all homozygotes subjects, whereas methanogens were detected in 62% of both cystic fibrosis heterozygotes and healthy subjects. Our qPCR results revealed a low frequency of sulfate-reducing bacteria in the homozygote (13%) and heterozygote (13%) patients with cystic fibrosis compared with healthy subjects (87.5%). This is a pioneer study showing that patients with cystic fibrosis exhibit significant reduction of H 2 -consuming microorganisms, which could increase hydrogen accumulation in the colon and the expulsion of this gas through non-microbial routes. © FEMS 2016.

Vx-809/Vx-770 treatment reduces inflammatory response to Pseudomonas aeruginosa in primary differentiated cystic fibrosis bronchial epithelial cells.

PubMed

Ruffin, Manon; Roussel, Lucie; Maillé, Émilie; Rousseau, Simon; Brochiero, Emmanuelle

2018-04-01

Cystic fibrosis patients exhibit chronic Pseudomonas aeruginosa respiratory infections and sustained proinflammatory state favoring lung tissue damage and remodeling, ultimately leading to respiratory failure. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with MAPK hyperactivation and increased cytokines expression, such as interleukin-8 [chemoattractant chemokine (C-X-C motif) ligand 8 (CXCL8)]. Recently, new therapeutic strategies directly targeting the basic CFTR defect have been developed, and ORKAMBI (Vx-809/Vx-770 combination) is the only Food and Drug Administration-approved treatment for CF patients homozygous for the F508del mutation. Here we aimed to determine the effect of the Vx-809/Vx-770 combination on the induction of the inflammatory response by fully differentiated primary bronchial epithelial cell cultures from CF patients carrying F508del mutations, following exposure to P. aeruginosa exoproducts. Our data unveiled that CFTR functional rescue with Vx-809/Vx-770 drastically reduces CXCL8 (as well as CXCL1 and CXCL2) transcripts and p38 MAPK phosphorylation in response to P. aeruginosa exposure through a CFTR-dependent mechanism. These results suggest that ORKAMBI has anti-inflammatory properties that could decrease lung inflammation and contribute to the observed beneficial impact of this treatment in CF patients.

Distribution of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutations in a Cohort of Patients Residing in Palestine.

PubMed

Siryani, Issa; Jama, Mohamed; Rumman, Nisreen; Marzouqa, Hiyam; Kannan, Moein; Lyon, Elaine; Hindiyeh, Musa

2015-01-01

Cystic fibrosis (CF) is an autosomal recessive inherited life-threatening disorder that causes severe damage to the lungs and the digestive system. In Palestine, mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that contributes to the clinical presentation of CF are ill defined. A cohort of thirty three clinically diagnosed CF patients from twenty one different Palestinian families residing in the central and southern part of Palestine were incorporated in this study. Sweat chloride testing was performed using the Sweat Chek Conductivity Analyzer (ELITECH Group, France) to confirm the clinical diagnosis of CF. In addition, nucleic acid from the patients' blood samples was extracted and the CFTR mutation profiles were assessed by direct sequencing of the CFTR 27 exons and the intron-exon boundaries. For patient's DNA samples where no homozygous or two heterozygous CFTR mutations were identified by exon sequencing, DNA samples were tested for deletions or duplications using SALSA MLPA probemix P091-D1 CFTR assay. Sweat chloride testing confirmed the clinical diagnosis of CF in those patients. All patients had NaCl conductivity >60 mmol/l. In addition, nine different CFTR mutations were identified in all 21 different families evaluated. These mutations were c.1393-1G>A, F508del, W1282X, G85E, c.313delA, N1303K, deletion exons 17a-17b-18, deletion exons 17a-17b and Q1100P. c.1393-1G>A was shown to be the most frequent occurring mutation among tested families. We have profiled the underling mutations in the CFTR gene of a cohort of 21 different families affected by CF. Unlike other studies from the Arab countries where F508del was reported to be the most common mutation, in southern/central Palestine, the c.1393-1G>A appeared to be the most common. Further studies are needed per sample size and geographic distribution to account for other possible CFTR genetic alterations and their frequencies. Genotype/phenotype assessments are also recommended and finally carrier frequency should be ascertained.

Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis *

PubMed Central

Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

2013-01-01

OBJECTIVE: To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). METHODS: This was a cross-sectional study involving adolescents and adults aged ≥ 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. RESULTS: We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. CONCLUSIONS: The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis. PMID:23670503

Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis.

PubMed

Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

2013-01-01

To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). This was a cross-sectional study involving adolescents and adults aged > 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis.

Year to year change in FEV1 in patients with cystic fibrosis and different mutation classes.

PubMed

De Boeck, K; Zolin, A

2017-03-01

In patients with cystic fibrosis, most treatments addressing the underlying basic defect are mutation or mutation class specific. These treatments are disease modifying if they lower the year to year change in lung function. We therefore calculated the current loss of lung function, measured by year to year change in forced expired volume in 1s in 11,417 patients included in the European Cystic Fibrosis Society Patient Registry. Whereas patients with at least one mutation of class IV or V have on average a lower year to year change, we did not find a difference between patients with a stop codon mutation, homozygous for F508del or at least one class III mutation. These data are useful background information to discuss the impact of different disease modifying treatments. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages*

PubMed Central

Deriy, Ludmila V.; Gomez, Erwin A.; Zhang, Guangping; Beacham, Daniel W.; Hopson, Jessika A.; Gallan, Alexander J.; Shevchenko, Pavel D.; Bindokas, Vytautas P.; Nelson, Deborah J.

2009-01-01

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933–944). Lysosomes and phagosomes in murine cftr−/− AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, ΔF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR ΔF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr−/−, as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung. PMID:19837664

The thermochemistry of 2,4-pentanedione revisited: observance of a nonzero enthalpy of mixing between tautomers and its effects on enthalpies of formation.

PubMed

Temprado, Manuel; Roux, Maria Victoria; Umnahanant, Patamaporn; Zhao, Hui; Chickos, James S

2005-06-30

The enthalpies of formation of pure liquid and gas-phase (Z)-4-hydroxy-3-penten-2-one and 2,4-pentanedione are examined in the light of some more recent NMR studies on the enthalpy differences between gas-phase enthalpies of the two tautomers. Correlation gas chromatography experiments are used to evaluate the vaporization enthalpies of the pure tautomers. Values of (51.2 +/- 2.2) and (50.8 +/- 0.6) kJ.mol(-1) are measured for pure 2,4-pentanedione and (Z)-4-hydroxy-3-penten-2-one, respectively. The value of (50.8 +/- 0.6) kJ.mol(-1) can be contrasted to a value of (43.2 +/- 0.2) kJ.mol(-1) calculated for pure (Z)-4-hydroxy-3-penten-2-one when the vaporization enthalpy is measured in a mixture of tautomers. The difference is attributed to an endothermic enthalpy of mixing that destabilizes the mixture relative to the pure components. Calculation of new enthalpies of formation for (Z)-4-hydroxy-3-penten-2-one and 2,4-pentanedione in both the gas, Delta(f)H degrees (m)(g) = (-378.2 +/- 1.2) and (-358.9 +/- 2.5) kJ.mol(-1), respectively, and liquid phases, Delta(f)H degrees (m)(l) = (-429.0 +/- 1.0) and (-410.1 +/- 1.2) kJ.mol(-1), respectively, results in enthalpy differences between the two tautomers both in the liquid and gas phases that are identical within experimental error, and in excellent agreement with recent gas-phase NMR studies.

Examination of in vivo mutagenicity of sodium arsenite and dimethylarsinic acid in gpt delta rats.

PubMed

Fujioka, Masaki; Gi, Min; Kawachi, Satoko; Tatsumi, Kumiko; Ishii, Naomi; Doi, Kenichiro; Kakehashi, Anna; Wanibuchi, Hideki

2016-11-01

Arsenic is a well-known human bladder and liver carcinogen, but its exact mechanism of carcinogenicity is not fully understood. Dimethylarsinic acid (DMA V ) is a major urinary metabolite of sodium arsenite (iAs III ) and induces urinary bladder cancers in rats. DMA V and iAs III are negative in in vitro mutagenicity tests. However, their in vivo mutagenicities have not been determined. The purpose of present study is to evaluate the in vivo mutagenicities of DMA V and iAs III in rat urinary bladder epithelium and liver using gpt delta F344 rats. Ten-week old male gpt delta F344 rats were randomized into 3 groups and administered 0, 92mg/L DMA V , or 87mg/L iAs III (each 50mg/L As) for 13weeks in the drinking water. In the mutation assay, point mutations are detected in the gpt gene by 6-thioguanine selection (gpt assay) and deletion mutations are identified in the red/gam genes by Spi - selection (Spi - assay). Results of the gpt and Spi - assays showed that DMA V and iAs III had no effects on the mutant frequencies or mutation spectrum in urinary bladder epithelium or liver. These findings indicate that DMA V and iAs III are not mutagenic in urinary bladder epithelium or liver in rats. Copyright © 2016. Published by Elsevier B.V.

Additive effect of multiple pharmacological chaperones on maturation of CFTR processing mutants

PubMed Central

Wang, Ying; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.

2007-01-01

The most common cause of CF (cystic fibrosis) is the deletion of Phe508 (ΔF508) in the CFTR [CF TM (transmembrane) conductance regulator] chloride channel. One major problem with ΔF508 CFTR is that the protein is defective in folding so that little mature protein is delivered to the cell surface. Expression of ΔF508 CFTR in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein. Unfortunately, the efficiency of corrector-induced maturation of ΔF508 CFTR is probably too low to have therapeutic value and approaches are needed to increase maturation efficiency. We postulated that expression of ΔF508 CFTR in the presence of multiple correctors that bound to different sites may have an additive effect on maturation. In support of this mechanism, we found that expression of P-glycoprotein (CFTR's sister protein) processing mutants in the presence of two compounds that bind to different sites (rhodamine B and Hoechst 33342) had an additive effect on maturation. Therefore we tested whether expression of ΔF508 CFTR in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. It was found that the combination of the quinazoline VRT-325 together with the thiazole corr-2b or bisaminomethylbithiazole corr-4a doubled the steady-state maturation efficiency of ΔF508 CFTR (approx. 40% of total CFTR was mature protein) compared with expression in the presence of a single compound. The additive effect of the correctors on ΔF508 CFTR maturation suggests that they directly interact at different sites of the protein. PMID:17535157

Breakthrough Therapies: Cystic Fibrosis (CF) Potentiators and Correctors

PubMed Central

Solomon, George M.; Marshall, Susan G.; Ramsey, Bonnie W.; Rowe, Steven M.

2015-01-01

Cystic Fibrosis is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene resulting in abnormal protein function. Recent advances of targeted molecular therapies and high throughput screening have resulted in multiple drug therapies that target many important mutations in the CFTR protein. In this review, we provide the latest results and current progress of CFTR modulators for the treatment of cystic fibrosis, focusing on potentiators of CFTR channel gating and Phe508del processing correctors for the Phe508del CFTR mutation. Special emphasis is placed on the molecular basis underlying these new therapies and emerging results from the latest clinical trials. The future directions for augmenting the rescue of Phe508del with CFTR modulators is also emphasized. PMID:26097168

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

Tezacaftor/Ivacaftor in Subjects with Cystic Fibrosis and F508del/F508del-CFTR or F508del/G551D-CFTR.

PubMed

Donaldson, Scott H; Pilewski, Joseph M; Griese, Matthias; Cooke, Jon; Viswanathan, Lakshmi; Tullis, Elizabeth; Davies, Jane C; Lekstrom-Himes, Julie A; Wang, Linda T

2018-01-15

Tezacaftor (formerly VX-661) is an investigational small molecule that improves processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in vitro, and improves CFTR function alone and in combination with ivacaftor. To evaluate the safety and efficacy of tezacaftor monotherapy and of tezacaftor/ivacaftor combination therapy in subjects with cystic fibrosis homozygous for F508del or compound heterozygous for F508del and G551D. This was a randomized, placebo-controlled, double-blind, multicenter, phase 2 study (NCT01531673). Subjects homozygous for F508del received tezacaftor (10 to 150 mg) every day alone or in combination with ivacaftor (150 mg every 12 h) in a dose escalation phase, as well as in a dosage regimen testing phase. Subjects compound heterozygous for F508del and G551D, taking physician-prescribed ivacaftor, received tezacaftor (100 mg every day). Primary endpoints were safety through Day 56 and change in sweat chloride from baseline through Day 28. Secondary endpoints included change in percent predicted FEV 1 (ppFEV 1 ) from baseline through Day 28 and pharmacokinetics. The incidence of adverse events was similar across treatment arms. Tezacaftor (100 mg every day)/ivacaftor (150 mg every 12 h) resulted in a 6.04 mmol/L decrease in sweat chloride and 3.75 percentage point increase in ppFEV 1 in subjects homozygous for F508del, and a 7.02 mmol/L decrease in sweat chloride and 4.60 percentage point increase in ppFEV 1 in subjects compound heterozygous for F508del and G551D from baseline through Day 28 (P < 0.05 for all). These results support continued clinical development of tezacaftor (100 mg every day) in combination with ivacaftor (150 mg every 12 h) in subjects with cystic fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT01531673).

Prevalence of hypoglycemia during oral glucose tolerance testing in adults with cystic fibrosis and risk of developing cystic fibrosis-related diabetes.

PubMed

Mannik, Lisa A; Chang, Kristy A; Annoh, Pascalyn Q K; Sykes, Jenna; Gilmour, Julie; Robert, Ronalee; Stephenson, Anne L

2018-04-18

Hypoglycemia in cystic fibrosis (CF) patients during the oral glucose tolerance test (OGTT) has been reported; however, these patients have not been well-characterized. Few studies have examined whether hypoglycemia during the OGTT increases the risk of developing CF-related diabetes (CFRD). Objectives of this study were to describe the characteristics of CF patients with hypoglycemia during the OGTT and to determine the incidence and time to development of CFRD in those with hypoglycemia. This cohort study included 466 adults with CF at the Toronto Adult CF Clinic between 1996 and 2015. Subjects were classified into two groups based on their plasma glucose (PG) level 2 h after a 75 g OGTT: hypoglycemia (PG ≤ 3.9 mmol/L) or no hypoglycemia (PG > 3.9 mmol/L). Clinical and demographic data were collected from the clinic visit closest to the OGTT. Differences between groups were assessed using Fisher's exact test or Mann-Whitney-Wilcoxon test. 138 patients (29.6%) experienced hypoglycemia during the OGTT. More males experienced hypoglycemia compared to no hypoglycemia (69.6% vs. 54.6% respectively; p = 0.003). Those who were heterozygous deltaF508 were more likely to experience hypoglycemia (p = 0.006). Subjects who experienced hypoglycemia were less likely to develop CFRD at ten years compared to no hypoglycemia (12.0% vs. 42.1%, respectively; p < 0.001). Hypoglycemia following OGTT is common in CF however the 10 year risk of developing CFRD in these patients was low. Males and those who were heterozygous deltaF508 were at higher risk for hypoglycemia. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Epithelial cell specific properties and genetic complementation in a delta F508 cystic fibrosis nasal polyp cell line.

PubMed

Kunzelmann, K; Lei, D C; Eng, K; Escobar, L C; Koslowsky, T; Gruenert, D C

1995-09-01

Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a "tight epithelium" in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, delta F508 homozygote, cell line that has tight junctions pre-crisis. The cells (sigma CFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl- transport as measured by efflux of radioactive Cl-, short circuit current (Isc), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K(+)- and Ca(+2)-dependent Cl- transport. Measurement of 36Cl- efflux following stimulation with A23187 and ionomycin indicate a Ca(+2)-dependent Cl- transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26 degrees C for 48 h there is no indication of cAMP-dependent Cl- as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Is there evidence for correct diagnosis in cystic fibrosis registries?

PubMed

Thomas, Muriel; Lemonnier, Lydie; Gulmans, Vincent; Naehrlich, Lutz; Vermeulen, François; Cuppens, Harry; Castellani, Carlo; Norek, Aleksandra; De Boeck, Kris

2014-05-01

Cystic fibrosis (CF) spans a wide spectrum. Therefore, benchmarking between registries implies comparing similar cohorts. Explore patient characteristics in Belgian (B), French (F), German (G) and Dutch (NL) registries (total N=13,122) and determine whether they fulfill predefined diagnostic criteria. Using as case definition sweat chloride >60mmol/L or 2 CFTR mutations identified, CF diagnosis was not documented in 2.8, 5.7, 6.5 and 21.6% of subjects in the F, B, NL, and G registries. Restricting CFTR mutation interpretation to 124 CF causing mutations in CFTR2, these numbers rose to 10.5, 10.4, 14.5 and 24.3% respectively. Excluding these subjects impacted on outcomes. The impact differed between countries; the largest changes seen were a decrease in % adults from 51.9 to 47.8% in G, a decrease in % pancreas sufficiency from 17.0 to 13.0 in F, an increase in % homozygous for F508del from 55.3 to 63.7 in NL and a decrease of % with sweat chloride ≤60mmol/L from 8.4 to 1.1 in B. CF diagnosis is not documented in 10 to 24% of patients included in CF registries. Excluding these patients for analyses leads to significant changes in outcomes. © 2013.

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.

CFTR gene mutations in isolated chronic obstructive pulmonary disease

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

Pignatti, P.F.; Bombien, C.; Marigo, C.

1994-09-01

In order to identify a possible hereditary predisposition to the development of chronic obstructive pulmonary disease (COPD), we have looked for the presence of cystic fibrosis transmembrane regulator (CFTR) gene DNA sequence modifications in 28 unrelated patients with no signs of cystic fibrosis. The known mutations in Italian CF patients, as well as the most frequent worldwide CF mutations, were investigated. In addition, a denaturing gradient gel electrophoresis analysis of about half of the coding sequence of the gene in 56 chromosomes from the patients and in 102 chromosomes from control individuals affected by other pulmonary diseases and from normalmore » controls was performed. Nine different CFTR gene mutations and polymorphisms were found in seven patients, a highly significant increase over controls. Two of the patients were compound heterozygotes. Two frequent CF mutations were detected: deletion F508 and R117H; two rare CF mutations: R1066C and 3667ins4; and five CF sequence variants: R75Q (which was also described as a disease-causing mutation in male sterility cases due to the absence of the vasa deferentia), G576A, 2736 A{r_arrow}G, L997F, and 3271+18C{r_arrow}T. Seven (78%) of the mutations are localized in transmembrane domains. Six (86%) of the patients with defined mutations and polymorphisms had bronchiectasis. These results indicate that CFTR gene mutations and sequence alterations may be involved in the etiopathogenesis of some cases of COPD.« less

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.

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

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 The British Pharmacological Society.

Comparison of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Ciliary Beat Frequency Activation by the CFTR Modulators Genistein, VRT-532, and UCCF-152 in Primary Sinonasal Epithelial Cultures

PubMed Central

Conger, Bryant T.; Zhang, Shaoyan; Skinner, Daniel; Hicks, Stephen B.; Sorscher, Eric J.; Rowe, Steven M.; Woodworth, Bradford A.

2014-01-01

IMPORTANCE Pharmacologic activation of mucociliary clearance (MCC) represents an emerging therapeutic strategy for patients with chronic rhinosinusitis, even in the absence of congenital mutations of the CFTR gene. Drug discovery efforts have identified small molecules that activate the cystic fibrosis transmembrane conductance regulator (CFTR), including potentiators under development for treatment of cystic fibrosis. OBJECTIVE To evaluate the properties of CFTR modulators and their effects on ciliary beat frequency (CBF) in human sinonasal epithelium (HSNE). DESIGN Primary HSNE cultures (wild type and F508del/F508del) were used to compare stimulation of CFTR-mediated Cl− conductance and CBF by the CFTR modulators genistein, VRT-532, and UCCF-152. MAIN OUTCOMES AND MEASURES Increase in CFTR-dependent anion transport and CBF. RESULTS HSNE cultures were analyzed using pharmacologic manipulation of ion transport (change in short-circuit current [ΔISC]) and high-speed digital imaging (CBF). Activation of CFTR-dependent anion transport was significantly different among agonists (P < .001), with genistein exerting the greatest effect (mean [SD] ΔISC, genistein, 23.1 [1.8] µA/cm2 > VRT-532, 8.1 [1.0] µA/cm2 > UCCF-152, 3.4 [1.4] µA/cm2 > control, 0.7 [0.2] µA/cm2; Tukey-Kramer P < .05) in the absence of forskolin. Genistein and UCCF-152 augmented CBF (under submerged conditions) significantly better (Tukey-Kramer P < .05) than cells treated with VRT-532 or dimethyl sulfoxide vehicle control (mean [SD] fold change over baseline, genistein, 1.63 [0.06]; UCCF-152, 1.56 [0.06]; VRT-532, 1.38 [0.08]; control, 1.27 [0.02]). Activation of CBF was blunted in F508del/F508del HSNE cultures. CONCLUSIONS AND RELEVANCE The degree of CBF stimulation was not dependent on the magnitude of Cl− secretion, suggesting that different mechanisms of action may underlie MCC activation by these small molecule potentiators. Agents that activate both CFTR-dependent ISC and CBF are particularly attractive as therapeutics because they may address 2 independent pathways that contribute to deficient MCC in chronic rhinosinusitis. PMID:23949358

Comparison of cystic fibrosis transmembrane conductance regulator (CFTR) and ciliary beat frequency activation by the CFTR Modulators Genistein, VRT-532, and UCCF-152 in primary sinonasal epithelial cultures.

PubMed

Conger, Bryant T; Zhang, Shaoyan; Skinner, Daniel; Hicks, Stephen B; Sorscher, Eric J; Rowe, Steven M; Woodworth, Bradford A

2013-08-01

Pharmacologic activation of mucociliary clearance (MCC) represents an emerging therapeutic strategy for patients with chronic rhinosinusitis, even in the absence of congenital mutations of the CFTR gene. Drug discovery efforts have identified small molecules that activate the cystic fibrosis transmembrane conductance regulator (CFTR), including potentiators under development for treatment of cystic fibrosis. To evaluate the properties of CFTR modulators and their effects on ciliary beat frequency (CBF) in human sinonasal epithelium (HSNE). Primary HSNE cultures (wild type and F508del/F508del) were used to compare stimulation of CFTR-mediated Cl- conductance and CBF by the CFTR modulators genistein, VRT-532, and UCCF-152. Increase in CFTR-dependent anion transport and CBF. HSNE cultures were analyzed using pharmacologic manipulation of ion transport (change in short-circuit current [∆ISC]) and high-speed digital imaging (CBF). Activation of CFTR-dependent anion transport was significantly different among agonists (P < .001), with genistein exerting the greatest effect (mean [SD] ∆ISC, genistein, 23.1 [1.8] μA/cm2² > VRT-532, 8.1 [1.0] μA/cm² > UCCF-152, 3.4 [1.4] μA/cm² > control, 0.7 [0.2] μA/cm²; Tukey-Kramer P < .05) in the absence of forskolin. Genistein and UCCF-152 augmented CBF (under submerged conditions) significantly better (Tukey-Kramer P < .05) than cells treated with VRT-532 or dimethyl sulfoxide vehicle control (mean [SD] fold change over baseline, genistein, 1.63 [0.06]; UCCF-152, 1.56 [0.06]; VRT-532, 1.38 [0.08]; control, 1.27 [0.02]). Activation of CBF was blunted in F508del/F508del HSNE cultures. The degree of CBF stimulation was not dependent on the magnitude of Cl- secretion, suggesting that different mechanisms of action may underlie MCC activation by these small molecule potentiators. Agents that activate both CFTR-dependent ISC and CBF are particularly attractive as therapeutics because they may address 2 independent pathways that contribute to deficient MCC in chronic rhinosinusitis.

Vitamin K status in cystic fibrosis patients with liver cirrhosis.

PubMed

Krzyżanowska, Patrycja; Drzymała-Czyż, Sławomira; Pogorzelski, Andrzej; Duś-Żuchowska, Monika; Skorupa, Wojciech; Bober, Lyudmyla; Sapiejka, Ewa; Oralewska, Beata; Rohovyk, Nataliya; Moczko, Jerzy; Nowak, Jan; Wenska-Chyży, Ewa; Rachel, Marta; Lisowska, Aleksandra; Walkowiak, Jarosław

2017-06-01

The available data on the influence of liver cirrhosis on vitamin K status in CF patients is scarce. Therefore, the aims of the present study were to assess the prevalence of vitamin K deficiency in cirrhotic CF subjects and to determine whether it correlates with liver cirrhosis. The study group comprised of 27 CF patients with and 63 without liver cirrhosis. Vitamin K status was assessed using prothrombin induced by vitamin K absence (PIVKA-II) and the percentage of undercarboxylated osteocalcin (u-OC). PIVKA-II concentrations were higher in cirrhotic than in non-cirrhotic CF patients (median [1st-3rd quartile]: 3.2ng/ml [1.0-10.0] vs. 1.3ng/ml [0.2-2.6], p=0.0029). However, the differences in u-OC percentages between the studied groups did not reach the level of significance (49.4% [7.0-73.8] vs. 8.0% [2.6-59.1], p=0.0501). Based on multiple linear regression analysis the dose of vitamin K and F508del mutation were potentially defined as determinants of vitamin K deficiency. Liver cirrhosis was not documented to be an independent risk factor. In CF patients with liver cirrhosis vitamin K deficiency is not only more frequent, but also more severe. However, not liver cirrhosis, but the presence of a F508del CFTR mutation constitutes an independent risk factor for vitamin K deficiency. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

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

Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near positionmore » 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.« less

Four Novel p.N385K, p.V36A, c.1033–1034insT and c.1417–1418delCT Mutations in the Sphingomyelin Phosphodiesterase 1 (SMPD1) Gene in Patients with Types A and B Niemann-Pick Disease (NPD)

PubMed Central

Manshadi, Masoumeh Dehghan; Kamalidehghan, Behnam; Keshavarzi, Fatemeh; Aryani, Omid; Dadgar, Sepideh; Arastehkani, Ahoora; Tondar, Mahdi; Ahmadipour, Fatemeh; Meng, Goh Yong; Houshmand, Massoud

2015-01-01

Background: Types A and B Niemann-Pick disease (NPD) are autosomal-recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase due to mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene. Methods: In order to determine the prevalence and distribution of SMPD1 gene mutations, the genomic DNA of 15 unrelated Iranian patients with types A and B NPD was examined using PCR, DNA sequencing and bioinformatics analysis. Results: Of 8 patients with the p.G508R mutation, 5 patients were homozygous, while the other 3 were heterozygous. One patient was heterozygous for both the p.N385K and p.G508R mutations. Another patient was heterozygous for both the p.A487V and p.G508R mutations. Two patients (one homozygous and one heterozygous) showed the p.V36A mutation. One patient was homozygous for the c.1033–1034insT mutation. One patient was homozygous for the c.573delT mutation, and 1 patient was homozygous for the c.1417–1418delCT mutation. Additionally, bioinformatics analysis indicated that two new p.V36A and p.N385K mutations decreased the acid sphingomyelinase (ASM) protein stability, which might be evidence to suggest the pathogenicity of these mutations. Conclusion: with detection of these new mutations, the genotypic spectrum of types A and B NPD is extended, facilitating the definition of disease-related mutations. However, more research is essential to confirm the pathogenic effect of these mutations. PMID:25811928

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

Quorum Sensing Down-Regulation Counteracts the Negative Impact of Pseudomonas aeruginosa on CFTR Channel Expression, Function and Rescue in Human Airway Epithelial Cells

PubMed Central

Maillé, Émilie; Ruffin, Manon; Adam, Damien; Messaoud, Hatem; Lafayette, Shantelle L.; McKay, Geoffrey; Nguyen, Dao; Brochiero, Emmanuelle

2017-01-01

The function of cystic fibrosis transmembrane conductance regulator (CFTR) channels is crucial in human airways. However unfortunately, chronic Pseudomonas aeruginosa infection has been shown to impair CFTR proteins in non-CF airway epithelial cells (AEC) and to alter the efficiency of new treatments with CFTR modulators designed to correct the basic CFTR default in AEC from cystic fibrosis (CF) patients carrying the F508del mutation. Our aim was first to compare the effect of laboratory strains, clinical isolates, engineered and natural mutants to determine the role of the LasR quorum sensing system in CFTR impairment, and second, to test the efficiency of a quorum sensing inhibitor to counteract the deleterious impact of P. aeruginosa both on wt-CFTR and on the rescue of F508del-CFTR by correctors. We first report that exoproducts from either the laboratory PAO1 strain or a clinical ≪Early≫ isolate (from an early stage of infection) altered CFTR expression, localization and function in AEC expressing wt-CFTR. Genetic inactivation of the quorum-sensing LasR in PAO1 (PAO1ΔlasR) or in a natural clinical mutant (≪Late≫ CF-adapted clinical isolate) abolished wt-CFTR impairment. PAO1 exoproducts also dampened F508del-CFTR rescue by VRT-325 or Vx-809 correctors in CF cells, whereas PAO1ΔlasR had no impact. Importantly, treatment of P. aeruginosa cultures with a quorum sensing inhibitor (HDMF) prevented the negative effect of P. aeruginosa exoproducts on wt-CFTR and preserved CFTR rescue by correctors in CF AEC. These findings indicate that LasR-interfering strategies could be of benefits to counteract the deleterious effect of P. aeruginosa in infected patients. PMID:29177135

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells

NASA Astrophysics Data System (ADS)

Rich, Devra P.; Anderson, Matthew P.; Gregory, Richard J.; Cheng, Seng H.; Paul, Sucharita; Jefferson, Douglas M.; McCann, John D.; Klinger, Katherine W.; Smith, Alan E.; Welsh, Michael J.

1990-09-01

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (ΔF508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

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.

Liver cirrhosis and portal hypertension in cystic fibrosis.

PubMed

Fustik, Stojka

2013-01-01

As the expected survival improves in individuals with the cystic fibrosis (CF), so they may be faced with a number of medical complications. The aim of this study was to analyze the prevalence of liver cirrhosis in our CF population as well as the clinical and genetic characteristics of these patients. All patients older than 2 years (n = 96) were screened for liver disease. Liver cirrhosis was defined by ultrasonographic findings of distinct heterogeneity of liver parenchyma and nodular liver surface and/or by liver biopsy findings. Enlarged spleen, distended portal vein and abnormal portal venous flow indicated portal hypertension. Clinical and genotype data were analyzed. Sixteen patients were found to have liver cirrhosis, three of them with portal hypertension. All patients had pancreatic insufficiency. Nutritional status expressed as standard deviation score (Z score) for weight, height, and body mass index was as follows: zW = -0.40 +/- 1.24, zH = -0.83 +/- 1.02, and BMI = 20.1 +/- 2.3. CF patients with liver cirrhosis generally had mild-to-moderate lung disease, with average FVC and FEV1 values of 97.1 +/- 16.5% of predicted and 87.9 +/- 23.5% of predicted, respectively. Genetic analysis showed high frequency of F508del mutation in the group with cirrhosis (90.6%). The prevalence of liver cirrhosis in our CF population older than 2 years was 16.6%. Patients with pancreatic insufficiency and severe CFTR mutations, especially F508del, were exposed to higher risk of developing liver cirrhosis. Liver cirrhosis has no significant impact on the pulmonary function and the nutritional status, until the end-stage liver disease.

Implementation of newborn screening for cystic fibrosis in Norway. Results from the first three years.

PubMed

Lundman, Emma; Gaup, H Junita; Bakkeheim, Egil; Olafsdottir, Edda J; Rootwelt, Terje; Storrøsten, Olav Trond; Pettersen, Rolf D

2016-05-01

Norway introduced newborn screening for cystic fibrosis (CF) March 1, 2012. We present results from the first three years of the national newborn CF screening program. Positive primary screening of immunoreactive trypsinogen (IRT) was followed by DNA testing of the Cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infants with two CFTR mutations were reported for diagnostic follow-up. Of 181,859 infants tested, 1454 samples (0.80%) were assessed for CFTR mutations. Forty children (1:4546) had two CFTR mutations, of which only 21 (1:8660) were confirmed to have a CF diagnosis. The CFTR mutations differed from previously clinically diagnosed CF patients, and p.R117H outnumbered p.F508del as the most frequent mutation. One child with a negative IRT screening test was later clinically diagnosed with CF. The CF screening program identified fewer children with a conclusive CF diagnosis than expected. Our data suggest a revision of the IRT/DNA protocol. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Serum leptin and cytokines in whole blood in relation to clinical and nutritional status in cystic fibrosis.

PubMed

Schmitt-Grohé, Sabina; Hippe, Valerie; Igel, Michael; von Bergmann, Klaus; Posselt, Hans G; Krahl, Andreas; Smaczny, Christina; Wagner, Thomas O F; Nikolaizik, Wilfried; Lentze, Michael J; Zielen, Stefan

2006-08-01

Leptin plays an important role in the energy balance and may be affected by hormonal and metabolic derangement associated with chronic disease. The aim of this study was to assess the correlation between leptin, proinflammatory cytokines and nutritional status with regard to clinical status in homozygous delta F 508 cystic fibrosis patients. Patients with mild (Shwachman score 71-100 points, group A) disease were compared with those with moderate disease (Shwachman score 41-55 points, group B) and age-matched controls (group C, n = 22). Leptin was assessed by enzyme-linked immunosorbent assay and cytokines (interleukin-8, tumor necrosis factor alpha) before and after stimulation with 5 ng lipopolysaccharide by a chemiluminescent immunometric assay. Twenty-two patients were recruited for each group (median A/B/C forced expiratory volume in 1 second 80%/59%/-; median age 12/13.5/12.5 years). Leptin (median 3.25/2.65/3.3 pg/mL; P = 0.083) and body mass index were lower (group A/B/C 18.55/16.75/20.5 kg/m(2); P = 0.023), but dietary intake was significantly higher (group A/B/C 50.5/68/43 kcal/kg body weight; P = 0.026) in moderate disease. Cytokines before stimulation with lipopolysaccharide were highest in moderate disease, but there was no significant difference after stimulation (interleukin-8 median A/B/C before--15/25.1/8.0 pg/mL, P < 0.005; after--570.5/573.5/415.5 pg/mL, not significant; tumor necrosis factor alpha median A/B/C 43/56/30 pg/mL, P < 0.0001; 580/427/720.5 pg/mL, not significant.). There is a physiological regulation of leptin even in more advanced states of disease with significantly lower body mass index than controls. However, our data do not support the idea of elevated cytokine levels inducing anorexia in homozygous delta F 508 cystic fibrosis patients.

Analysis of 16 cystic fibrosis mutations in Mexican patients

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

Villalobos-Torres, C.; Rojas-Martinez, A.; Barrera-Saldana, H.A.

1997-04-14

We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation AF508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for AF508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3849 + 10 kb C{r_arrow}T, N1303K, S549N, and 621 + 1 G{r_arrow}T) were detected, andmore » these accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used. 20 refs., 2 tabs.« less

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.

Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics.

PubMed

Strug, Lisa J; Gonska, Tanja; He, Gengming; Keenan, Katherine; Ip, Wan; Boëlle, Pierre-Yves; Lin, Fan; Panjwani, Naim; Gong, Jiafen; Li, Weili; Soave, David; Xiao, Bowei; Tullis, Elizabeth; Rabin, Harvey; Parkins, Michael D; Price, April; Zuberbuhler, Peter C; Corvol, Harriet; Ratjen, Felix; Sun, Lei; Bear, Christine E; Rommens, Johanna M

2016-10-15

Cystic fibrosis is realizing the promise of personalized medicine. Recent advances in drug development that target the causal CFTR directly result in lung function improvement, but variability in response is demanding better prediction of outcomes to improve management decisions. The genetic modifier SLC26A9 contributes to disease severity in the CF pancreas and intestine at birth and here we assess its relationship with disease severity and therapeutic response in the airways. SLC26A9 association with lung disease was assessed in individuals from the Canadian and French CF Gene Modifier consortia with CFTR-gating mutations and in those homozygous for the common Phe508del mutation. Variability in response to a CFTR-directed therapy attributed to SLC26A9 genotype was assessed in Canadian patients with gating mutations. A primary airway model system determined if SLC26A9 shows modification of Phe508del CFTR function upon treatment with a CFTR corrector. In those with gating mutations that retain cell surface-localized CFTR we show that SLC26A9 modifies lung function while this is not the case in individuals homozygous for Phe508del where cell surface expression is lacking. Treatment response to ivacaftor, which aims to improve CFTR-channel opening probability in patients with gating mutations, shows substantial variability in response, 28% of which can be explained by rs7512462 in SLC26A9 (P = 0.0006). When homozygous Phe508del primary bronchial cells are treated to restore surface CFTR, SLC26A9 likewise modifies treatment response (P = 0.02). Our findings indicate that SLC26A9 airway modification requires CFTR at the cell surface, and that a common variant in SLC26A9 may predict response to CFTR-directed therapeutics.

Patterns of haplotypes for 92 cystic fibrosis mutations: Variability, association and recurrence

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

Morral, N.; Llevadot, R.; Estivill, X.

1994-09-01

Most CFTR mutations are very uncommon among the cystic fibrosis population, with frequencies of less than 1%, and many are found only in specific areas. We have analyzed 92 CF mutations for several markers (4 microsatellites and 3 other polymorphisms) scattered in the CFTR gene. Haplotypes associated with these mutations can be used as a framework in the screening of chromosomes carrying unknown mutations. The association between mutation and haplotype reduces the number of mutations it is necessary to search for to a maximum of 16 for the same haplotype. Only mutations {triangle}F508, G542X and N1303K are associated with moremore » than one haplotype as a result of slippage at more than one microsatellite loci, suggesting that these three are the most ancient CF mutations. Recurrence has been found for at least 7 mutations: H199Y, R347P, L558S, R553X, 2184insA, 3272-26A{r_arrow}G, 3849+10kbC{r_arrow}T and R1162X. Also microsatellite analysis of chromosomes of several ethnic origins (Czech, Italian, Russian, Slovac and Spanish) suggested that possibility of three or more independent origins for mutations R334W, R347P, R1162X, and 3849+10kbC{r_arrow}T, which was confirmed by analysis of markers flanking these mutations.« less

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.

Clinical expression of patients with the D1152H CFTR mutation.

PubMed

Terlizzi, Vito; Carnovale, Vincenzo; Castaldo, Giuseppe; Castellani, Carlo; Cirilli, Natalia; Colombo, Carla; Corti, Fabiola; Cresta, Federico; D'Adda, Alice; Lucarelli, Marco; Lucidi, Vincenzina; Macchiaroli, Annamaria; Madarena, Elisa; Padoan, Rita; Quattrucci, Serena; Salvatore, Donatello; Zarrilli, Federica; Raia, Valeria

2015-07-01

Discordant results were reported on the clinical expression of subjects bearing the D1152H CFTR mutation, and also for the small number of cases reported so far. A retrospective review of clinical, genetic and biochemical data was performed from individuals homozygous or compound heterozygous for the D1152H mutation followed in 12 Italian cystic fibrosis (CF) centers. 89 subjects carrying at least D1152H on one allele were identified. 7 homozygous patients had very mild clinical expression. Over half of the 74 subjects compound heterozygous for D1152H and a I-II-III class mutation had borderline or pathological sweat test and respiratory or gastrointestinal symptoms; one third had pulmonary bacteria colonization and 10/74 cases had complications (i.e. diabetes, allergic bronchopulmonary aspergillosis, and hemoptysis). However, their clinical expression was less severe as compared to a group of CF patients homozygous for the F508del mutation. Finally, 8 subjects compound heterozygous for D1152H and a IV-V class mutation showed very mild disease. The natural history of subjects bearing the D1152H mutation is widely heterogeneous and is influenced by the mutation in trans. Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Cyclic crack growth behavior of reactor pressure vessel steels in light water reactor environments

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

Van Der Sluys, W.A.; Emanuelson, R.H.

1986-01-01

During normal operation light water reactor (LWR) pressure vessels are subjected to a variety of transients resulting in time varying stresses. Consequently, fatigue and environmentally assisted fatigue are growth mechanisms relevant to flaws in these pressure vessels. In order to provide a better understanding of the resistance of nuclear pressure vessel steels to flaw growth process, a series of fracture mechanics experiments were conducted to generate data on the rate of cyclic crack growth in SA508-2 and SA533b-1 steels in simulated 550/sup 0/F boiling water reactor (BWR) and 550/sup 0/F pressurized water reactor (PWR) environments. Areas investigated over the coursemore » of the test program included the effects of loading frequency and r ratio (Kmin-Kmax) on crack growth rate as a function of the stress intensity factor (deltaK) range. In addition, the effect of sulfur content of the test material on the cyclic crack growth rate was studied. Cyclic crack growth rates were found to be controlled by deltaK, R ratio, and loading frequency. The sulfur impurity content of the reactor pressure vessel steels studied had a significant effect on the cyclic crack growth rates. The higher growth rates were always associated with materials of higher sulfur content. For a given level of sulfur, growth rates were in a 550/sup 0/F simulated BWR environment than in a 550/sup 0/F simulated PWR environment. In both environments cyclic crack growth rates were a strong function of the loading frequency.« less

Activation of the Cystic Fibrosis Transmembrane Conductance Regulator by the Flavonoid Quercetin

PubMed Central

Pyle, Louise C.; Fulton, Jennifer C.; Sloane, Peter A.; Backer, Kyle; Mazur, Marina; Prasain, Jeevan; Barnes, Stephen; Clancy, J. P.; Rowe, Steven M.

2010-01-01

Therapies to correct the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) folding defect require sensitive methods to detect channel activity in vivo. The β2 adrenergic receptor agonists, which provide the CFTR stimuli commonly used in nasal potential difference assays, may not overcome the channel gating defects seen in ΔF508 CFTR after plasma membrane localization. In this study, we identify an agent, quercetin, that enhances the detection of surface ΔF508 CFTR, and is suitable for nasal perfusion. A screen of flavonoids in CFBE41o− cells stably transduced with ΔF508 CFTR, corrected to the cell surface with low temperature growth, revealed that quercetin stimulated an increase in the short-circuit current. This increase was dose-dependent in both Fisher rat thyroid and CFBE41o− cells. High concentrations inhibited Cl− conductance. In CFBE41o− airway cells, quercetin (20 μg/ml) activated ΔF508 CFTR, whereas the β2 adrenergic receptor agonist isoproterenol did not. Quercetin had limited effects on cAMP levels, but did not produce detectable phosphorylation of the isolated CFTR R-domain, suggesting an activation independent of channel phosphorylation. When perfused in the nares of Cftr+ mice, quercetin (20 μg/ml) produced a hyperpolarization of the potential difference that was absent in Cftr−/− mice. Finally, quercetin-induced, dose-dependent hyperpolarization of the nasal potential difference was also seen in normal human subjects. Quercetin activates CFTR-mediated anion transport in respiratory epithelia in vitro and in vivo, and may be useful in studies intended to detect the rescue of ΔF508 CFTR by nasal potential difference. PMID:20042712

Strategies for the etiological therapy of cystic fibrosis.

PubMed

Maiuri, Luigi; Raia, Valeria; Kroemer, Guido

2017-11-01

Etiological therapies aim at repairing the underlying cause of cystic fibrosis (CF), which is the functional defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein owing to mutations in the CFTR gene. Among these, the F508del CFTR mutation accounts for more than two thirds of CF cases worldwide. Two somehow antinomic schools of thought conceive CFTR repair in a different manner. According to one vision, drugs should directly target the mutated CFTR protein to increase its plasma membrane expression (correctors) or improve its ion transport function (potentiators). An alternative strategy consists in modulating the cellular environment and proteostasis networks in which the mutated CFTR protein is synthesized, traffics to its final destination, the plasma membrane, and is turned over. We will analyze distinctive advantages and drawbacks of these strategies in terms of their scientific and clinical dimensions, and we will propose a global strategy for CF research and development based on a reconciliatory approach. Moreover, we will discuss the utility of preclinical biomarkers that may guide the personalized, patient-specific implementation of CF therapies.

Strategies for the etiological therapy of cystic fibrosis

PubMed Central

Maiuri, Luigi; Raia, Valeria; Kroemer, Guido

2017-01-01

Etiological therapies aim at repairing the underlying cause of cystic fibrosis (CF), which is the functional defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein owing to mutations in the CFTR gene. Among these, the F508del CFTR mutation accounts for more than two thirds of CF cases worldwide. Two somehow antinomic schools of thought conceive CFTR repair in a different manner. According to one vision, drugs should directly target the mutated CFTR protein to increase its plasma membrane expression (correctors) or improve its ion transport function (potentiators). An alternative strategy consists in modulating the cellular environment and proteostasis networks in which the mutated CFTR protein is synthesized, traffics to its final destination, the plasma membrane, and is turned over. We will analyze distinctive advantages and drawbacks of these strategies in terms of their scientific and clinical dimensions, and we will propose a global strategy for CF research and development based on a reconciliatory approach. Moreover, we will discuss the utility of preclinical biomarkers that may guide the personalized, patient-specific implementation of CF therapies. PMID:28937684

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function*

PubMed Central

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

2014-01-01

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile508, Asn509, and Asp511) in communicating with converter domain residue Arg759. We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle. PMID:24627474

Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop.

PubMed

Ehrhardt, Annette; Chung, W Joon; Pyle, Louise C; Wang, Wei; Nowotarski, Krzysztof; Mulvihill, Cory M; Ramjeesingh, Mohabir; Hong, Jeong; Velu, Sadanandan E; Lewis, Hal A; Atwell, Shane; Aller, Steve; Bear, Christine E; Lukacs, Gergely L; Kirk, Kevin L; Sorscher, Eric J

2016-01-22

In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of nasal potential difference in cftr knockout and F508del-CFTR mice.

PubMed

Saussereau, Emilie Lyne; Roussel, Delphine; Diallo, Siradiou; Debarbieux, Laurent; Edelman, Aleksander; Sermet-Gaudelus, Isabelle

2013-01-01

Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V(TE)) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V(TE) in CF mice must be well characterized for correct interpretation. We performed V(TE) measurements in large-scale studies of two mouse models of CF--B6;129 cftr knockout and FVB F508del-CFTR--and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice. We determined the typical V(TE) values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl(-) solution was considered to indicate a normal response. These data will make it possible to interpret changes in nasal V(TE) in mouse models of CF, in future preclinical studies.

Protein disulphide isomerase is required for signal peptide peptidase-mediated protein degradation.

PubMed

Lee, Seong-Ok; Cho, Kwangmin; Cho, Sunglim; Kim, Ilkwon; Oh, Changhoon; Ahn, Kwangseog

2010-01-20

The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2-induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA-mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate-binding mutant of PDI, but not the catalytically inactive mutant, dominant-negatively inhibited US2-mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP-mediated degradation of CD3delta but not Derlin-1-dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP-mediated ER-associated degradation machinery.

Role of SRC-3delta4 in the Progression and Metastasis of Castration-Resistant Prostate Cancer

DTIC Science & Technology

2014-12-01

tyrosine phosphorylation of SRC-3∆4, which was inhibited by the treatment with EGFR inhibitor AG1478. Mutation of Y1159 to phenylalanine (Y1159F...Y1159 to phenylalanine (Y1159F) greatly reduced SRC-3∆4/AR interaction that is stimulated by EGF. Figure 7 Overexpression of SRC-3∆4 promoted...adhesion turnover and matrix metalloproteinase expression. Cancer research 68, 5460-5468. 6. Chung, A.C., Zhou, S., Liao, L ., Tien, J.C., Greenberg

CFTR Genotype and Maximal Exercise Capacity in Cystic Fibrosis: A Cross-sectional Study.

PubMed

Radtke, Thomas; Hebestreit, Helge; Gallati, Sabina; Schneiderman, Jane E; Braun, Julia; Stevens, Daniel; Hulzebos, Erik Hj; Takken, Tim; Boas, Steven R; Urquhart, Don S; Lands, Larry C; Tejero, Sergio; Sovtic, Aleksandar; Dwyer, Tiffany; Petrovic, Milos; Harris, Ryan A; Karila, Chantal; Savi, Daniela; Usemann, Jakob; Mei-Zahav, Meir; Hatziagorou, Elpis; Ratjen, Felix; Kriemler, Susi

2018-02-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human skeletal muscle cells. Variations of CFTR dysfunction among patients with cystic fibrosis may be an important determinant of maximal exercise capacity in cystic fibrosis. Previous studies on the relationship between CFTR genotype and maximal exercise capacity are scarce and contradictory. This study was designed to explore factors influencing maximal exercise capacity, expressed as peak oxygen uptake (V.O2peak), with a specific focus on CFTR genotype in children and adults with cystic fibrosis. In an international, multicenter, cross-sectional study, we collected data on CFTR genotype and cardiopulmonary exercise tests in patients with cystic fibrosis who were ages 8 years and older. CFTR mutations were classified into functional classes I–V. The final analysis included 726 patients (45% females; age range, 8–61 yr; forced expiratory volume in 1 s, 16 to 123% predicted) from 17 cystic fibrosis centers in North America, Europe, Australia, and Asia, all of whom had both valid maximal cardiopulmonary exercise tests and complete CFTR genotype data. Overall, patients exhibited exercise intolerance (V.O2peak, 77.3 ± 19.1% predicted), but values were comparable among different CFTR classes. We did not detect an association between CFTR genotype functional classes I–III and either V.O2peak (percent predicted) (adjusted β = −0.95; 95% CI, −4.18 to 2.29; P = 0.57) or maximum work rate (Wattmax) (adjusted β = −1.38; 95% CI, −5.04 to 2.27; P = 0.46) compared with classes IV–V. Those with at least one copy of a F508del-CFTR mutation and one copy of a class V mutation had a significantly lower V.O2peak (β = −8.24%; 95% CI, −14.53 to −2.99; P = 0.003) and lower Wattmax (adjusted β = −7.59%; 95% CI, −14.21 to −0.95; P = 0.025) than those with two copies of a class II mutation. On the basis of linear regression analysis adjusted for relevant confounders, lung function and body mass index were associated with V.O2peak. CFTR functional genotype class was not associated with maximal exercise capacity in patients with cystic fibrosis overall, but those with at least one copy of a F508del-CFTR mutation and a single class V mutation had lower maximal exercise capacity.

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

[Historical compilation of cystic fibrosis].

PubMed

Navarro, Salvador

2016-01-01

Cystic fibrosis is the most common life-shortening recessively inherited disorder in the Caucasian population. The genetic mutation that most frequently provokes cystic fibrosis (ΔF508) appeared at least 53,000years ago. For many centuries, the disease was thought to be related to witchcraft and the "evil eye" and it was only in 1938 that Dorothy H. Andersen characterized this disorder and suspected its genetic origin. The present article reviews the pathological discoveries and diagnostic and therapeutic advances made in the last 75 years. The review ends with some considerations for the future. Copyright © 2015 Elsevier España, S.L.U. and AEEH y AEG. All rights reserved.

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

[Dehydration and metabolic alkalosis: an unusual presentation of cystic fibrosis in an infant].

PubMed

Aranzamendi, Roberto J; Breitman, Fanny; Asciutto, Carolina; Delgado, Norma; Castaños, Claudio

2008-10-01

Cystic fibrosis (CF) may present during neonatal period with classic clinic symptoms related to the disease. The severity of the disease is multifactorial, one of the factors depends on the level of activity of the CFTR protein, which is related with the mutation type that affects the patient. An infant is presented who developed recurrent episodes of vomiting, anorexia, weight loss, dehydration and electrolyte abnormalities, such as metabolic alkalosis, hyponatremia, hypokalemia and hypochloremia. CF was diagnosed after the third episode showing an unusual and not very publicized presentation of the disease. Mutations !F 508 and 2789+5G-A were found. CF should be considered in patients of any age, but particularly in infants, presenting with anorexia, vomiting, failure to thrive, that are associated with recurrent episodes of hyponatremic hypochloremic, dehydration with metabolic alkalosis unexplained by other causes, even in the absence of respiratory or gastrointestinal symptoms or failure to thrive.

The cystic fibrosis transmembrane conductance regulator (CFTR) and its stability.

PubMed

Meng, Xin; Clews, Jack; Kargas, Vasileios; Wang, Xiaomeng; Ford, Robert C

2017-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for the disease cystic fibrosis (CF). It is a membrane protein belonging to the ABC transporter family functioning as a chloride/anion channel in epithelial cells around the body. There are over 1500 mutations that have been characterised as CF-causing; the most common of these, accounting for ~70 % of CF cases, is the deletion of a phenylalanine at position 508. This leads to instability of the nascent protein and the modified structure is recognised and then degraded by the ER quality control mechanism. However, even pharmacologically 'rescued' F508del CFTR displays instability at the cell's surface, losing its channel function rapidly and it is rapidly removed from the plasma membrane for lysosomal degradation. This review will, therefore, explore the link between stability and structure/function relationships of membrane proteins and CFTR in particular and how approaches to study CFTR structure depend on its stability. We will also review the application of a fluorescence labelling method for the assessment of the thermostability and the tertiary structure of CFTR.

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.

Antenatal testing for cystic fibrosis in Cuba, 1988-2011.

PubMed

Collazo, Teresa; López, Ixchel; Clark, Yulia; Piloto, Yaixa; González, Laura; Gómez, Manuel; García, Marileivis; Reyes, Lidice; Rodríguez, Fidel

2014-01-01

INTRODUCTION Cystic fibrosis is a multisystem autosomal recessive disease with wide variability in clinical severity. It is incurable and characterized by elevated and premature mortality, as well as poor quality of life. Its frequency, lethality and devastating impact on both the physical and psychological wellbeing of patients and their families, make it a serious health problem. Its frequency in Cuba is 1 in 9862 live births, where marked molecular heterogeneity of the CFTR gene makes molecular diagnosis difficult. Six mutations have been identified that together enable molecular characterization of only 55.5% of cystic fibrosis chromosomes. This paper presents national results of antenatal diagnostic testing, using direct and indirect methods, for detection of cystic fibrosis. OBJECTIVE Characterize the Cuban public health system's experience with antenatal molecular testing for cystic fibrosis from 1988 through 2011. METHODS A retrospective descriptive study was conducted with results of antenatal diagnostic testing of amniotic fluid, performed nationwide from 1988 through 2011, for 108 fetuses of couples with some risk of having children affected by cystic fibrosis, who requested testing. Polymerase chain reaction detected mutations p.F508del, p.G542X, p.R1162X, p.R334W, p.R553X and c.3120+1G>A, and markers XV2C and KM19. Data were analyzed using absolute frequencies and percentages, and presented in tables. RESULTS For 93 cases (86.1%), testing for cystic fibrosis was done using direct analysis of mutations p.F508del, p.G542X, p.R1162X, p.R334W, p.R553X and c.3120+1G>A; five cases (4.6%) were tested indirectly using markers XV2C/Taq I and KM19/Pst I; and 10 (9.3%) were tested using a combination of the two methods. A total of 72 diagnoses (66.7% of studies done) were concluded, of which there were 20 healthy fetuses, 16 affected, 27 carrier, and 9 who were either healthy or carriers of an unknown mutation. CONCLUSIONS Direct or indirect molecular study was successfully used in over half of antenatal tests requested by couples throughout Cuba at risk of having children affected by cystic fibrosis, which is of great social value because of CF's burden on affected persons and their families.

High-temperature elastic-plastic and creep properties for SA533 Grade B Class I and SA508 materials

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

Reddy, G.B.; Ayres, D.J.

1982-12-01

High temperature elastic-plastic and creep properties are presented for SA533 Grade B Class I and SA508 Class II materials. These properties are derived from tests conducted at Combustion Engineering Material and Metallurgical Laboratories and cover the temperature range of 70/sup 0/F to 1200/sup 0/F.

Quantifying Acoustic Uncertainty Due to Marine Mammals and Fish Near the Shelfbreak Front off Cape Hatteras

DTIC Science & Technology

2014-09-30

and Fish Near the Shelfbreak Front off Cape Hatteras James F. Lynch MS #11, Woods Hole Oceanographic Institution Woods Hole, MA 02543 Phone...508) 289-2230 Fax: (508) 457-2194 e-mail: jlynch@whoi.edu Glen Gawarkiewicz MS#21, Woods Hole Oceanographic Institution Woods Hole, MA 02543...Phone: (508) 289-2913 Fax: (508) 457-2181 e-mail: gleng@whoi.edu Ying-Tsong Lin MS #11, Woods Hole Oceanographic Institution Woods Hole, MA 02543

Primary hyperoxaluria type 1: is genotyping clinically helpful?

PubMed

Leumann, Ernst; Hoppe, Bernd

2005-05-01

There is some controversy about the value of mutation analysis in the management of primary hyperoxaluria type 1 (PH1). About 50 different mutations of the AGXT gene encoding the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) are currently known. The three most common mutations in the Western population account for less than half of the mutant alleles, and no simple screening test is available. Does the genotype help in diagnosis, prognosis and therapy? Definitive diagnosis is indispensable if liver transplantation is considered and can under certain circumstances be established by mutation analysis, but a liver biopsy is still necessary to determine AGT activity in a number of cases. Prognosis is difficult to assess due to a large clinical variation, despite identical mutations. Although the homozygous 508G>A (Gly170Arg) mutation appears to be associated with a better (and 33insC with a worse) prognosis, there are too many exceptions for precise prediction. Pyridoxine responsiveness can be anticipated in some genotypes (508G>A (Gly170Arg) and 454T>A (Phe153Ile)), but it should still be tested for in all patients. Genetic testing is thus clinically helpful but has clear limitations.

Changing Incidence of Cystic Fibrosis in Wisconsin, USA

PubMed Central

Bersie, Rachel; Hoffman, Gary; Rock, Michael; Baker, Mei; Farrell, Philip M.; Simpson, Pippa; Levy, Hara

2015-01-01

Summary Rationale Previous investigations of cystic fibrosis (CF) incidence in Massachusetts, Colorado, and Minnesota (USA) yielded contradictory results, particularly regarding allele p.Phe508del; the racial compositions of the cohorts were not reported. Objectives To clarify discrepancies in reported incidence with the ultimate goal of improving screening and quality of care, we assessed CF incidence, stratified by race and mutations in cystic fibrosis transmembrane conductance regulator (CFTR), in Wisconsin (USA) from 1994 to 2011. Methods Data on patients diagnosed with CF (N=283), CFTR genotypes, CF carriers, and birth rate were collected. All data were categorized by racial background of the birth mother and the incidence of CF births was accordingly adjusted. Spearman’s nonparametric rank correlation and Fisher’s exact test were performed for continuous and categorical variables, respectively. Trends over time were fitted with a cubic spline. Results We detected a trending increase in CF cases (range within all data 1.67–2.98 per 10,000 births per year), homozygous p.Phe508del cases (0.57–1.79 per 10,000), heterozygous p.Phe508del cases (0.29–1.55 per 10,000), and cases lacking p. Phe508del (0–0.45 per 10,000). Both the number of cases lacking the p.Phe508del mutation per year and the number of cases lacking p.Phe508del per 10,000 births significantly increased (P=0.05) from 1994 to 2011; the increase in overall incidence was not significant. The number of carriers identified through newborn screening significantly increased within the non-Hispanic Black (P=0.0.021) and Hispanic (P=0.003) populations. Conclusion The racial composition of the CF cohort is changing in Wisconsin, possibly influencing disease detection, care, and outcome. PMID:26258862

Changing incidence of cystic fibrosis in Wisconsin, USA.

PubMed

Parker-McGill, Katelyn; Nugent, Melodee; Bersie, Rachel; Hoffman, Gary; Rock, Michael; Baker, Mei; Farrell, Philip M; Simpson, Pippa; Levy, Hara

2015-11-01

Previous investigations of cystic fibrosis (CF) incidence in Massachusetts, Colorado, and Minnesota (USA) yielded contradictory results, particularly regarding allele p.Phe508del; the racial compositions of the cohorts were not reported. To clarify discrepancies in reported incidence with the ultimate goal of improving screening and quality of care, we assessed CF incidence, stratified by race and mutations in cystic fibrosis transmembrane conductance regulator (CFTR), in Wisconsin (USA) from 1994 to 2011. Data on patients diagnosed with CF (N = 283), CFTR genotypes, CF carriers, and birth rate were collected. All data were categorized by racial background of the birth mother and the incidence of CF births was accordingly adjusted. Spearman's nonparametric rank correlation and Fisher's exact test were performed for continuous and categorical variables, respectively. Trends over time were fitted with a cubic spline. We detected a trending increase in CF cases (range within all data 1.67-2.98 per 10,000 births per year), homozygous p.Phe508del cases (0.57-1.79 per 10,000), heterozygous p.Phe508del cases (0.29-1.55 per 10,000), and cases lacking p.Phe508del (0-0.45 per 10,000). Both the number of cases lacking the p.Phe508del mutation per year and the number of cases lacking p.Phe508del per 10,000 births significantly increased (P = 0.05) from 1994 to 2011; the increase in overall incidence was not significant. The number of carriers identified through newborn screening significantly increased within the non-Hispanic Black (P = 0.0.021) and Hispanic (P = 0.003) populations. The racial composition of the CF cohort is changing in Wisconsin, possibly influencing disease detection, care, and outcome. © 2015 Wiley Periodicals, Inc.

Modulating the folding of P-glycoprotein and cystic fibrosis transmembrane conductance regulator truncation mutants with pharmacological chaperones.

PubMed

Wang, Ying; Loo, Tip W; Bartlett, M Claire; Clarke, David M

2007-03-01

Cystic fibrosis transmembrane conductance regulator (CFTR) and P-glycoprotein (P-gp) are ATP-binding cassette (ABC) transporters that have two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Defective folding of CFTR lacking phenylalanine 508 (DeltaPhe508) in NBD1 is the most common cause of cystic fibrosis. The Phe508 position seems to be universally important in ABC transporters because deletion of the equivalent residue (Tyr490) in P-gp also inhibits maturation of the protein. The pharmacological chaperone VRT-325 can repair the DeltaPhe508-type folding defects in P-gp or CFTR. VRT-325 may repair the folding defects by promoting dimerization of the two NBDs or by promoting folding of the TMDs. To distinguish between these two mechanisms, we tested the ability of VRT-325 to promote folding of truncation mutants lacking one or both NBDs. Sensitivity to glycosidases was used as an indirect indicator of folding. It was found that VRT-325 could promote maturation of truncation mutants lacking NBD2. Truncation mutants of CFTR or P-gp lacking both NBDs showed deficiencies in core-glycosylation that could be partially reversed by carrying out expression in the presence of VRT-325. The results show that dimerization of the two NBDs to form a "nucleotide-sandwich" structure or NBD interactions with the TMDs are not essential for VRT-325 enhancement of folding. Instead, VRT-325 can promote folding of the TMDs alone. The ability of VRT-325 to promote core-glycosylation of the NBD-less truncation mutants suggests that one mechanism whereby the compound enhances folding is by promoting proper insertion of TM segments attached to the glycosylated loops so that they adopt an orientation favorable for glycosylation.

Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

PubMed

Vernon, Robert M; Chong, P Andrew; Lin, Hong; Yang, Zhengrong; Zhou, Qingxian; Aleksandrov, Andrei A; Dawson, Jennifer E; Riordan, John R; Brouillette, Christie G; Thibodeau, Patrick H; Forman-Kay, Julie D

2017-08-25

Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Ivacaftor treatment of cystic fibrosis patients with the G551D mutation: a review of the evidence.

PubMed

Kotha, Kavitha; Clancy, John P

2013-10-01

Cystic fibrosis (CF) is a recessive disorder caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. CFTR protein is a chloride and bicarbonate channel that is critical for normal epithelial ion transport and hydration of epithelial surfaces. Current CF care is supportive, but recent breakthroughs have occurred with the advent of novel therapeutic strategies that assist the function of mutant CFTR proteins. The development and key clinical trial results of ivacaftor, a small molecule that targets gating defects in disease-causing CFTR mutations including G551D CFTR, are summarized in this review. The G551D mutation is reasonably common in the CF patient population and produces a CFTR protein that localizes normally to the plasma membrane, but fails to open in response to cellular cues. Ivacaftor treatment produces dramatic improvements in lung function, weight, lung disease stability, patient-reported outcomes, and CFTR biomarkers in patients with CF harboring the G551D CFTR mutation compared with placebo controls and patients with two copies of the common F508del CFTR mutation. The unprecedented success of ivacaftor treatment for the G551D CF patient population has generated excitement in the CF care community regarding the expansion of its use to other CF patient populations with primary or secondary gating defects.

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

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.

Nature and Recurrence of AVPR2 Mutations in X-linked Nephrogenic Diabetes Insipidus

PubMed Central

Bichet, Daniel G.; Birnbaumer, Mariel; Lonergan, Michèle; Arthus, Marie-Françoise; Rosenthal, Walter; Goodyer, Paul; Nivet, Hubert; Benoit, Stéphane; Giampietro, Philip; Simonetti, Simonetta; Fish, Alfred; Whitley, Chester B.; Jaeger, Philippe; Gertner, Joseph; New, Maria; DiBona, Francis J.; Kaplan, Bernard S.; Robertson, Gary L.; Hendy, Geoffrey N.; Fujiwara, T. Mary; Morgan, Kenneth

1994-01-01

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine-vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. We analyzed 31 independent NDI families to determine the nature and recurrence of AVPR2 mutations. Twenty-one new putative disease-causing mutations were identified: 113delCT, 253del35, 255del9, 274insG, V88M, R106C, 402delCT, C112R, Y124X, S126F, W164S, S167L, 684delTA, 804insG, W284X, A285P, W293X, R337X, and three large deletions or gene rearrangements. Five other mutations—R113W, Y128S, R137H, R181C, and R202C—that previously had been reported in other families were detected. There was evidence for recurrent mutation for four mutations (R113W, R137H, S167L, and R337X). Eight de novo mutation events were detected (274insG, R106C, Y128S, 167L [twice], R202C, 684delTA, and R337X). The origins were maternal (one), grandmaternal (one), and grandpaternal (six). In the 31 NDI families and 6 families previously reported by us, there is evidence both for mutation hot spots for nucleotide substitutions and for small deletions and insertions. More than half (58%) of the nucleotide substitutions in 26 families could be a consequence of 5-methylcytosine deamination at a CpG dinucleotide. Most of the small deletions and insertions could be attributed to slipped mispairing during DNA replication. PMID:8037205

Stable isotope ratios of marijuana. I. Carbon and nitrogen stable isotopes describe growth conditions.

PubMed

West, Jason B; Hurley, Janet M; Ehleringer, James R

2009-01-01

There remains significant uncertainty in illicit marijuana cultivation. We analyzed the delta(13)C and delta(15)N of 508 domestic samples from known U.S.A. counties, 31 seized from a single location, 5 samples grown in Mexico and Colombia, and 10 northwest border seizures. For a subset, inflorescences and leaves were analyzed separately. These data revealed a strong correspondence, with inflorescences having slightly higher delta(13)C and delta(15)N values than leaves. A framework for interpreting these results is introduced and evaluated. Samples identified as outdoor-grown by delta(13)C were generally recorded as such by the Drug Enforcement Administration (DEA). DEA-classified indoor-grown samples had the most negative delta(13)C values, consistent with indoor cultivation, although many were also in the outdoor-grown domain. Delta(15)N indicated a wide range of fertilizers across the dataset. Samples seized at the single location suggested multiple sources. Northwest border delta(13)C values suggested indoor growth, whereas for the Mexican and Colombian samples they indicated outdoor growth.

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator

PubMed Central

Chang, Xiu-bao; Mengos, April; Hou, Yue-xian; Cui, Liying; Jensen, Timothy J.; Aleksandrov, Andrei; Riordan, John R.; Gentzsch, Martina

2009-01-01

Summary The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated Δ F508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway. PMID:18682497

Analysis of Leigh syndrome mutations in the yeast SURF1 homolog reveals a new member of the cytochrome oxidase assembly factor family.

PubMed

Bestwick, Megan; Jeong, Mi-Young; Khalimonchuk, Oleh; Kim, Hyung; Winge, Dennis R

2010-09-01

Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F(249)T and Y(344)D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G(137)E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G(137)E Shy1 mutant phenocopied shy1Delta cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G(137)E Shy1 mutant revealed Coa2, Cox10, and a novel factor designated Coa4. Coa2 and Cox10 are previously characterized CcO assembly factors. Coa4 is a twin CX(9)C motif mitochondrial protein localized in the intermembrane space and associated with the inner membrane. Cells lacking Coa4 are depressed in CcO activity but show no impairment in Cox1 maturation or formation of the Shy1-stabilized Cox1 assembly intermediate. To glean insights into the functional role of Coa4 in CcO biogenesis, an unbiased suppressor screen of coa4Delta cells was conducted. Respiratory function of coa4Delta cells was restored by the overexpression of CYC1 encoding cytochrome c. Cyc1 is known to be important at an ill-defined step in the assembly and/or stability of CcO. This new link to Coa4 may begin to further elucidate the role of Cyc1 in CcO biogenesis.

Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice.

PubMed

Liu, Yu-Min; Liu, Wei; Jia, Jun-Shuang; Chen, Bang-Zhu; Chen, Heng-Wei; Liu, Yu; Bie, Ya-Nan; Gu, Peng; Sun, Yan; Xiao, Dong; Gu, Wei-Wang

2018-05-25

Hairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs. Generation of PLCD1 sgRNAs and Cas 9 mRNA was performed as described (Shao in Nat Protoc 9:2493-2512, 2014). PLCD1-modified mice (F0) were generated via co-microinjection of PLCD1-sgRNA and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Homozygous PLCD1-deficient mice (F1) were obtained by intercrossing of F0 mice with the similar mutation. PLCD1-modified mice (F0) showed progressive hair loss after birth and the genotype of CRISPR/Cas9-induced mutations in exon 2 of PLCD1 locus, suggesting the sgRNA is effective to cause mutations that lead to hair growth defect. Homozygous PLCD1-deficient mice (F1) displayed baldness in abdomen and hair sparse in dorsa. Histological abnormalities of the reduced number of hair follicles, irregularly arranged and curved hair follicles, epidermal hyperplasia and disturbed differentiation of epidermis were observed in the PLCD1-deficient mice. Moreover, the expression level of PLCD1 was significantly decreased, while the expression levels of other genes (i.e., Krt1, Krt5, Krt13, loricrin and involucrin) involved in the differentiation of hair follicle were remarkerably increased in skin tissues of PLCD1-deficient mice. In conclusion, we achieve PLCD1 KO mice by CRISPR/Cas9 technology, which provide a new animal model for hair development research, although homozygotes don't display completely hairless phenotype as expected.

mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

PubMed

Felício, V; Ramalho, A S; Igreja, S; Amaral, M D

2017-03-01

Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Management of male infertility due to congenital bilateral absence of vas deferens should not ignore the diagnosis of cystic fibrosis.

PubMed

Grzegorczyk, V; Rives, N; Sibert, L; Dominique, S; Macé, B

2012-10-01

Microsurgical or percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection (ICSI) are proposed to overcome male infertility due to congenital bilateral absence of vas deferens (CBAVD). CBAVD has been associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and consequently, genetic counselling has to be addressed before beginning ICSI procedure. However, management of male infertility due to CBAVD should not ignore a mild form of cystic fibrosis. We describe the case of cystic fibrosis late diagnosis performed in a 49-year-old infertile men with CBAVD. CFTR molecular testing detected two mutations F508del and A455E corresponding to a cystic fibrosis genotype. Pneumological evaluation revealed a severe obstructive respiratory disease, bronchiectasis and high sweat chloride levels. Symptoms consistent with a cystic fibrosis have to be identified in infertile men with CBAVD before beginning assisted reproductive procedures. © 2012 Blackwell Verlag GmbH.

76 FR 53144 - Agency Information Collection Activities: Form I-508 and Form I-508F, Extension of a Currently...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-25

..., Waiver of Rights, Privileges, Exemptions and Immunities. The Department of Homeland Security, U.S... Immunities. (3) Agency form number, if any, and the applicable component of the Department of Homeland...

Comparison of vectorial ion transport in primary murine airway and human sinonasal air-liquid interface cultures, models for studies of cystic fibrosis, and other airway diseases.

PubMed

Zhang, Shaoyan; Fortenberry, James A; Cohen, Noam A; Sorscher, Eric J; Woodworth, Bradford A

2009-01-01

The purpose of this study was to compare vectorial ion transport within murine trachea, murine nasal septa, and human sinonasal cultured epithelium. Our hypothesis is that murine septal epithelium, rather than trachea, will more closely mimic the electrophysiology properties of human sinonasal epithelium. Epithelium from murine trachea, murine septa, and human sinonasal tissue were cultured at an air-liquid interface to confluence and full differentiation. A limited number of homozygous dF508 epithelia were also cultured. Monolayers were mounted in modified Ussing chambers to investigate pharmacologic manipulation of ion transport. The change in forskolin-stimulated current (delta-I(SC), expressed as micro-A/cm(2)) in murine septal (n = 19; 16.84 +/- 2.09) and human sinonasal (n = 18; 12.15 +/- 1.93) cultures was significantly increased over murine tracheal cultures (n = 15; 6.75 +/- 1.35; p = 0.035 and 0.0005, respectively). Forskolin-stimulated I(SC) was inhibited by the specific cystic fibrosis transmembrane regulator (CFTR) inhibitor INH-172 (5 microM). No forskolin-stimulated I(SC) was shown in cultures of dF508 homozygous murine septal epithelium (n = 3). Murine septal I(SC) was largely inhibited by amiloride (12.03 +/- 0.66), whereas human sinonasal cultures had a very limited response (0.70 +/- 0.47; p < 0.0001). The contribution of CFTR to stimulated chloride current as measured by INH-172 was highly significantly different between all groups (murine septa, 19.51 +/- 1.28; human sinonasal, 11.12 +/- 1.58; murine trachea, 4.85 +/- 0.49; p < 0.0001). Human sinonasal and murine septal epithelial cultures represent a useful model for studying CFTR activity and may provide significant advantages over lower airway tissues for investigating upper and lower respiratory pathophysiology.

Plasma lipidomics reveals potential prognostic signatures within a cohort of cystic fibrosis patients

PubMed Central

Ollero, Mario; Astarita, Giuseppe; Guerrera, Ida Chiara; Sermet-Gaudelus, Isabelle; Trudel, Stéphanie; Piomelli, Daniele; Edelman, Aleksander

2011-01-01

Cystic fibrosis (CF) is associated with abnormal lipid metabolism. We have recently shown variations in plasma levels of several phosphatidylcholine (PC) and lysophopshatidylcholine (LPC) species related to disease severity in CF patients. Here our goal was to search for blood plasma lipid signatures characteristic of CF patients bearing the same mutation (F508del) and different phenotypes, and to study their correlation with forced expiratory volume in 1 s (FEV1) and Pseudomonas aeruginosa chronic infection, evaluated at the time of testing (t = 0) and three years later (t = 3). Samples from 44 F508del homozygotes were subjected to a lipidomic approach based on LC-ESI-MS. Twelve free fatty acids were positively correlated with FEV1 at t = 0 (n = 29). Four of them (C20:3n-9, C20:5n-3, C22:5n-3, and C22:6n-3) were also positively correlated with FEV1 three years later, along with PC(32:2) and PC(36:4) (n = 31). Oleoylethanolamide (OEA) was negatively correlated with FEV1 progression (n = 17). Chronically infected patients at t = 0 showed lower PC(32:2), PC(38:5), and C18:3n-3 and higher cholesterol, cholesterol esters, and triacylglycerols (TAG). Chronically infected patients at t = 3 showed significantly lower levels of LPC(18:0). These results suggest a potential prognostic value for some lipid signatures in, to our knowledge, the first longitudinal study aimed at identifying lipid biomarkers for CF. PMID:21335323

delta. -aminolevulinic acid dehydratase deficiency can cause. delta. -aminolevulinate auxotrophy in Escherichia coli

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

O'Neill, G.P.; Michelsen, U.; Soll, D.

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required {delta}-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar tomore » a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduce ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. Analysis of another class of ALA-requiring mutants showed that the auxotrophy of the hem-205 mutant could be relieved by either methionine or cysteine and that the mutation maps in the cysG gene, which encodes uroporphyrinogen III methylase. The properties of these nonleaky ALA-requiring strains suggest that ALA is involved more extensively in E. coli intermediary metabolism than has been appreciated to date.« less

76 FR 31972 - Agency Information Collection Activities: Form I-508 and Form I-508F, Extension of a Currently...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-02

..., Waiver of Rights, Privileges, Exemptions and Immunities; OMB Control No. 1615-0025. The Department of..., Privileges, Exemptions and Immunities. (3) Agency form number, if any, and the applicable component of the...

Microstructural Characterization of a Directionally-Solidified Ni-33 (at. %)Al-31Cr-3Mo Eutectic Alloy as a Function of Withdrawal Rate

NASA Technical Reports Server (NTRS)

Raj, S. V.; Locci, I. E.; Whittenberger, J. D.; Salem, J. A.

2000-01-01

The Ni-33 (at. %)Al-3lCr-3Mo eutectic alloy was directionally-solidified (DS) at different rates, V(sub I), varying between 2.5 to 508 mm/ h. Detailed qualitative and quantitative metallographic and chemical analyses were conducted on the directionally-solidified rods. The microstructures consisted of eutectic colonies with parallel lamellar NiAl/(Cr,Mo) plates for solidification rates at and below 12.7 mm/ h. Cellular eutectic microstructures were observed at higher solidification rates, where the plates exhibited a radial pattern. The microstructures were demonstrated to be fairly uniform throughout a 100 mm length of the DS zone by quantitative metallography. The average cell size, bar-d, decreased with increasing growth rate to a value of 125 microns at 508 mm/ h according to the relation bar-d (microns) approx. = 465 V(sup -0.22, sub I), where V(sub I) is in mm/ h. Both the average NiAl plate thickness, bar-Delta(sub NiAl), and the interlamellar spacing, bar-lambda, were observed to be constant for V(sub I) less than or = 50.8 mm/ h but decreased with increasing growth rate above this value as 0.93 bar-Delta(sub NiAl)(microns) = 61.2 V(sup -0.93, sub I) and bar-lambda (microns) = 47.7 V(sup -0.64, sub I), respectively. The present results are detailed on a microstructural map. Keywords Optical microscopy, microstructure, compounds intermetallic, directional solidification

Cystic Fibrosis in the African Diaspora.

PubMed

Stewart, Cheryl; Pepper, Michael S

2017-01-01

Identifying mutations that cause cystic fibrosis (CF) is important for making an early, unambiguous diagnosis, which, in turn, is linked to better health and a greater life expectancy. In patients of African descent, a molecular diagnosis is often confounded by the fact that the majority of investigations undertaken to identify causative mutations have been conducted on European populations, and CF-causing mutations tend to be population specific. We undertook a survey of published data with the aim of identifying causative CF mutations in patients of African descent in the Americas. We found that 1,584 chromosomes had been tested in only 6 countries, of which 876 alleles (55.3%) still remained unidentified. There were 59 mutations identified. Of those, 41 have been shown to cause CF, 17 have no associated functional studies, and one (R117H) is of varying clinical consequence. The most common mutations identified in the patients of African descent were: ΔF508 (29.4% identified in the United States, Colombia, Brazil, and Venezuela); 3120 + 1G>A (8.4% identified in Brazil, the United States, and Colombia); G85E (3.8% identified in Brazil); 1811 + 1.6kbA>G (3.7% identified in Colombia); and 1342 - 1G>C (3.1% identified in the United States). The majority of the mutations identified (81.4%) have been described in just one country. Our findings indicate that there is a need to fully characterize the spectrum of CF mutations in the diaspora to improve diagnostic accuracy for these patients and facilitate treatment.

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.

Detection of CFTR function and modulation in primary human nasal cell spheroids.

PubMed

Brewington, John J; Filbrandt, Erin T; LaRosa, F J; Ostmann, Alicia J; Strecker, Lauren M; Szczesniak, Rhonda D; Clancy, John P

2018-01-01

Expansion of CFTR modulators to patients with rare/undescribed mutations will be facilitated by patient-derived models quantifying CFTR function and restoration. We aimed to generate a personalized model system of CFTR function and modulation using non-surgically obtained nasal epithelial cells (NECs). NECs obtained by curettage from healthy volunteers and CF patients were expanded and grown in 3-dimensional culture as spheroids, characterized, and stimulated with cAMP-inducing agents to activate CFTR. Spheroid swelling was quantified as a proxy for CFTR function. NEC spheroids recapitulated characteristics of pseudostratified respiratory epithelia. When stimulated with forskolin/IBMX, spheroids swelled in the presence of functional CFTR, and shrank in its absence. Spheroid swelling quantified mutant CFTR restoration in F508del homozygous cells using clinically available CFTR modulators. NEC spheroids hold promise for understanding rare CFTR mutations and personalized modulator testing to drive evaluation for CF patients with common, rare or undescribed mutations. Portions of this data have previously been presented in abstract form at the 2016 meetings of the American Thoracic Society and the 2016 North American Cystic Fibrosis Conference. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Delta-Beta Thalassaemia in a Pathan Family.

PubMed

Ahmad, Saqib Qayyum; Zafar, Saerah Iffat; Malik, Hamid Saeed; Ahmed, Suhaib

2017-11-01

Delta-beta-thalassaemia (δβ-thalassaemia) is a rare type of thalassaemia which mostly results from deletion of δ and β genes with preservation of γ genes. δβ-thalassaemia is classified into (δβ)+ and (δβ)0 types. The (δβ)0-thalassemia is further divided into GγAγ(δβ)0-thalassaemia and Gγ(Aγδβ)0-thalassaemia. In heterozygous state, (δβ)0mutations give rise to phenotype resembling β-thalassaemia trait but with raised Hb-F, ranging from 5 to 20%, without a rise in Hb-A2. In homozygotes, the clinical picture is usually that of thalassaemia intermedia and the patients have 100% Hb-F. Workup of a 1-year child suffering from pallor, chronic ill health, and splenomegaly referred to our laboratory with the suspicion of β-thalassaemia, ultimately resulted in a diagnosis on polymerase chain reaction as having homozygous inversion/deletion Gγ(Aγδβ)0-thalassaemia. Her family members were also investigated.

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.

Analysis of MSH3 in endometrial cancers with defective DNA mismatch repair.

PubMed

Swisher, E M; Mutch, D G; Herzog, T J; Rader, J S; Kowalski, L D; Elbendary, A; Goodfellow, P J

1998-01-01

To clarify the origin of defective mismatch repair (MMR) in sporadic endometrial cancers with microsatellite instability (MSI), a thorough mutation analysis was performed on the human mismatch repair gene MSH3. Twenty-eight MSI-positive endometrial cancers were investigated for mutations in the human mismatch repair gene MSH3 using single-strand conformation variant (SSCV) analysis of all 24 exons. All variants were sequenced. Loss of heterozygosity was investigated at all MSH3 polymorphisms discovered. A subset of tumors were investigated for methylation of the 5' promoter region of MSH3 using Southern blot hybridization. An identical single-base deletion (delta A) predicted to result in a truncated proteins was discovered in six tumors (21.4%). This deletion occurs in a string of eight consecutive adenosine residues (A8). Because simple repeat sequences are unstable in cells with defective MMR, the observed mutation may be an effect, rather than a cause, of MSI. Evidence of inactivation of the second MSH3 allele in tumors with the delta A mutation would strongly support a causal role for these MSH3 mutations. However, there was no evidence of a second mutation, loss of sequences, or methylation of the promoter region in any of the tumors with the delta A mutation. Although the delta A mutation is a frequent event in sporadic MSI-positive endometrial cancers, it may not be causally associated with defective DNA MMR.

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.

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

PubMed

Voisin, Grégory; Bouvet, Guillaume F; Legendre, Pierre; Dagenais, André; Massé, Chantal; Berthiaume, Yves

2014-09-01

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients. Copyright © 2014 the American Physiological Society.

Mechanosensitive activation of CFTR by increased cell volume and hydrostatic pressure but not shear stress.

PubMed

Vitzthum, Constanze; Clauss, Wolfgang G; Fronius, Martin

2015-11-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that is essential for electrolyte and fluid homeostasis. Preliminary evidence indicates that CFTR is a mechanosensitive channel. In lung epithelia, CFTR is exposed to different mechanical forces such as shear stress (Ss) and membrane distention. The present study questioned whether Ss and/or stretch influence CFTR activity (wild type, ∆F508, G551D). Human CFTR (hCFTR) was heterologously expressed in Xenopus oocytes and the response to the mechanical stimulus and forskolin/IBMX (FI) was measured by two-electrode voltage-clamp experiments. Ss had no influence on hCFTR activity. Injection of an intracellular analogous solution to increase cell volume alone did not affect hCFTR activity. However, hCFTR activity was augmented by injection after pre-stimulation with FI. The response to injection was similar in channels carrying the common mutations ∆F508 and G551D compared to wild type hCFTR. Stretch-induced CFTR activation was further assessed in Ussing chamber measurements using Xenopus lung preparations. Under control conditions increased hydrostatic pressure (HP) decreased the measured ion current including activation of a Cl(-) secretion that was unmasked by the CFTR inhibitor GlyH-101. These data demonstrate activation of CFTR in vitro and in a native pulmonary epithelium in response to mechanical stress. Mechanosensitive regulation of CFTR is highly relevant for pulmonary physiology that relies on ion transport processes facilitated by pulmonary epithelial cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Complete Genome Sequence of Pseudomonas chlororaphis subsp. aurantiaca Reveals a Triplicate Quorum-Sensing Mechanism for Regulation of Phenazine Production

PubMed Central

Morohoshi, Tomohiro; Yamaguchi, Takahito; Xie, Xiaonan; Wang, Wen-zhao; Takeuchi, Kasumi; Someya, Nobutaka

2017-01-01

Pseudomonas chlororaphis subsp. aurantiaca StFRB508 regulates phenazine production through N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing. Two sets of AHL-synthase and AHL-receptor genes, phzI/phzR and aurI/aurR, have been identified from the incomplete draft genome of StFRB508. In the present study, the complete genome of StFRB508, comprising a single chromosome of 6,997,933 bp, was sequenced. The complete genome sequence revealed the presence of a third quorum-sensing gene set, designated as csaI/csaR. An LC-MS/MS analysis revealed that StFRB508 produced six types of AHLs, with the most important AHL being N-(3-hydroxyhexanoyl)-l-homoserine lactone (3-OH-C6-HSL). PhzI mainly catalyzed the biosynthesis of 3-OH-C6-HSL, while AurI and CsaI catalyzed that of N-hexanoyl-l-homoserine lactone and N-(3-oxohexanoyl)-l-homoserine lactone, respectively. A mutation in phzI decreased phenazine production, whereas that in aurI or csaI did not. A phzI aurI csaI triple mutant (508ΔPACI) did not produce phenazine. Phenazine production by 508ΔPACI was stimulated by exogenous AHLs and 3-OH-C6-HSL exerted the strongest effects on phenazine production at the lowest concentration tested (0.1 μM). The plant protection efficacy of 508ΔPACI against an oomycete pathogen was lower than that of wild-type StFRB508. These results demonstrate that the triplicate quorum-sensing system plays an important role in phenazine production by and the biocontrol activity of StFRB508. PMID:28239068

Thermodynamic properties of {Delta}H{sub f 298}{degree}, S{sub 298}{degree}, and C{sub p}(T) for 2-fluoro-2-methylpropane, {Delta}H{sub f 298}{degree} of fluorinated ethanes, and group additivity for fluoroalkanes

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

Yamada, Takahiro; Bozzelli, J.W.

1999-09-09

G2(MP2) composite calculations are performed to obtain thermodynamic properties ({Delta}H{sub f 298}{degree}, S{sub 298}{degree} and C{sub p}(T)'s) of 2-fluoro-2-methylpropane. {Delta}H{sub f 298}{degree} is calculated from the G2(MP2) calculated enthalpy of reaction ({Delta}H{sub rxn 298}{degree}) and use of isodesmic reactions. Standard entropy (S{sub 298}{degree} in cal/(mol{center{underscore}dot}K)) and heat capacities (C{sub p}(T)'s in cal/(mol{center{underscore}dot}K)) are calculated using the rigid-rotor--harmonic-oscillator approximation with direct integration over energy levels of the intermolecular rotation potential energy curve. These thermodynamic properties are used to estimate data for the C/C3/F group. Enthalpies of formation ({Delta}H{sub f 298}{degree} in kcal/mol) for 1,2-difluoroethane ({minus}102.7), 1,1,2-trifluoroethane ({minus}156.9), 1,1,2,2- and 1,1,1,2-tetrafluoroethane (209.6more » and 213.3), and pentafluoroethane ({minus}264.1), are calculated using total energies obtained from G2(MP2) composite ab initio methods. Isodesmic reactions with existing literature values of {Delta}H{sub f 298}{degree} for ethane, 1-fluoroethane, 1,1-difjuoroethane and 1,1,1-trifluoroethane are used. Fluorine/fluorine interaction terms, F/F, 2F/F, 3F/F, 2F/2F, and 3F/2F, where ``/'' indicates interaction for alkane compounds, for {Delta}H{sub f 298}{degree} are reevaluated based on {Delta}H{sub f 298}{degree} of the above five fluoroethanes. Thermodynamic properties ({Delta}H{sub f 298}{degree}, S{sub 298}{degree} and C{sub p}(T)'s (300 {le} T/K {le} 1500)) for fluorinated carbon groups, C/C3F, C/C2/F/H, C/C2/F2, are calculated using data from ab initio methods and existing literature data. Fluorine-methyl (alkyl) group additivity corrections for gauche interactions are also evaluated.« less

Cystic fibrosis transmembrane conductance regulator regulates epithelial cell response to Aspergillus and resultant pulmonary inflammation.

PubMed

Chaudhary, Neelkamal; Datta, Kausik; Askin, Frederic B; Staab, Janet F; Marr, Kieren A

2012-02-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR(-/-) mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease.

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis

PubMed Central

Masvidal, Laia; Igreja, Susana; Ramos, Maria D; Alvarez, Antoni; de Gracia, Javier; Ramalho, Anabela; Amaral, Margarida D; Larriba, Sara; Casals, Teresa

2014-01-01

The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657+5G>A; 2562T>G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657+5G>A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT/qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression. PMID:24129438

A Low-Cost and Simple Genetic Screening for Cystic Fibrosis Provided by the Brazilian Public Health System.

PubMed

Rispoli, Thaiane; Martins de Castro, Simone; Grandi, Tarciana; Prado, Mayara; Filippon, Letícia; Dornelles da Silva, Cláudia Maria; Vargas, José Eduardo; Rossetti, Lucia Maria Rosa

2018-05-03

Cystic fibrosis newborn screening was implemented in Brazil by the Public Health System in 2012. Because of cost, only 1 mutation was tested - p.Phe508del. We developed a robust low-cost genetic test for screening 11 CFTR gene mutations with potential use in developing countries. Copyright © 2018 Elsevier Inc. All rights reserved.

Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

NASA Astrophysics Data System (ADS)

Ebner, Andreas; Nikova, Dessy; Lange, Tobias; Häberle, Johannes; Falk, Sabine; Dübbers, Angelika; Bruns, Reimer; Hinterdorfer, Peter; Oberleithner, Hans; Schillers, Hermann

2008-09-01

CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl-) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients.

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 create a shunt pathway that restricts the amount of toxic secondary BAs that enter the liver. PMID:22370478

Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase

NASA Technical Reports Server (NTRS)

Vitart, V.; Christodoulou, J.; Huang, J. F.; Chazin, W. J.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.

Sarcolemmal alpha and gamma sarcoglycan protein deficiencies in Turkish siblings with a novel missense mutation in the alpha sarcoglycan gene.

PubMed

Diniz, Gulden; Tosun Yildirim, Hulya; Akinci, Gulcin; Hazan, Filiz; Ozturk, Aysel; Yararbas, Kanay; Tukun, Ajlan

2014-06-01

The sarcoglycan alpha gene, also known as the adhalin gene, is located on chromosome 17q21; mutations in this gene are associated with limb-girdle muscular dystrophy type 2D. We describe two Turkish siblings with findings consistent with limb-girdle muscular dystrophy type 2D. The evaluation excluded a dystrophinopathy, which is the most common form of muscular dystrophy. Both siblings had very high levels of creatinine phosphokinase and negative molecular tests for deletions and duplications of the dystrophin gene. The older boy presented at 8 years of age with an inability to climb steps and an abnormal gait. His younger brother was 5 years old and had similar symptoms. The muscle biopsy evaluation was performed only in the older brother. The muscle biopsy showed dystrophic features as well as a deficiency in the expression of two different glycoproteins: the alpha sarcoglycan and the gamma sarcoglycan. Sarcolemmal expressions of dystrophin and other sarcoglycans (beta and delta) were diffusely present. DNA analysis demonstrated the presence of previously unknown homozygous mutations [c.226 C > T (p.L76 F)] in exon 3 in the sarcoglycan alpha genes of both siblings. Similar heterozygous point mutations at the same locus were found in both parents, but the genes of beta, delta, and gamma sarcoglycan were normal in the remaining family members. We describe two siblings with limb-girdle muscular dystrophy type 2D with a novel missense mutation. These patients illustrate that the differential diagnosis of muscular dystrophies is impossible with clinical findings alone. Therefore, a muscle biopsy and DNA analysis remain essential methods for diagnosis of muscle diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Plant delta 15N correlates with the transpiration efficiency of nitrogen acquisition in tropical trees.

PubMed

Cernusak, Lucas A; Winter, Klaus; Turner, Benjamin L

2009-11-01

Based upon considerations of a theoretical model of (15)N/(14)N fractionation during steady-state nitrate uptake from soil, we hypothesized that, for plants grown in a common soil environment, whole-plant delta(15)N (deltaP) should vary as a function of the transpiration efficiency of nitrogen acquisition (F(N)/v) and the difference between deltaP and root delta(15)N (deltaP - deltaR). We tested these hypotheses with measurements of several tropical tree and liana species. Consistent with theoretical expectations, both F(N)/v and deltaP - deltaR were significant sources of variation in deltaP, and the relationship between deltaP and F(N)/v differed between non-N(2)-fixing and N(2)-fixing species. We interpret the correlation between deltaP and F(N)/v as resulting from variation in mineral nitrogen efflux-to-influx ratios across plasma membranes of root cells. These results provide a simple explanation of variation in delta(15)N of terrestrial plants and have implications for understanding nitrogen cycling in ecosystems.

Evaluation of mutation screening as a first line test for the diagnosis of the primary hyperoxalurias.

PubMed

Rumsby, Gill; Williams, Emma; Coulter-Mackie, Marion

2004-09-01

A definitive diagnosis of primary hyperoxaluria type 1 (PH1) and primary hyperoxaluria type 2 (PH2) requires the measurement of alanine:glyoxylate aminotransferase (AGT) and glyoxylate reductase (GR) activities, respectively, in a liver biopsy. We have evaluated a molecular genetic approach for the diagnosis of these autosomal-recessive diseases. Polymerase chain reaction (PCR) was used to detect three common mutations in the AGXT gene (c.33_34insC, c.508G>A, and c.731T>C) and one, c.103delG, in the GRHPR gene in DNA samples from 365 unrelated individuals referred for diagnosis of PH1 and/or PH2 by liver enzyme analysis. One or more of these mutations was found in 183 (68.8%) biopsy proven cases of PH1 and PH2 with a test negative predictive value of 62% and 2%, respectively. 102 (34.1%) patients were homozygous or compound heterozygous, making a molecular diagnosis possible. Age of onset and presenting features were similar in patients homozygous for any of the four mutations. Of the AGXT homozygotes, only the c.508G>A mutant was associated with significant AGT catalytic activity and in two of these activity was in the low normal range, possibly reflecting variation in mitochondrial content of the biopsy as this particular mutation is associated with mitochondrial mistargeting. Limited mutation analysis can provide a useful first line test for PH1 and PH2 in patients in whom primary hyperoxaluria is suspected and in whom secondary causes have been excluded. Those patients in whom a single mutation, or no mutation, is found can then be selectively targeted for liver biopsy.

CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients

PubMed Central

Ziętkiewicz, Ewa; Rutkiewicz, Ewa; Pogorzelski, Andrzej; Klimek, Barbara; Voelkel, Katarzyna; Witt, Michał

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers. PMID:24586523

CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.

PubMed

Ziętkiewicz, Ewa; Rutkiewicz, Ewa; Pogorzelski, Andrzej; Klimek, Barbara; Voelkel, Katarzyna; Witt, Michał

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.

The magnitude of ivacaftor effects on fluid secretion via R117H-CFTR channels: Human in vivo measurements

PubMed Central

Char, Jessica E.; Dunn, Colleen; Davies, Zoe; Milla, Carlos; Moss, Richard B.; Wine, Jeffrey J.

2017-01-01

We optically measured effects of orally available ivacaftor (Kalydeco®) on sweat rates of identified glands in 3 R117H subjects, each having a unique set of additional mutations, and compared them with 5 healthy control subjects tested contemporaneously. We injected β-adrenergic agonists intradermally to stimulate CFTR-dependent ‘C-sweat’ and methacholine to stimulate ‘M-sweat’, which persists in CF subjects. We focused on an R117H-7T/F508del subject who produced quantifiable C-sweat off ivacaftor and was available for 1 blinded, 3 off ivacaftor, and 3 on ivacaftor tests, allowing us to estimate in vivo fold-increase in sweat rates produced by ivacaftor’s effect on the open probability (PO) of R117H-CFTR. Measured sweat rates must be corrected for sweat losses. With estimated sweat losses of 0.023 to 0.08 nl·gland-1·min-1, ivacaftor increased the average C-sweat rates 3–7 fold, and estimated function as % of WT were 4.1–12% off ivacaftor and 21.9–32% on ivacaftor (larger values reflect increased loss estimates). Based on single tests, an R117H-7T/ R117H-7T subject showed 6–9% WT function off ivacaftor and 28–43% on ivacaftor. Repeat testing of an R117H-5T/F508del subject detected only trace responding to ivacaftor. We conclude that in vivo, R117H PO is strongly increased by ivacaftor, but channel number, mainly determined by variable deletion of exon 10, has a marked influence on outcomes. PMID:28419121

Risk factors for lung function decline in a large cohort of young cystic fibrosis patients.

PubMed

Cogen, Jonathan; Emerson, Julia; Sanders, Don B; Ren, Clement; Schechter, Michael S; Gibson, Ronald L; Morgan, Wayne; Rosenfeld, Margaret

2015-08-01

To identify novel risk factors and corroborate previously identified risk factors for mean annual decline in FEV1% predicted in a large, contemporary, United States cohort of young cystic fibrosis (CF) patients. Retrospective observational study of participants in the EPIC Observational Study, who were Pseudomonas-negative and ≤12 years of age at enrollment in 2004-2006. The associations between potential demographic, clinical, and environmental risk factors evaluated during the baseline year and subsequent mean annual decline in FEV1 percent predicted were evaluated using generalized estimating equations. The 946 participants in the current analysis were followed for a mean of 6.2 (SD 1.3) years. Mean annual decline in FEV1% predicted was 1.01% (95%CI 0.85-1.17%). Children with one or no F508del mutations had a significantly smaller annual decline in FEV1 compared to F508del homozygotes. In a multivariable model, risk factors during the baseline year associated with a larger subsequent mean annual lung function decline included female gender, frequent or productive cough, low BMI (<66th percentile, median in the cohort), ≥1 pulmonary exacerbation, high FEV1 (≥115% predicted, in the top quartile), and respiratory culture positive for methicillin-sensitive Staphylococcus aureus, methicillin-resistant S. aureus, or Stenotrophomonas maltophilia. We have identified a range of risk factors for FEV1 decline in a large cohort of young, CF patients who were Pa negative at enrollment, including novel as well as previously identified characteristics. These results could inform the design of a clinical trial in which rate of FEV1 decline is the primary endpoint and identify high-risk groups that may benefit from closer monitoring. © 2015 Wiley Periodicals, Inc.

Cystic Fibrosis Transmembrane Conductance Regulator Regulates Epithelial Cell Response to Aspergillus and Resultant Pulmonary Inflammation

PubMed Central

Chaudhary, Neelkamal; Datta, Kausik; Askin, Frederic B.; Staab, Janet F.

2012-01-01

Rationale: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. Objectives: To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. Methods: A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. Measurements and Main Results: Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR−/− mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. Conclusions: Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease. PMID:22135344

Coronary oscillatory flow amplitude is more affected by perfusion pressure than ventricular pressure.

PubMed

Krams, R; Sipkema, P; Westerhof, N

1990-06-01

In this study on the isolated, maximally vasodilated, blood-perfused cat heart we investigated the relation between left ventricular developed pressure (delta Piv) and coronary oscillatory flow amplitude (diastolic minus systolic flow, delta F) at different levels of constant perfusion pressure (Pp). We hypothesized that the effect of cardiac contraction on the phasic flow results from the changing elastic properties of cardiac muscle. The coronary vessel compartment can, as can the left ventricular lumen compartment, be described by a time-varying elastance. This concept predicts that the effect of left ventricular pressure on delta F is small, whereas the effect of Pp is considerable. Both the waterfall model and the intramyocardial pump model predict the inverse. The relation between delta Piv and delta F at a Pp of 10 kPa is delta F = (4.71 +/- 3.08).delta Piv + 337 +/- 75 (slope in ml.min-1.100 g-1.kPa-1 and intercept in ml.min-1.100 g-1; n = 7); the relation between (constant levels of) Pp and delta F at a constant delta Piv of 10 kPa is delta F = 51.Pp + 211 (slope in ml.min-1.100 g-1.kPa-1 and intercept in ml.min-1.100 g-1; n = 6). The differences in slope are best predicted by the time-varying elastance concept.

TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.

PubMed

Ramalingam, Sivaprakash; Annaluru, Narayana; Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

2014-01-01

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

HER2 isoforms co-expression differently tunes mammary tumor phenotypes affecting onset, vasculature and therapeutic response

PubMed Central

Balboni, Tania; Ianzano, Marianna L.; Laranga, Roberta; Landuzzi, Lorena; Giusti, Veronica; Ceccarelli, Claudio; Santini, Donatella; Taffurelli, Mario; Di Oto, Enrico; Asioli, Sofia; Amici, Augusto; Pupa, Serenella M.; De Giovanni, Carla; Tagliabue, Elda; Iezzi, Manuela; Nanni, Patrizia; Lollini, Pier-Luigi

2017-01-01

Full-length HER2 oncoprotein and splice variant Delta16 are co-expressed in human breast cancer. We studied their interaction in hybrid transgenic mice bearing human full-length HER2 and Delta16 (F1 HER2/Delta16) in comparison to parental HER2 and Delta16 transgenic mice. Mammary carcinomas onset was faster in F1 HER2/Delta16 and Delta16 than in HER2 mice, however tumor growth was slower, and metastatic spread was comparable in all transgenic mice. Full-length HER2 tumors contained few large vessels or vascular lacunae, whereas Delta16 tumors presented a more regular vascularization with numerous endothelium-lined small vessels. Delta16-expressing tumors showed a higher accumulation of i.v. injected doxorubicin than tumors expressing full-length HER2. F1 HER2/Delta16 tumors with high full-length HER2 expression made few large vessels, whereas tumors with low full-length HER2 and high Delta16 contained numerous small vessels and expressed higher levels of VEGF and VEGFR2. Trastuzumab strongly inhibited tumor onset in F1 HER2/Delta16 and Delta16 mice, but not in full-length HER2 mice. Addiction of F1 tumors to Delta16 was also shown by long-term stability of Delta16 levels during serial transplants, in contrast full-length HER2 levels underwent wide fluctuations. In conclusion, full-length HER2 leads to a faster tumor growth and to an irregular vascularization, whereas Delta16 leads to a faster tumor onset, with more regular vessels, which in turn could better transport cytotoxic drugs within the tumor, and to a higher sensitivity to targeted therapeutic agents. F1 HER2/Delta16 mice are a new immunocompetent mouse model, complementary to patient-derived xenografts, for studies of mammary carcinoma onset, prevention and therapy. PMID:28903354

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

More about the Viking hypothesis of origin of the delta32 mutation in the CCR5 gene conferring resistance to HIV-1 infection.

PubMed

Lucotte, Gérard; Dieterlen, Florent

2003-11-01

The chemokine receptor CCR5 constitutes the major coreceptor for the HIV-1, because a mutant allele of the CCR5 gene named delta32 was shown to provide to homozygotes a strong resistance against infection. In the present study the frequency of the delta32 allele was collected in 36 European populations and in Cyprus, and the highest allele frequencies were found in Nordic countries. We constructed an allele map of delta32 frequencies in Europe; the map is in accordance to the Vikings hypothesis of the origin of the mutation and his dissemination during the eighth to the tenth centuries.

Skn-1a/Oct-11 and {Delta}Np63{alpha} exert antagonizing effects on human keratin expression

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

Lena, Anna Maria; Cipollone, Rita; Amelio, Ivano

2010-10-29

Research highlights: {yields} Skn-1a markedly downregulates {Delta}Np63-driven K14 expression. {yields} {Delta}Np63 inhibits Skn-1a-mediated K10 expression. {yields} {Delta}Np63, mutated in SAM domain, is less effecting in K10 downregulation. {yields} Immunolocalization in human skin of the two transcription factors is partially overlapping. {yields} The antagonistic effects of Skn-1a and p63 is through competition for overlapping responsive elements or through an indirect interaction. -- Abstract: The formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation. Here, we report the reciprocal effect on keratin expression of {Delta}Np63, pivotal in normal epidermal morphogenesis and maintenance, and Skn-1a/Oct-11, a POUmore » transcription factor that triggers and regulates the differentiation of keratinocytes. The expression of Skn-1a markedly downregulated {Delta}Np63-driven K14 expression in luciferase reporter assays. The extent of downregulation was comparable to the inhibition of Skn-1a-mediated K10 expression upon expression of {Delta}Np63. {Delta}Np63, mutated in the protein-protein interaction domain (SAM domain; mutated in human ectodermal dysplasia syndrome), was significantly less effecting in downregulating K10, raising the possibility of a direct interaction among Skn-1a and {Delta}Np63. Immunolocalization in human skin biopsies revealed that the expression of the two transcription factors is partially overlapping. Co-immunoprecipitation experiments did not, however, demonstrate a direct interaction between {Delta}Np63 and Skn-1a, suggesting that the antagonistic effects of Skn-1a and p63 on keratin promoter transactivation is probably through competition for overlapping binding sites on target gene promoter or through an indirect interaction.« less

Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels.

PubMed

Cukras, Catherine A; Jeliazkova, Iana; Nichols, Colin G

2002-06-01

All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.

Cystic Fibrosis Transmembrane Conductance Regulator is an Epithelial Cell Receptor for Clearance of Pseudomonas aeruginosa from the Lung

NASA Astrophysics Data System (ADS)

Pier, Gerald B.; Grout, Martha; Zaidi, Tanweer S.

1997-10-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant Δ F508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

PubMed Central

Smith, Laura; Litman, Paul; Kohli, Ekta; Amick, Joseph; Page, Richard C.; Misra, Saurav

2013-01-01

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na+/H+ exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8–15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization. PMID:23636454

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem.

PubMed Central

Ananvoranich, S; Lafontaine, D A; Perreault, J P

1999-01-01

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway. PMID:10037808

Synthesis of a potent and selective (18)F-labeled delta-opioid receptor antagonist derived from the Dmt-Tic pharmacophore for positron emission tomography imaging.

PubMed

Ryu, Eun Kyoung; Wu, Zhanhong; Chen, Kai; Lazarus, Lawrence H; Marczak, Ewa D; Sasaki, Yusuke; Ambo, Akihiro; Salvadori, Severo; Ren, Chuancheng; Zhao, Heng; Balboni, Gianfranco; Chen, Xiaoyuan

2008-03-27

Identification and pharmacological characterization of two new selective delta-opioid receptor antagonists, derived from the Dmt-Tic pharmacophore, of potential utility in positron emission tomography (PET) imaging are described. On the basis of its high delta selectivity, H-Dmt-Tic--Lys(Z)-OH (reference compound 1) is a useful starting point for the synthesis of (18)F-labeled compounds prepared by the coupling of N-succinimidyl 4-[ (18)F]fluorobenzoate ([(18)F]SFB) with Boc-Dmt-Tic--Lys(Z)-OH under slightly basic conditions at 37 degrees C for 15 min, deprotection with TFA, and HPLC purification. The total synthesis time was 120 min, and the decay-corrected radiochemical yield of [(18)F]- 1 was about 25-30% ( n = 5) starting from [(18)F]SFB ( n = 5) with an effective specific activity about 46 GBq/micromol. In vitro autoradiography studies showed prominent uptake of [ (18)F]- 1 in the striatum and cortex with significant blocking by 1 and UFP-501 (selective delta-opioid receptor antagonist), suggesting high specific binding of [(18)F]- 1 to delta-opioid receptors. Noninvasive microPET imaging studies revealed the absence of [(18)F]- 1 in rat brain, since it fails to cross the blood-brain barrier. This study demonstrates the suitability of [ (18)F]- 1 for imaging peripheral delta-opioid receptors.

In silico analysis of novel mutations in maple syrup urine disease patients from Iran.

PubMed

Abiri, Maryam; Karamzadeh, Razieh; Mojbafan, Marziyeh; Alaei, Mohammad Reza; Jodaki, Atefeh; Safi, Masomeh; Kianfar, Soodeh; Bandehi Sarhaddi, Ameneh; Noori-Daloii, Mohammad Reza; Karimipoor, Morteza; Zeinali, Sirous

2017-02-01

Maple Syrup Urine Disease (MSUD) is a rare autosomal recessive disorder of branched-chain amino acid (BCAA) metabolism. The disease is mainly caused by mutations either in the BCKDHA, BCKDHB, DBT or DLD genes encoding components of the E1α, E1β, E2 and E3 subunits of branched-chain α-keto acid dehydrogenase complex (BCKDC), respectively. BCKDC is a mitochondrial enzyme which is responsible for the normal breakdown of BCAA. The rate of consanguineous marriage in Iran is 38.6 %, so the prevalence of autosomal recessive disorders is higher in comparison to other countries. Consanguinity increases the chance of the presence of pathogenic mutations in a homoallelic state. This phenomenon has made homozygosity mapping a powerful tool for finding the probable causative gene in heterogeneous disorders like IEM (Inborn Errors of Metabolism). In this study, two sets of multiplex polymorphic STR (Short Tandem Repeat) markers linked to the above-mentioned genes were selected to identify the probable pathogenic gene in the studied families. The families who showed a homozygous haplotype for the STR markers of the BCKDHB gene were subsequently sequenced. Four novel mutations including c.633 + 1G > A, c.988G > A, c.833_834insCAC, and a homozygous deletion of whole exon 3 c. (274 + 1_275-1) _(343 + 1_344-1), as well as one recently reported (c. 508G > T) mutation have been identified. Interestingly, three families shared a common haplotype structure along with the c. 508G > T mutation. Also, four other families revealed another similar haplotype with c.988G > A mutation. Founder effect can be a suggestive mechanism for the disease. Additionally, structural models of MSUD mutations have been performed to predict the pathogenesis of the newly identified variants.

A Specimen Size Effect on the Fatigue Crack Growth Rate Threshold of IN 718

NASA Technical Reports Server (NTRS)

Garr, K. R.; Hresko, G. C., III

1998-01-01

Fatigue crack growth rate (FCGR) tests were conducted on IN 718 in the solution annealed and aged condition at room temperature in accordance with E647-87. As part of each test, the FCGR threshold was measured using the decreasing Delta K method. A new heat of material was being tested and some of this material was sent to a different laboratory which wanted to use a specimen with a 127 mm width. Threshold data previously had been established on specimens with a width of 50.8 mm. As a check of the laboratory, tests were conducted at room temperature and R equal to 0.1 for comparison with the earlier data. The results were a threshold significantly higher than previously observed. Interchanging of specimen sizes and laboratories showed that the results were not due to a heat-to-heat or lab-to-lab variation. The results to be presented here are those obtained at the original laboratory. Growth rates were measured using the electric potential drop technique at R values of 0.1, 0.7, and 0.9. Compact tension specimen sizes with planer dimensions of 25.4 mm, 50.8 mm, and 127 mm were used. Crack growth rates at threshold were generally below 2.5 X 10(exp -8) mm / cycle. Closure measurements were made on some of the specimens by a manual procedure using a clip gage. When the crack growth rate data for the specimens tested at R equal to 0.1 were plotted as a function of applied Delta K, the thresholds varied with specimen width. The larger the width, the higher the threshold. The thresholds varied from 6.5 MPa-m(exp 1/2) for the 25.4 mm specimen to 15.4 MPa-m(exp 1/2) for the 127 mm specimen. At R equal to 0.7, the 25.4 mm and 50.8 mm specimens had essentially the same threshold, about 2.9 MPa-m(exp 1/2)while the 127 mm specimen had a threshold of 4.5 MPa-m(exp 1/2). When plotted as a function of effective Delta K, the R equal to 0.1 data are essentially normalized. Various aspects of the test procedure will be discussed as well as the results of analysis of the data using some different closure models.

A Size Effect on the Fatigue Crack Growth Rate Threshold of Alloy 718

NASA Technical Reports Server (NTRS)

Garr, K. R.; Hresko, G. C., III

1998-01-01

Fatigue crack growth rate (FCGR) tests were conducted on Alloy 718 in the solution annealed and aged condition at room temperature. In each test, the FCGR threshold was measured using the decreasing (Delta)K method. Initial testing was at two facilities, one of which used C(T) specimens with W = 127 mm. Previous data at the other facility had been obtained with specimens with W = 50.8 mm. A comparison of test results at R = 0.1 showed that the threshold for the 127 mm specimen was considerably higher than that of the 50.8 mm specimen. A check showed that this difference was not due to a heat-to-heat or lab-to-lab variation. Additional tests were conducted on specimens with W = 25.4 mm and at other R values. Data for the various specimens is presented along with parameters usually used to describe threshold behavior.

Protein disulphide isomerase is required for signal peptide peptidase-mediated protein degradation

PubMed Central

Lee, Seong-Ok; Cho, Kwangmin; Cho, Sunglim; Kim, Ilkwon; Oh, Changhoon; Ahn, Kwangseog

2010-01-01

The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2-induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA-mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate-binding mutant of PDI, but not the catalytically inactive mutant, dominant-negatively inhibited US2-mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP-mediated degradation of CD3δ but not Derlin-1-dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP-mediated ER-associated degradation machinery. PMID:19942855

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.

Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype.

PubMed

Spring, K; Cross, S; Li, C; Watters, D; Ben-Senior, L; Waring, P; Ahangari, F; Lu, S L; Chen, P; Misko, I; Paterson, C; Kay, G; Smorodinsky, N I; Shiloh, Y; Lavin, M F

2001-06-01

ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Atm-DeltaSRI, was designed as a model of one of the most common deletion mutations (7636del9) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556-2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-DeltaSRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm(-/-) mice. The life span of Atm-DeltaSRI mice was significantly longer than that of Atm(-/-) mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-DeltaSRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-DeltaSRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm(-/-) mice were observed in older Atm-DeltaSRI animals. Thus, expression of mutant protein in Atm-DeltaSRI knock-in mice gives rise to a discernibly different phenotype to Atm(-/-) mice, which may account for the heterogeneity seen in A-T patients with different mutations.

Renal proximal tubule function is preserved in Cftrtm2camΔF508 cystic fibrosis mice

PubMed Central

Kibble, J D; Balloch, K J D; Neal, A M; Hill, C; White, S; Robson, L; Green, R; Taylor, C J

2001-01-01

Changes in proximal tubule function have been reported in cystic fibrosis patients. The aim of this study was to investigate proximal tubule function in the Cftrtm2camΔF508 cystic fibrosis (CF) mouse model. A range of techniques were used including renal clearance studies, in situ microperfusion, RT-PCR and whole-cell patch clamping. Renal Na+ clearance was similar in wild-type (1.4 ± 0.3 μl min−1, number of animals, N= 12) and CF mice (1.6 ± 0.4 μl min−1, N= 7) under control conditions. Acute extracellular volume expansion resulted in significant natriuresis in wild-type (7.0 ± 0.8 μl min−1, N= 8) and CF mice (9.3 ± 1.4 μl min−1, N= 9); no difference between genotypes was observed. In situ microperfusion revealed that fluid absorptive rate (Jv) was similar under control conditions between wild-type (2.2 ± 0.4 nl mm−1 min−1, n= 10) and CF mice (1.9 ± 0.3 nl mm−1 min−1, n= 11). Addition of a forskolin-dibutyryl cAMP (db-cAMP) cocktail to the perfusate caused no significant change in Jv in either wild-type (2.6 ± 0.7 nl mm−1 min−1, n= 10) or Cftrtm2camΔF508 mice (2.0 ± 0.5 nl mm−1 min−1, n= 10). CFTR expression was confirmed in samples of outer cortex using RT-PCR. However, no evidence for functional CFTR was obtained when outer cortical cells were stimulated with protein kinase A or forskolin-db-cAMP using whole-cell patch clamping. In conclusion, no functional deficit in proximal tubule function was found in Cftrtm2camΔF508 mice. This may be a consequence of a lack of whole-cell cAMP-dependent Cl− conductance in mouse proximal tubule cells. PMID:11306663

Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse.

PubMed

Walters, C L; Blendy, J A

2001-12-01

Addiction is a complex process that relies on the ability of an organism to integrate positive and negative properties of drugs of abuse. Therefore, studying the reinforcing as well as aversive components of drugs of abuse in a single model system will enable us to understand the role of final common mediators, such as cAMP response element-binding protein (CREB), in the addiction process. To this end, we analyzed mice with a mutation in the alpha and Delta isoforms of the CREB gene. Previously we have shown that CREB(alphaDelta) mutant mice in a mixed genetic background show attenuated signs of physical dependence, as measured by the classic signs of withdrawal. We have generated a uniform genetically stable F1 hybrid (129SvEv/C57BL/6) mouse line harboring the CREB mutation. We have found the functional activity of CREB in these F1 hybrid mice to be dramatically reduced compared with their wild-type littermates. These mice maintain a reduced withdrawal phenotype after chronic morphine. We are now poised to examine a number of complex behavioral phenotypes related to addiction in a well defined CREB-deficient mouse model. We demonstrate that the aversive properties of morphine are still present in CREB mutant mice despite a reduction of physical withdrawal. On the other hand, these mice do not respond to the reinforcing properties of morphine in a conditioned place preference paradigm. In contrast, CREB mutant mice demonstrate an enhanced response to the reinforcing properties of cocaine compared with their wild-type controls in both conditioned place preference and sensitization behaviors. These data may provide the first paradigm for differential vulnerability to various drugs of abuse.

Pattern Specificity in the Effect of Prior [delta]f on Auditory Stream Segregation

ERIC Educational Resources Information Center

Snyder, Joel S.; Weintraub, David M.

2011-01-01

During repeating sequences of low (A) and high (B) tones, perception of two separate streams ("streaming") increases with greater frequency separation ([delta]f) between the A and B tones; in contrast, a prior context with large [delta]f results in less streaming during a subsequent test pattern. The purpose of the present study was to…

Cover-gas seal program. Test report - sodium dip-seal wetting study. [at 450/sup 0/F

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

Carnevali, R.

1977-10-20

This report documents the tests conducted to find a reliable surface preparation method of treating the CRBRP dip seal blade (SA508 Class 2 steel) to insure its sodium wettability at 450F or less. Two techniques were established which depressed the sodium wetting temperature of SA 508, Class 2 dip seal blade material to 375F. These techniques were depositing an approx. 60 x 10/sup -6/ inch layer of tin on the blade surface by a brush-on plating process, and, by cleaning the blade surface with ultrasonics while it is immersed in sodium. The tin plating technique is recommended as the initialmore » and primary surface preparation method and ultrasonics as a rewetting and backup technique. This work was conducted in support of the Sodium Dip Seal Feature Test, DRS 32.05.« less

Fatigue crack growth in SA508-CL2 steel in a high temperature, high purity water environment

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

Gerber, T.L.; Heald, J.D.; Kiss, E.

1974-10-01

Fatigue crack growth tests were conducted with 1 in. plate specimens of SA508-CL 2 steel in room temperature air, 550$sup 0$F air and in a 550$sup 0$F, high purity, water environment. Zero-tension load controlled tests were run at cyclic frequencies as low as 0.037 CPM. Results show that growth rates in the simulated Boiling Water Reactor (BWR) water environment are faster than growth rates observed in 550$sup 0$F air and these rates are faster than the room temperature rate. In the BWR water environment, lowering the cyclic frequency from 0.37 to 0.037 CPM caused only a slight increase in themore » fatigue crack growth rate. All growth rates measured in these tests were below the upper bound design curve presented in Section XI of the ASME Code. (auth)« less

New insights into ETS-10 and titanate quantum wire: a comprehensive characterization.

PubMed

Jeong, Nak Cheon; Lee, Young Ju; Park, Jung-Hyun; Lim, Hyunjin; Shin, Chae-Ho; Cheong, Hyeonsik; Yoon, Kyung Byung

2009-09-16

The titanate quantum wires in ETS-10 crystals remain intact during ion exchange of the pristine cations (Na(+)(0.47) + K(+)(0.53)) with M(n+) ions (M(n+) = Na(+), K(+), Mg(2+), Ca(2+), Sr(2+), Ba(2+), Pb(2+), Cd(2+), Zn(2+)) and during reverse exchange of the newly exchanged cations with Na(+). The binding energies of O(1s) and Ti(2p) decrease as the electronegativity of the cation decreases, and they are inversely proportional to the negative partial charge of the framework oxygen [-delta(O(f))]. At least five different oxygen species were identified, and their binding energies (526.1-531.9 eV) indicate that the titanate-forming oxides are much more basic than those of aluminosilicate zeolites (530.2-533.3 eV), which explains the vulnerability of the quantum wire to acids and oxidants. The chemical shifts of the five NMR-spectroscopically nonequivalent Si sites, delta(I(A)), delta(I(B)), delta(II(A)), delta(II(B)), and delta(III), shift downfield as -delta(O(f)) increases, with slopes of 2.5, 18.6, 133.5, 216.3, and 93.8 ppm/[-delta(O(f))], respectively. The nonuniform responses of the chemical shifts to -delta(O(f)) arise from the phenomenon that the cations in the 12-membered-ring channels shift to the interiors of the cages surrounded by four seven-membered-ring windows. On the basis of the above, we assign delta(I(A)), delta(I(B)), delta(II(A)), and delta(II(B)) to the chemical shifts arising from Si(12,12), Si(12,7), Si(7,12), and Si(7,7) atoms, respectively. The frequency of the longitudinal stretching vibration of the titanate quantum wire increases linearly and the bandwidth decreases nonlinearly with increasing -delta(O(f)), indicating that the titanate quantum wire resembles a metallic carbon nanotube. As the degree of hydration increases, the vibrational frequency shifts linearly to higher frequencies while the bandwidth decreases. We identified another normal mode of vibration of the quantum wire, which vibrates in the region of 274-280 cm(-1). In the dehydrated state, the band-gap energy and the first absorption maximum shift to lower energies as -delta(O(f)) increases, indicating the oxide-to-titanium(IV) charge-transfer nature of the transitions.

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 combination with ivacaftor provided a benefit for patients with cystic fibrosis homozygous for the Phe508del CFTR mutation. (Funded by Vertex Pharmaceuticals and others; TRAFFIC and TRANSPORT ClinicalTrials.gov numbers, NCT01807923 and NCT01807949.).

Detection and Characteristics of Rifampicin-Resistant Isolates of Mycobacterium tuberculosis.

PubMed

Cherednichenko, A G; Dymova, M A; Solodilova, O A; Petrenko, T I; Prozorov, A I; Filipenko, M L

2016-03-01

Genotyping and analysis the drug resistance of 59 isolates of M. tuberculosis obtained from patients living in Altai Territory were performed using a BACTEC MGIT 960 fluorometric system by means of VNTR typing (variable number tandem repeat), PCR-RFLP analysis, and sequence analysis. The occurrence frequency was highest for isolates of the Beijing family (n=30, 50.8%). Analysis of mutation spectrum in the rpoB gene associated with rifampicin resistance revealed the major mutation (codon 531 of the rpoB gene) in 93% samples, which allows us to use rapid test systems.

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

Sevast'yanov, E A; Sadekova, E Kh

The Bulgarian mathematicians Sendov, Popov, and Boyanov have well-known results on the asymptotic behaviour of the least deviations of 2{pi}-periodic functions in the classes H{sup {omega}} from trigonometric polynomials in the Hausdorff metric. However, the asymptotics they give are not adequate to detect a difference in, for example, the rate of approximation of functions f whose moduli of continuity {omega}(f;{delta}) differ by factors of the form (log(1/{delta})){sup {beta}}. Furthermore, a more detailed determination of the asymptotic behaviour by traditional methods becomes very difficult. This paper develops an approach based on using trigonometric snakes as approximating polynomials. The snakes of ordermore » n inscribed in the Minkowski {delta}-neighbourhood of the graph of the approximated function f provide, in a number of cases, the best approximation for f (for the appropriate choice of {delta}). The choice of {delta} depends on n and f and is based on constructing polynomial kernels adjusted to the Hausdorff metric and polynomials with special oscillatory properties. Bibliography: 19 titles.« less

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.

PubMed

Lin, Hsin Hung; Pan, Yih Jiuan; Hsu, Shen Hsing; Van, Ru Chuan; Hsiao, Yi Yuong; Chen, Jiun Hsien; Pan, Rong Long

2005-10-15

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

PubMed Central

Senior, Alan E.; Muharemagi, Alma; Wilke-Mounts, Susan

2008-01-01

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, competent in nucleotide binding, and had normal N-terminal sequence. In F1-subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiment showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming, and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector. PMID:17176112

ABCB1-1Delta polymorphism can predict hematologic toxicity in dogs treated with vincristine.

PubMed

Mealey, K L; Fidel, J; Gay, J M; Impellizeri, J A; Clifford, C A; Bergman, P J

2008-01-01

Dogs that harbor the naturally occurring ABCB1-1Delta polymorphism experience increased susceptibility to avermectin-induced neurological toxicosis as a result of deficient P-glycoprotein function. Whether or not the ABCB1-1Delta polymorphism affects susceptibility to toxicity of other P-glycoprotein substrate drugs has not been studied. Dogs that possess the ABCB1-1Delta mutation are more likely to develop hematologic toxicity associated with vincristine than ABCB1 wild-type dogs. Thirty-four dogs diagnosed with lymphoma were included in this study. Cheek swab samples were obtained from dogs diagnosed with lymphoma that were to be treated with vincristine. DNA was extracted from cheek swabs and the ABCB1 genotype was determined. Hematologic adverse drug reactions were recorded for each dog and graded according to the Veterinary Comparative Oncology Group's criteria for adverse event reporting (Consensus Document). In order to avoid possible bias, ABCB1 genotype results for a particular patient were not disclosed to oncologists until an initial adverse event report had been submitted. Dogs heterozygous or homozygous for the ABCB1-1Delta mutation were significantly more likely to develop hematologic toxicity, specifically neutropenia (P= .0005) and thrombocytopenia (P= .0001), after treatment with vincristine than ABCB1 wild-type dogs. At currently recommended dosages (0.5-0.7 mg/M(2)), vincristine is likely to cause hematologic toxicity in dogs with the ABCB1-1Delta mutation, resulting in treatment delays and unacceptable morbidity and mortality. Assessing the ABCB1-1Delta genotype before vincristine administration and decreasing the dosage may prevent toxicity and treatment delays resulting from neutropenia or thrombocytopenia.

Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid.

PubMed

Qin, Y M; Marttila, M S; Haapalainen, A M; Siivari, K M; Glumoff, T; Hiltunen, J K

1999-10-01

The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly(16) and Gly(329) of the S. cerevisiae A and B domains, corresponding to Gly(16), which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aDelta) and pYE352::ScMFE-2(bDelta) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aDeltabDelta) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Delta)) and mutational variants of the A and B domains (Ct MFE- 2(h2DeltaaDelta), Ct MFE- 2(h2DeltabDelta), and Ct MFE- 2(h2DeltaaDeltabDelta)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.

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 visits up to and including week 24. All randomly assigned patients who were exposed to any amount of study drug, with treatment assignment as assigned were included in primary and other efficacy analyses. All patients who were exposed to any amount of study drug, with treatment assignment as treated, were included in the safety analysis. This study was registered with ClinicalTrials.gov, number NCT02514473. Between July 23, 2015, and Sept 20, 2016, a total of 206 patients were enrolled and randomly assigned to receive lumacaftor and ivacaftor (n=104) or placebo (n=102). Two randomly assigned patients were never dosed with study drug (one in the placebo arm due to ineligibility arising from a streptococcal throat infection and one in the lumacaftor and ivacaftor arm due to withdrawal based on refusal to provide blood tests) and were not included in the analyses. 103 patients received at least one dose of lumacaftor and ivacaftor and 101 patients received at least one dose of placebo. For the primary endpoint, the average absolute change in LCI 2·5 from baseline over all study visits up to and including the week 24 visit, least squares mean difference was -1·09 units (95% CI -1·43 to -0·75, p<0·0001) for lumacaftor and ivacaftor versus placebo. For the key secondary endpoint of sweat chloride concentration, the least squares mean difference versus placebo was -20·8 mmol/L (95% CI -23·4 to -18·2, average absolute change at day 15/week 4; p<0·0001). The least squares mean difference compared with placebo in absolute change in ppFEV 1 from all on-treatment study visits until week 24 was 2·4 (95% CI 0·4-4·4, p=0·0182). 196 (96%) of 204 patients reported adverse events, most of which were mild (87 [43%]) or moderate (98 [48%]). Treatment was discontinued due to adverse events in three (3%) of 103 patients in the lumacaftor and ivacaftor group and two (2%) of 101 patients in the placebo group. Serious adverse events were reported in 13 (13%) of 103 patients in the lumacaftor and ivacaftor group and 11 (11%) of 101 patients in the placebo group. Treatment with lumacaftor and ivacaftor was associated with statistically significant improvements in lung function, as measured by LCI 2·5 and ppFEV 1 , versus placebo in patients aged 6-11 years with cystic fibrosis homozygous for F508del-CFTR. The overall safety profile was consistent with previous phase 3 studies of lumacaftor and ivacaftor. Vertex Pharmaceuticals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of Delta5-fatty acid desaturase from the cellular slime mold dictyostelium discoideum.

PubMed

Saito, T; Ochiai, H

1999-10-01

cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project. Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase. The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain. The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina. We have confirmed its function as Delta5-desaturase by over expression mutation in D. discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae. Analysis of the lipids from transformed D. discoideum and yeast demonstrated the accumulation of Delta5-desaturated products. This is the first report concering fatty acid desaturase in cellular slime molds.

Generation and characterization of Dyt1 DeltaGAG knock-in mouse as a model for early-onset dystonia.

PubMed

Dang, Mai T; Yokoi, Fumiaki; McNaught, Kevin St P; Jengelley, Toni-Ann; Jackson, Tehone; Li, Jianyong; Li, Yuqing

2005-12-01

A trinucleotide deletion of GAG in the DYT1 gene that encodes torsinA protein is implicated in the neurological movement disorder of Oppenheim's early-onset dystonia. The mutation removes a glutamic acid in the carboxy region of torsinA, a member of the Clp protease/heat shock protein family. The function of torsinA and the role of the mutation in causing dystonia are largely unknown. To gain insight into these unknowns, we made a gene-targeted mouse model of Dyt1 DeltaGAG to mimic the mutation found in DYT1 dystonic patients. The mutated heterozygous mice had deficient performance on the beam-walking test, a measure of fine motor coordination and balance. In addition, they exhibited hyperactivity in the open-field test. Mutant mice also showed a gait abnormality of increased overlap. Mice at 3 months of age did not display deficits in beam-walking and gait, while 6-month mutant mice did, indicating an age factor in phenotypic expression as well. While striatal dopamine and 4-dihydroxyphenylacetic acid (DOPAC) levels in Dyt1 DeltaGAG mice were similar to that of wild-type mice, a 27% decrease in 4-hydroxy, 3-methoxyphenacetic acid (homovanillic acid) was detected in mutant mice. Dyt1 DeltaGAG tissues also have ubiquitin- and torsinA-containing aggregates in neurons of the pontine nuclei. A sex difference was noticed in the mutant mice with female mutant mice exhibiting fewer alterations in behavioral, neurochemical, and cellular changes. Our results show that knocking in a Dyt1 DeltaGAG allele in mouse alters their motor behavior and recapitulates the production of protein aggregates that are seen in dystonic patients. Our data further support alterations in the dopaminergic system as a part of dystonia's neuropathology.

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

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

Magari, Masaki; Kanehiro, Yuichi; Todo, Kagefumi

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

Do non-steroidal anti-inflammatory drugs influence the steroid hormone milieu in male athletes?

PubMed

Di Luigi, L; Rossi, C; Sgrò, P; Fierro, V; Romanelli, F; Baldari, C; Guidetti, L

2007-10-01

Prostaglandins modulate the hypothalamus-pituitary-adrenal and -gonadal axis pathways. We explored the effects of a single course of treatment with acetylsalicylic acid (ASA), an inhibitor of prostaglandin synthesis, on the steroid milieu in athletes. Morning plasma cortisol (F), dehydroepiandrosterone sulphate, free-testosterone, testosterone (T) and their ratios were evaluated before and after the administration of either ASA or placebo in twelve male athletes, when affected by minor musculoskeletal trauma and, as control, after a five/six week wash-out in healthy conditions respectively. One tablet of ASA (800 mg), or placebo, was administered two times daily for 10 days during treatment. All the volunteers suspended exercise training during treatment. The results revealed that compared to placebo, plasma F was significantly lower after ASA treatment (p = 0.023). Furthermore, the comparison of hormone's absolute and percentage of variations (Delta and Delta%) between ASA and placebo treatment showed significant differences respectively for DeltaF (p = 0.045), for DeltaT (p = 0.047), for DeltaT/F (p = 0.042), for DeltaF% (p = 0.04) and for DeltaT% (p = 0.049). Our data suggest that in comparison to placebo, a short-term ASA treatment is able to influence the plasma steroid milieu in athletes. Due to the observed variability of the individual hormonal patterns, further research is required to substantiate these findings.

Contributions of Torpedo nicotinic acetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function.

PubMed

Xie, Y; Cohen, J B

2001-01-26

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.

A randomized placebo-controlled trial of miglustat in cystic fibrosis based on nasal potential difference.

PubMed

Leonard, Anissa; Lebecque, Patrick; Dingemanse, Jasper; Leal, Teresinha

2012-05-01

Preclinical data suggest that miglustat could restore the function of the cystic fibrosis transmembrane conductance regulator gene in cystic fibrosis cells. Single-center, randomized, double-blind, placebo-controlled, crossover Phase II study in 11 patients (mean±SD age, 26.3±7.7 years) homozygous for the F508del mutation received oral miglustat 200 mgt.i.d. or placebo for two 8-day cycles separated by a 14-day washout period. The primary endpoint was the change in total chloride secretion (TCS) assessed by nasal potential difference. No statistically significant changes in TCS, sweat chloride values or FEV(1) were detected. Pharmacokinetic and safety were similar to those observed in patients with other diseases exposed to miglustat. There was no evidence of a treatment effect on any nasal potential difference variable. Further studies with miglustat need to adequately address criteria for assessment of nasal potential difference. Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Synergy of cAMP and calcium signaling pathways in CFTR regulation

PubMed Central

Bozoky, Zoltan; Ahmadi, Saumel; Milman, Tal; Kim, Tae Hun; Du, Kai; Di Paola, Michelle; Pasyk, Stan; Pekhletski, Roman; Keller, Jacob P.; Bear, Christine E.; Forman-Kay, Julie D.

2017-01-01

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport. PMID:28242698

A new role for bicarbonate secretion in cervico-uterine mucus release.

PubMed

Muchekehu, Ruth W; Quinton, Paul M

2010-07-01

Cervical mucus thinning and release during the female reproductive cycle is thought to rely mainly on fluid secretion. However, we now find that mucus released from the murine reproductive tract critically depends upon concurrent bicarbonate (HCO(3)(-)) secretion. Prostaglandin E(2) (PGE(2))- and carbachol-stimulated mucus release was severely inhibited in the absence of serosal HCO(3)(-), HCO(3)(-) transport, or functional cystic fibrosis transmembrane conductance regulator (CFTR). In contrast to mucus release, PGE(2)- and carbachol-stimulated fluid secretion was not dependent on bicarbonate or on CFTR, but was completely blocked by niflumic acid. We found stimulated mucus release was severely impaired in the cystic fibrosis F508 reproductive tract, even though stimulated fluid secretion was preserved. Thus, CFTR mutations and/or poor bicarbonate secretion may be associated with reduced female fertility associated with abnormal mucus and specifically, may account for the increased viscosity and lack of cyclical changes in cervical mucus long noted in women with cystic fibrosis.

A new role for bicarbonate secretion in cervico-uterine mucus release

PubMed Central

Muchekehu, Ruth W; Quinton, Paul M

2010-01-01

Cervical mucus thinning and release during the female reproductive cycle is thought to rely mainly on fluid secretion. However, we now find that mucus released from the murine reproductive tract critically depends upon concurrent bicarbonate (HCO3−) secretion. Prostaglandin E2 (PGE2)- and carbachol-stimulated mucus release was severely inhibited in the absence of serosal HCO3−, HCO3− transport, or functional cystic fibrosis transmembrane conductance regulator (CFTR). In contrast to mucus release, PGE2- and carbachol-stimulated fluid secretion was not dependent on bicarbonate or on CFTR, but was completely blocked by niflumic acid. We found stimulated mucus release was severely impaired in the cystic fibrosis ΔF508 reproductive tract, even though stimulated fluid secretion was preserved. Thus, CFTR mutations and/or poor bicarbonate secretion may be associated with reduced female fertility associated with abnormal mucus and specifically, may account for the increased viscosity and lack of cyclical changes in cervical mucus long noted in women with cystic fibrosis. PMID:20478977

Newborn screening for cystic fibrosis: Polish 4 years' experience with CFTR sequencing strategy.

PubMed

Sobczyńska-Tomaszewska, Agnieszka; Ołtarzewski, Mariusz; Czerska, Kamila; Wertheim-Tysarowska, Katarzyna; Sands, Dorota; Walkowiak, Jarosław; Bal, Jerzy; Mazurczak, Tadeusz

2013-04-01

Newborn screening for cystic fibrosis (NBS CF) in Poland was started in September 2006. Summary from 4 years' experience is presented in this study. The immunoreactive trypsin/DNA sequencing strategy was implemented. The group of 1,212,487 newborns were screened for cystic fibrosis during the programme. We identified a total of 221 CF cases during this period, including, 4 CF cases were reported to be omitted by NBS CF. Disease incidence in Poland based on the programme results was estimated as 1/4394 and carrier frequency as 1/33. The frequency of the F508del was similar (62%) to population data previously reported. This strategy allowed us to identify 29 affected infants with rare genotypes. The frequency of some mutations (eg, 2184insA, K710X) was assessed in Poland for the first time. Thus, sequencing assay seems to be accurate method for screening programme using blood spots in the Polish population.

Differential phosphorylation patterns of P-glycoprotein reconstituted into a proteoliposome system: insight into additional unconventional phosphorylation sites.

PubMed

Lelong-Rebel, Isabelle H; Cardarelli, Carol O

2005-01-01

Membrane vesicles from the multidrug-resistant KB-V1 and KB-C1 cell lines overexpressing P-glycoprotein (Pgp), responsible for pleiotropic chemotherapeutic agents resistance, were solubilized with octyl-glucoside (OG-EX) and further fractionated on DEAE-sepharose column with increased concentrations of NaCl. The fraction containing Pgp (F3) was reconstituted into proteoliposomes (F3-PLP). Comparisons of the phosphorylation levels of Pgp achieved throughout the purification and reconstitution steps were addressed in this study. The [delta32 P] ATP-driven phosphorylation of Pgp was strongly increased in OG-EX, decreased in F3 and not detected in F3-PLP, when compared to Pgp phosphorylation in native plasma membrane vesicles. [delta32 P]ATP-phosphorylation of Pgp in F3-PLP could be restored by exogenously added PKC or by the catalytic sub-unit of PKA. The vanadate-induced hyperphosphorylation effect on Pgp by [delta32 P]ATP observed with plasma membrane vesicles was maintained in OG-EX, but was lost in F3 and did not enable labelling in F3-PLP. Enhancement of [delta32 P]-labelling of native Pgp via [delta32 P]ATP combined with GTP was maintained and also triggered phosphorylation of purified/reconstituted Pgp in F3-PLP as well. Altogether, our data suggest differential phosphorylation patterns of the transporter linked to environmental molecular composition (lipids, presence of detergent) and structure (unfolded versus embedded). In addition, restoration by GTP of Pgp phosphorylation by [delta32 P]ATP in the frame of F3-PLP suggests intra-molecular modulations and hints that other phosphorylation sites and processes, different from the classic ones involving PKC and/or PKA, may participate in the transporter's mechanism.

Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae.

PubMed Central

Basrai, M A; Kingsbury, J; Koshland, D; Spencer, F; Hieter, P

1996-01-01

A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and kinetochore mutation ndc10-42 or ctf13-30 show a synthetic conditional phenotype. Both spt4-138 and spt4 delta strains exhibit synergistic chromosome instability in combination with CEN DNA mutations and show in vitro defects in microtubule binding to minichromosomes. These results indicate that Spt4p plays a role in chromosome segregation. The results of in vivo genetic interactions with mutations in kinetochore proteins and CEN DNA and of in vitro biochemical assays suggest that Spt4p is important for kinetochore function. PMID:8649393

Influence of long-time stress relief treatments on the dynamic fracture toughness properties of ASME SA508 C1 2a and ASME SA533 GR B C12 pressure vessel steels

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

Logsdon, W.A.

1982-03-01

Dynamic fracture toughness tests were performed on materials which had been subjected to one of three long-time post weld type stress relief heat treatments: 48 hours at 1000/degree/F (538/degree/C), 24 hours at 1125/degree/F (607/degree/C), and 48 hours at 1125/degree/F (607/degree/C). Linear elastic K/sub Id/ results were obtained at low temperatures while J-integral techniques were utilized to evaluate dynamic fracture toughness over the transition and upper shelf temperature ranges. Tensile, Charpy impact, and drop weight nil-ductility transition tests as well as room temperature, air environment fatigue crack growth rate tests (SA508 Cl 2a only) were also performed. The fracture toughness ofmore » both materials exceeded the ASME specified minimum reference toughness K/sub IR/ curve. 17 refs.« less

Nocturnal frontal lobe epilepsy caused by a mutation in the GATOR1 complex gene NPRL3.

PubMed

Korenke, Georg-Christoph; Eggert, Marlene; Thiele, Holger; Nürnberg, Peter; Sander, Thomas; Steinlein, Ortrud K

2016-03-01

Mutations in NPRL3, one of three genes that encode proteins of the mTORC1-regulating GATOR1 complex, have recently been reported to cause cortical dysplasia with focal epilepsy. We have now analyzed a multiplex epilepsy family by whole exome sequencing and identified a frameshift mutation (NM_001077350.2; c.1522delG; p.E508Rfs*46) within exon 13 of NPRL3. This truncating mutation causes an epilepsy phenotype characterized by early childhood onset of mainly nocturnal frontal lobe epilepsy. The penetrance in our family was low (three affected out of six mutation carriers), compared to families with either ion channel- or DEPDC5-associated familial nocturnal frontal lobe epilepsy. The absence of apparent structural brain abnormalities suggests that mutations in NPRL3 are not necessarily associated with focal cortical dysplasia but might be able to cause epilepsy by different, yet unknown pathomechanisms. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Gaucher disease: A G[sup +1][yields]A[sup +1] IVS2 splice donor site mutation causing exon 2 skipping in the acid [beta]-glucosidase mRNA

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

He, Guo-Shun; Grabowski, G.A.

1992-10-01

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent Jewish genetic disease. About 30 identified missense mutations are causal to the defective activity of acid [beta]-glucosidase in this disease. cDNAs were characterized from a moderately affected 9-year-old Ashkenazi Jewish Gaucher disease type 1 patient whose 80-years-old, enzyme-deficient, 1226G (Asn[sup 370][yields]Ser [N370S]) homozygous grandfather was nearly asymptomatic. Sequence analyses revealed four populations of cDNAs with either the 1226G mutation, an exact exon 2 ([Delta] EX2) deletion, a deletion of exon 2 and the first 115 bp of exon 3 ([Delta] EX2-3), or a completely normal sequence. Aboutmore » 50% of the cDNAs were the [Delta] EX2, the [Delta] EX2-3, and the normal cDNAs, in a ratio of 6:3:1. Specific amplification and characterization of exon 2 and 5[prime] and 3[prime] intronic flanking sequences from the structural gene demonstrated clones with either the normal sequence or with a G[sup +1][yields]A[sup +1] transition at the exon 2/intron 2 boundary. This mutation destroyed the splice donor consensus site (U1 binding site) for mRNA processing. This transition also was present at the corresponding exon/intron boundary of the highly homologous pseudogene. This new mutation, termed [open quotes]IVS2 G[sup +1],[close quotes] is the first in the Ashkenazi Jewish population. The occurrence of this [open quotes]pseudogene[close quotes]-type mutation in the structural gene indicates the role of acid [beta]-glucosidase pseudogene and structural gene rearrangements in the pathogenesis of this disease. 33 refs., 8 figs., 1 tab.« less

Time-dependent correlation of cerebral blood flow with oxygen metabolism in activated human visual cortex as measured by fMRI.

PubMed

Lin, Ai-Ling; Fox, Peter T; Yang, Yihong; Lu, Hanzhang; Tan, Li-Hai; Gao, Jia-Hong

2009-01-01

The aim of this study was to investigate the relationship between relative cerebral blood flow (delta CBF) and relative cerebral metabolic rate of oxygen (delta CMRO(2)) during continuous visual stimulation (21 min at 8 Hz) with fMRI biophysical models by simultaneously measuring of BOLD, CBF and CBV fMRI signals. The delta CMRO(2) was determined by both a newly calibrated single-compartment model (SCM) and a multi-compartment model (MCM) and was in agreement between these two models (P>0.5). The duration-varying delta CBF and delta CMRO(2) showed a negative correlation with time (r=-0.97, P<0.001); i.e., delta CBF declines while delta CMRO(2) increases during continuous stimulation. This study also illustrated that without properly calibrating the critical parameters employed in the SCM, an incorrect and even an opposite appearance of the flow-metabolism relationship during prolonged visual stimulation (positively linear coupling) can result. The time-dependent negative correlation between flow and metabolism demonstrated in this fMRI study is consistent with a previous PET observation and further supports the view that the increase in CBF is driven by factors other than oxygen demand and the energy demands will eventually require increased aerobic metabolism as stimulation continues.

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.

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae.

PubMed

Saparbaev, M; Prakash, L; Prakash, S

1996-03-01

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

The Integration of Delta Prime (f)in a Multidimensional Space

NASA Technical Reports Server (NTRS)

Farassat, F.

1999-01-01

Consideration is given to the thickness noise term of the Ffowcs Williams-Hawkings equation when the time derivative is taken explicitly. An interpretation is presented of the integral I = function phi(x)delta-prime(f) dx, where it is initially assumed that the absolute value of Del-f is not equal to 1 on the surface f = 0.

Halogenated 2,5-pyrrolidinediones: synthesis, bacterial mutagenicity in Ames tester strain TA-100 and semi-empirical molecular orbital calculations.

PubMed

Freeman, B A; Wilson, R E; Binder, R G; Haddon, W F

2001-02-20

The chloroimide 3,3-dichloro-4-(dichloromethylene)-2,5-pyrrolidinedione, a tetrachloroitaconimide, is the principal mutagen produced by chlorination of simulated poultry chiller water. It is the second most potent mutagenic disinfection by-product of chlorination ever reported. Six of seven new synthetic analogs of this compound are direct-acting mutagens in Ames tester strain TA-100. Computed energies of the lowest unoccupied molecular orbital (E(LUMO)) and of the radical anion stability (DeltaH(f)(rad)-DeltaH(f)) from MNDO-PM3 for the chloroimides show a quantitative correlation with the Ames TA-100 bacterial mutagenicity values. The molar mutagenicities of these direct acting mutagenic imides having an exocyclic double bond fit the same linear correlation (lnM(m) vs. E(LUMO); lnM(m) vs. DeltaH(f)(rad)--DeltaH(f)) as the chlorinated 2(5H)-furanones, including the potent mutagen MX, 3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone, a by-product of water chlorination and paper bleaching with chlorine. Mutagenicity data for related haloimides having endocyclic double bonds are also given. For the same number of chlorine atoms, the imides with endocyclic double bonds have significantly higher Ames mutagenicity compared to their structural analogs with exocyclic double bonds, but do not follow the same E(LUMO) or DeltaH(f)(rad)-DeltaH(f) correlation as the exocyclic chloroimides and the chlorinated 2(5H)-furanones.

Hydrodynamic Force on a Cylinder Oscillating at Low Frequency

NASA Technical Reports Server (NTRS)

Berg, Robert F.; Yao, Minwu; Panzarella, Charles H.

2007-01-01

The hydrodynamic force on a cylinder oscillating transversely to its axis is a nonlinear function of the displacement amplitude x0. We report measurements and numerical calculations of the force at frequencies low enough that delta > R, where delta is the viscous penetration length and R is the cylinder radius. For small amplitudes, the numerically calculated Fourier transform of the force per unit length, F(sub small), agrees with Stokes' analytical calculation. For larger amplitudes, the force per unit length found by both calculation and measurement is F = F(sub small)C (x(sub 0)/delta,R/delta). The complex function C depends only weakly on R/delta, indicating that x0/delta is more appropriate as a scaling variable than the Keulegan-Carpenter number KC = pi*x(sub 0)/R. The measurements used a torsion oscillator driven at frequencies from 1 to 12 Hz while immersed in dense xenon. The oscillator comprised cylinders with an effective radius of R = 13.4 micron and oscillation amplitudes as large as x(sub 0)/delta = 4 (corresponding to KC as large as 71). The calculations used similar conditions except that the amplitudes were as large as x0/delta = 28.

Effects of recharge and discharge on delta2H and delta18O composition and chloride concentration of high arsenic/fluoride groundwater from the Datong Basin, northern China.

PubMed

Xie, Xianjun; Wang, Yanxin; Su, Chunli; Duan, Mengyu

2013-02-01

To better understand the effects of recharge and discharge on the hydrogeochemistry of high levels of arsenic (As) and fluoride (F) in groundwater, environmental isotopic composition (delta2H and delta18O) and chloride (Cl) concentrations were analyzed in 29 groundwater samples collected from the Datong Basin. High arsenic groundwater samples (As > 50 micog/L) were found to be enriched in lighter isotopic composition that ranged from -92 to -78 per thousand for deuterium (delta2H) and from -12.5 to -9.9 per thousand for oxygen-18 (delta18O). High F-containing groundwater (F > 1 mg/L) was relatively enriched in heavier isotopic composition and varied from -90 to -57 per thousand and from -12.2 to -6.7 per thousand for delta2H and delta18O, respectively. High chloride concentrations and delta18O values were primarily measured in groundwater samples from the northern and southwestern portions of the study area, indicating the effect of evaporation on groundwater. The observation of relatively homogenized and low delta18O values and chloride concentrations in groundwater samples from central part of the Datong Basin might be a result of fast recharge by irrigation returns, which suggests that irrigation using arsenic-contaminated groundwater affected the occurrence of high arsenic-containing groundwater in the basin.

Ion Channel Modulators in Cystic Fibrosis.

PubMed

Gentzsch, Martina; Mall, Marcus A

2018-05-08

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and remains one of the most common life-shortening genetic diseases affecting the lung and other organs. CFTR functions as a cAMP-dependent anion channel that transports chloride and bicarbonate across epithelial surfaces and disruption of these ion transport processes plays a central role in the pathogenesis of CF. These findings provided the rationale for pharmacological modulation of ion transport, either by targeting mutant CFTR or alternative ion channels that can compensate for CFTR dysfunction, as a promising therapeutic approach. High throughput screening has supported the development of CFTR modulator compounds. CFTR correctors are designed to improve defective protein processing, trafficking and cell surface expression, whereas potentiators increase the activity of mutant CFTR at the cell surface. The approval of the first potentiator ivacaftor for the treatment of patients with specific CFTR mutations and, more recently the corrector lumacaftor in combination with ivacaftor for patients homozygous for the common F508del mutation, were major breakthroughs on the path to causal therapies for all patients with CF. In this review, we focus on recent developments and remaining challenges of CFTR-directed therapies, as well as modulators of other ion channels such as alternative chloride channels and the epithelial sodium channel (ENaC) as additional targets in CF lung disease. Further, we discuss how patient-derived precision medicine models may aid the translation of emerging next generation ion channel modulators from the laboratory to the clinic and tailor their use for optimal therapeutic benefits in individual patients with CF. Copyright © 2018. Published by Elsevier Inc.

Stimulated electromagnetic emission and plasma line during pump wave frequency stepping near 4th electron gyroharmonic at HAARP

NASA Astrophysics Data System (ADS)

Grach, Savely; Sergeev, Evgeny; Shindin, Alexey; Mishin, Evgeny; Watkins, Brenton

Concurrent observations of stimulated (secondary) electromagnetic emissions (SEE) and incoherent plasma line (PL) backscatter from the MUIR radar during HF pumping of the ionosphere by the HAARP heating facility (62.4(°) °N, 145.15(°) W, magnetic inclination α = 75.8^circ) with the pump wave (PW) frequency sweeps about the fourth electron gyroharmonic (4f_c) are presented. The PW frequency f0 was changed every 0.2 s in a 1-kHz step, i.e. with the rate of r_{f_0}=5 kHz/s. PW was transmitted at the magnetic zenith (MZ). Prior to sweeping, PW was transmitted continuously (CW) during 2 min at f_0 = 5730 kHz <4f_c to create the “preconditioned” ionosphere with small-scale magnetic field-aligned irregularities. During CW pumping, a typical SEE spectrum for f_0<4f_c, containing the prominent downshifted maxiμm (DM) shifted by Delta f_{DM} = f_{DM}-f_0approx-9 kHz, developed in 5-10 s after PW turn on. The PL echoes were observed during 2-3 s from the range dsim 220 km corresponding to the altitude slightly above PW reflection height. After sim5 s the PL echoes descended to dsim 210-212 km corresponding to the height h = d / (sinalpha) by sim 7 km below the height where f_0 = 4f_c. During frequency sweeps, two upshifted features appeared in the SEE spectrum for f_0> 4f_c, namely BUM_S and BUM_D. The former (stationary broad upshifted maxiμm) peaks at Delta f_{BUMs} approx f0 - nfc (d) + 15-20 kHz and is a typical SEE spectral feature. The latter, the dynamic BUM_D at smaller Delta f, is observed only at high pump powers (ERP=1.7 GW) and corresponds to artificial descending plasma layers created in the F-region ionosphere [1]. In the experiment in question, the BUM_D was present for f_0> f^*, where f^* was 5805-5815 kHz during stepping up and sim 10 kHz less for stepping down, and located 8-10 km below the background F-layer. The miniμm DM which indicated that f_0=4f_c=f_{uh} in the background ionospheric plasma, was sim 5760 kHz. The PL was observed only for f_0< f^* and mainly from altitudes h where f_0 <4f_c. The height h decreased with increasing f_0 in accordance with the altitude dependence 4f_c(h), the difference Delta f_g = f_0 - 4f_c was kept constant during either sweeping up [-(4-8 kHz)] or sweeping down [-(18-22 kHz)]. This corresponds to the difference between the altitude where f_0=4f_c and the PL generation altitude by Delta h sim 1.5-3 km and 7-8 km, respectively. During stepping up, the PL was observed also from the ranges where f_0 > 4f_c. In this case we obtained Delta f_g sim 8-13 kHz corresponding to Delta h sim - 4 km. The PL has never been observed for f_0>f^*$. \\ 1. Sergeev E., Grach S., et al. //Phys. Rev. Lett., 110 (2013), 065002.

Drosophila melanogaster auxilin regulates the internalization of Delta to control activity of the Notch signaling pathway

PubMed Central

Hagedorn, Elliott J.; Bayraktar, Jennifer L.; Kandachar, Vasundhara R.; Bai, Ting; Englert, Dane M.; Chang, Henry C.

2006-01-01

We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain–containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch. PMID:16682530

Genotyping Brahman cattle for generalised glycogenosis.

PubMed

Dennis, J A; Healy, P J; Reichmann, K G

2002-05-01

To develop procedures for genotyping Brahman cattle for loss-of-function alleles within the acidic alpha-glucosidase gene and to assess the risk of generalised glycogenosis in Australian Brahman cattle. PCR assays for three loss-of-function alleles were designed to exploit internal restriction sites within acidic alpha-glucosidase amplicons that are independent of allelic variants at the mutant sites. Genotyping 8529 clinically normal Brahmans between August 1996 and August 2001 revealed 16.4% were heterozygous for the more common of the two mutations (1057deltaTA, often referred to as the 'E7' mutation) that cause generalised glycogenosis in this breed. The less common 1783T mutation (often referred to as the 'E13' mutation) was restricted to descendants of one imported bull, and was not detected in 600 randomly selected Brahmans. Prior to definition of these two disease-causing mutations, 640 (18%), and 14 (0.4%), of 3559 clinically normal Brahmans analysed between January 1994 and December 1996, were heterozygous, and homozygous, respectively, for a silent polymorphism (2223G-->A) that is associated with generalised glycogenosis. In addition to the 1057deltaTA and 1783T mutations, approximately 15% of Brahmans were found to be heterozygous for a single base substitution in exon 9 (1351T, commonly referred to as the 'E9' mutation) that significantly reduces acidic alpha-glucosidase activity, but has not been associated with clinical disease. These three loss-of-function alleles were found in Brahmans imported, or selected for import, from the USA. The PCR procedures reported here represent a significant improvement in reliability and accuracy over previous published methods. Utilisation of these PCR/restriction enzyme based assays will facilitate precise selection against the 1057deltaTA and 1783T alleles, and consequently reduce the incidence of generalised glycogenosis in registered and commercial Brahman herds.

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae.

PubMed

Galli, Alvaro; Cervelli, Tiziana; Schiestl, Robert H

2003-05-01

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.

Assessing thermochemical data

NASA Astrophysics Data System (ADS)

Holmes, John L.; Aubry, Christiane; Wang, Xian

2007-11-01

This paper describes, with examples, a critical assessment of thermochemical data for some small molecules and free radicals. The available heats of formation, [Delta]fH° (all 298 K values). for simple alkyl hydroperoxides and di-alkyl peroxides were compared and new data are provided. The [Delta]fH° values, all ±5 kJ/mol, are: CH3OOH, -135; CH3CH2OOH, -168; n-C3H7OOH, -189; s-C3H7OOH, -205; t-C4H9OOH, -240; CH3OOCH3, -132; CH3CH2OOCH3, -165; C2H5OOC2H5, -198; n-C3H7OOn-C3H7, -240; s-C3H7OOs-C3H7, -272; t-C4H9OOt-C4H9, -342. These are consistent with established O-O bond dissociation energies and with additivity considerations. [Delta]fH° values for the corresponding alkoxy radicals are also addressed. A similar survey was applied to the homologous n-alkyl aldehydes, C2 to C8, for which recommended [Delta]fH° values, all ±1.5 kJ/mol, are: -166.5, -189, -207.5, -227, -248, -268 and -289, respectively. Particular attention was given to [Delta]fH°(CH3CO) = -10.3 ± 1.8 kJ/mol. The current NIST WebBook datum, [Delta]fH°(CS) = 280.3 kJ/mol, is arguably the best value, being consistent with related thermochemical data. Finally the [Delta]fH° values for the allylic free radicals CH2CHCH2, 174 ± 3 kJ/mol, CH2CHCH(OH), 4.5 ± 4 kJ/mol, and (CH2CH)2C(OH), 37 ± 4 kJ/mol, derived from experimental data and results of computational chemistry are described, together with some related homolytic bond strengths.

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D.

PubMed

Bolz, H; von Brederlow, B; Ramírez, A; Bryda, E C; Kutsche, K; Nothwang, H G; Seeliger, M; del C-Salcedó Cabrera, M; Vila, M C; Molina, O P; Gal, A; Kubisch, C

2001-01-01

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa (RP). So far, six loci (USH1A-USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A for USH1B and the gene encoding harmonin for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G-->C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, Delta M1281 and IVS51+5G-->A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.

Improved modification for the density-functional theory calculation of thermodynamic properties for C-H-O composite compounds.

PubMed

Liu, Min Hsien; Chen, Cheng; Hong, Yaw Shun

2005-02-08

A three-parametric modification equation and the least-squares approach are adopted to calibrating hybrid density-functional theory energies of C(1)-C(10) straight-chain aldehydes, alcohols, and alkoxides to accurate enthalpies of formation DeltaH(f) and Gibbs free energies of formation DeltaG(f), respectively. All calculated energies of the C-H-O composite compounds were obtained based on B3LYP6-311++G(3df,2pd) single-point energies and the related thermal corrections of B3LYP6-31G(d,p) optimized geometries. This investigation revealed that all compounds had 0.05% average absolute relative error (ARE) for the atomization energies, with mean value of absolute error (MAE) of just 2.1 kJ/mol (0.5 kcal/mol) for the DeltaH(f) and 2.4 kJ/mol (0.6 kcal/mol) for the DeltaG(f) of formation.

Four novel connexin 32 mutations in X-linked Charcot-Marie-Tooth disease. Phenotypic variability and central nervous system involvement.

PubMed

Karadima, Georgia; Koutsis, Georgios; Raftopoulou, Maria; Floroskufi, Paraskewi; Karletidi, Karolina-Maria; Panas, Marios

2014-06-15

Charcot-Marie-Tooth (CMT) disease, the most common hereditary neuropathy, is clinically and genetically heterogeneous. X-linked CMT (CMTX) is usually caused by mutations in the gap junction protein b 1 gene (GJB1) coding for connexin 32 (Cx32). The clinical manifestations of CMTX are characterized by significant variability, with some patients exhibiting central nervous system (CNS) involvement. We report four novel mutations in GJB1, c.191G>A (p.Cys64Tyr), c.508G>T (p.Val170Phe), c.778A>G (p.Lys260Glu) and c.300C>G (p.His100Gln) identified in four unrelated Greek families. These mutations were characterized by variable phenotypic expression, including a family with the Roussy-Lévy syndrome, and three of them were associated with mild clinical CNS manifestations. Copyright © 2014. Published by Elsevier B.V.

Influence of illuminants on the color distribution of shade guides.

PubMed

Park, Ji-Hoon; Lee, Yong-Keun; Lim, Bum-Soon

2006-12-01

Although a shade tab in a shade guide is matched to a natural tooth in the order of value, hue, and chroma, there are limited data on the color distribution of currently available shade guides sorted by these 3 parameters. Furthermore, spectrophotometric color measurements of shade tabs differ depending on the standard illuminant employed. The purpose of this study was to determine the color distributions of 2 shade guides in value (CIE L( *)), chroma (C( *)(ab)) and hue angle (h(o)) scale relative to the standard illuminants D(65), A, and F2. Color of shade tabs (n=36) from 2 shade guides (Vita Lumin and Chromascop) were measured, and the distributions for CIE L( *), C( *)(ab) and h(o) values were compared. Color differences of shade tabs depending on the illuminant were calculated. The distributions of the ratios of CIE L( *) and C( *)(ab) values of each shade tab compared with the lowest value tab or the lowest chroma tab were determined. The data for the value, chroma, and hue angle within each shade guide were analyzed with a 2-way ANOVA with the factors of shade designation and type of illuminant (alpha=.05). Color difference caused by change of illuminant was analyzed with a 2-way ANOVA with the factors of shade designation and pair of illuminants compared (alpha=.05). The Scheffe multiple comparison test was performed as a post hoc test. CIE L( *), C( *)(ab) and h(o) values were influenced by shade designation and type of illuminant in both shade guides. Color difference caused by change of the illuminant was influenced by the shade designation and pair of illuminants compared. The order of mean color differences of 16 Vita Lumin shade tabs by pairs of illuminants compared was as follows: DeltaE( *)(ab) (D(65)/F2) = 1.63 3.7). Color distribution of 2 shade guides by the value and chroma was not logical.

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress.

PubMed

Voloshin, Olga; Gocheva, Yana; Gutnick, Marina; Movshovich, Natalia; Bakhrat, Anya; Baranes-Bachar, Keren; Bar-Zvi, Dudy; Parvari, Ruti; Gheber, Larisa; Raveh, Dina

2010-06-01

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.

[Changes in index-F and index-delta 4P in normal pregnancy, labor and the puerperium].

PubMed

Kamada, T

1984-04-01

Index-F and index-delta 4P (cortisol and progesterone which are not bound to corticosteroid-binding globulin (CBG) in the umbilical cord vein and the maternal blood were determined during pregnancy, at delivery and puerperium. Index-F and index-delta 4P were calculated as the total cortisol or total progesterone X% unbound to CBG divided by 100. The level of index-F showed a gradual rise during pregnancy, and in late pregnancy reached about 1.5 times as high as that of non-pregnant women, whereas the total cortisol level was about 3.3 times. Near delivery, index-F was almost completely stable, but at delivery, it increased suddenly in proportion to the rise in the total cortisol level. This rise is probably due to stress. In the umbilical cord vein blood, the level of index-F was 1.5 times higher than that in the maternal plasma before delivery; however the total cortisol level was lower than that of the maternal plasma. The levels of both index-delta 4P and total progesterone showed a gradual increase during pregnancy in parallel, and each value in late pregnancy was about 4.5 to 4.9 times that of early pregnant women. At or near delivery, the level of index-delta 4P was almost stable and no decrease occurred. In the umbilical cord vein plasma, the levels of index-delta 4P and total progesterone were extremely high. However, the meaning of these results isn't clear.

Disrupting the Myosin Converter-Relay Interface Impairs Drosophila Indirect Flight Muscle Performance

PubMed Central

Ramanath, Seemanti; Wang, Qian; Bernstein, Sanford I.; Swank, Douglas M.

2011-01-01

Structural interactions between the myosin converter and relay domains have been proposed to be critical for the myosin power stroke and muscle power generation. We tested this hypothesis by mutating converter residue 759, which interacts with relay residues I508, N509, and D511, to glutamate (R759E) and determined the effect on Drosophila indirect flight muscle mechanical performance. Work loop analysis of mutant R759E indirect flight muscle fibers revealed a 58% and 31% reduction in maximum power generation (PWL) and the frequency at which maximum power (fWL) is generated, respectively, compared to control fibers at 15°C. Small amplitude sinusoidal analysis revealed a 30%, 36%, and 32% reduction in mutant elastic modulus, viscous modulus, and mechanical rate constant 2πb, respectively. From these results, we infer that the mutation reduces rates of transitions through work-producing cross-bridge states and/or force generation during strongly bound states. The reductions in muscle power output, stiffness, and kinetics were physiologically relevant, as mutant wing beat frequency and flight index decreased about 10% and 45% compared to control flies at both 15°C and 25°C. Thus, interactions between the relay loop and converter domain are critical for lever-arm and catalytic domain coordination, high muscle power generation, and optimal Drosophila flight performance. PMID:21889448

Exocrine cell-derived microparticles in response to lipopolysaccharide promote endocrine dysfunction in cystic fibrosis.

PubMed

Constantinescu, Andrei Alexandru; Gleizes, Céline; Alhosin, Mahmoud; Yala, Elhassan; Zobairi, Fatiha; Leclercq, Alexandre; Stoian, Gheorghe; Mitrea, Ioan Liviu; Prévost, Gilles; Toti, Florence; Kessler, Laurence

2014-03-01

Diabetes in cystic fibrosis (CF) is a result of exocrine pancreas alteration followed by endocrine dysfunction at a later stage. Microparticles (MPs) are plasma membrane fragments shed from stimulated or damaged cells that act as cellular effectors. Our aim was to identify a new form of interaction between exocrine and endocrine pancreatic cells mediated by exocrine MPs, in the context of recurrent infection in CF. MPs from either human exocrine CFTRΔF508-mutated (CFPAC-1) cells or exocrine normal pancreatic (PANC-1) cells were collected after treatment by LPS from Pseudomonas aeruginosa and applied to rat endocrine normal insulin-secreting RIN-m5F cells. MP membrane integration in target cells was established by confocal microscopy and flow cytometry using PKH26 lipid probe. Apoptosis, lysosomal activity, insulin secretion were measured after 18 h. MP-mediated NF-κB activation was measured in HEK-Blue reporter cells by SEAP reporter gene system and in RIN-m5F cells by Western blot. In endocrine normal cells, CFTR inhibition was achieved using Inhibitor-172. Compared to PANC-1, MPs from CFPAC-1 significantly reduced insulin secretion and lysosomal activity in RIN-m5F. MPs induced NF-κB activation by increasing the level of IκB phosphorylation. Moreover, the inhibition of NF-κB activation using specific inhibitors was associated with a restored insulin secretion. Interestingly, CFTR inhibition in normal RIN-m5F cells promoted apoptosis and decreased insulin secretion. During recurrent infections associated with CF, exocrine MPs may contribute to endocrine cell dysfunction via NF-κB pathways. Membrane CFTR dysfunction is associated with decreased insulin secretion. © 2013. Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. All rights reserved.

Recessive Twinkle mutations in early onset encephalopathy with mtDNA depletion.

PubMed

Hakonen, Anna H; Isohanni, Pirjo; Paetau, Anders; Herva, Riitta; Suomalainen, Anu; Lönnqvist, Tuula

2007-11-01

Twinkle is a mitochondrial replicative helicase, the mutations of which have been associated with autosomal dominant progressive external ophthalmoplegia (adPEO), and recessively inherited infantile onset spinocerebellar ataxia (IOSCA). We report here a new phenotype in two siblings with compound heterozygous Twinkle mutations (A318T and Y508C), characterized by severe early onset encephalopathy and signs of liver involvement. The clinical manifestations included hypotonia, athetosis, sensory neuropathy, ataxia, hearing deficit, ophthalmoplegia, intractable epilepsy and elevation of serum transaminases. The liver showed mtDNA depletion, whereas the muscle mtDNA was only slightly affected. Alpers-Huttenlocher syndrome has previously been associated with mutations of polymerase gamma, a replicative polymerase of mtDNA. We show here that recessive mutations of the close functional partner of the polymerase, the Twinkle helicase, can also manifest as early encephalopathy with liver involvement, a phenotype reminiscent of Alpers syndrome, and are a new genetic cause underlying tissue-specific mtDNA depletion.

Genome-wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2

PubMed Central

Wright, Fred A.; Strug, Lisa J.; Doshi, Vishal K.; Commander, Clayton W.; Blackman, Scott M.; Sun, Lei; Berthiaume, Yves; Cutler, David; Cojocaru, Andreea; Collaco, J. Michael; Corey, Mary; Dorfman, Ruslan; Goddard, Katrina; Green, Deanna; Kent, Jack W.; Lange, Ethan M.; Lee, Seunggeun; Li, Weili; Luo, Jingchun; Mayhew, Gregory M.; Naughton, Kathleen M.; Pace, Rhonda G.; Paré, Peter; Rommens, Johanna M.; Sandford, Andrew; Stonebraker, Jaclyn R.; Sun, Wei; Taylor, Chelsea; Vanscoy, Lori L.; Zou, Fei; Blangero, John; Zielenski, Julian; O’Neal, Wanda K.; Drumm, Mitchell L.; Durie, Peter R.; Knowles, Michael R.; Cutting, Garry R.

2012-01-01

A combined genome-wide association and linkage study was used to identify loci causing variation in CF lung disease severity. A significant association (P=3. 34 × 10-8) near EHF and APIP (chr11p13) was identified in F508del homozygotes (n=1,978). The association replicated in F508del homozygotes (P=0.006) from a separate family-based study (n=557), with P=1.49 × 10-9 for the three-study joint meta-analysis. Linkage analysis of 486 sibling pairs from the family-based study identified a significant QTL on chromosome 20q13.2 (LOD=5.03). Our findings provide insight into the causes of variation in lung disease severity in CF and suggest new therapeutic targets for this life-limiting disorder. PMID:21602797

Discovery of Multitarget Agents Active as Broad-Spectrum Antivirals and Correctors of Cystic Fibrosis Transmembrane Conductance Regulator for Associated Pulmonary Diseases.

PubMed

Tassini, Sabrina; Sun, Liang; Lanko, Kristina; Crespan, Emmanuele; Langron, Emily; Falchi, Federico; Kissova, Miroslava; Armijos-Rivera, Jorge I; Delang, Leen; Mirabelli, Carmen; Neyts, Johan; Pieroni, Marco; Cavalli, Andrea; Costantino, Gabriele; Maga, Giovanni; Vergani, Paola; Leyssen, Pieter; Radi, Marco

2017-02-23

Enteroviruses (EVs) are among the most frequent infectious agents in humans worldwide and represent the leading cause of upper respiratory tract infections. No drugs for the treatment of EV infections are currently available. Recent studies have also linked EV infection with pulmonary exacerbations, especially in cystic fibrosis (CF) patients, and the importance of this link is probably underestimated. The aim of this work was to develop a new class of multitarget agents active both as broad-spectrum antivirals and as correctors of the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding defect responsible for >90% of CF cases. We report herein the discovery of the first small molecules able to simultaneously act as correctors of the F508del-CFTR folding defect and as broad-spectrum antivirals against a panel of EVs representative of all major species.

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.

Study of lee-side flows over conically cambered Delta wings at supersonic speeds, part 2

NASA Technical Reports Server (NTRS)

Wood, Richard M.; Watson, Carolyn B.

1987-01-01

An experimental investigation was performed in which surface pressure data, flow visualization data, and force and moment data were obtained on four conical delta wing models which differed in leading edge camber only. Wing leading edge camber was achieved through a deflection of the outboard 30% of the local wing semispan of a reference 75 deg swept flat delta wing. The four wing models have leading edge deflection angles delta sub F of 0, 5, 10, and 15 deg measured streamwise. Data for the wings with delta sub F = 10 and 15 deg showed that hinge line separation dominated the lee-side wing loading and prohibited the development of leading edge separation on the deflected portion of wing leading edge. However, data for the wing with delta sub F = 5 deg showed that at an angle of attack of 5 deg, a vortex was positioned on the deflected leading edge with reattachment at the hinge line. Flow visualization results were presented which detail the influence of Mach number, angle of attack, and camber on the lee-side flow characteristics of conically cambered delta wings. Analysis of photographic data identified the existence of 12 distinctive lee-side flow types.

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 drug. 340 patients continued treatment with lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h; 176 patients who had received placebo in the TRANSPORT or TRAFFIC studies initiated treatment with lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h, the commercially available dose, for which data are presented. The most common adverse events were infective pulmonary exacerbations, cough, increased sputum, and haemoptysis. Modest blood pressure increases seen in TRAFFIC and TRANSPORT were also observed in PROGRESS. For patients continuing treatment, the mean change from baseline in ppFEV 1 was 0·5 (95% CI -0·4 to 1·5) at extension week 72 and 0·5 (-0·7 to 1·6) at extension week 96; change in BMI was 0·69 (0·56 to 0·81) at extension week 72 and 0·96 (0·81 to 1·11) at extension week 96. The annualised pulmonary exacerbation rate in patients continuing treatment through extension week 96 (0·65, 0·56 to 0·75) remained lower than the placebo rate in TRAFFIC and TRANSPORT. The annualised rate of ppFEV 1 decline was reduced in lumacaftor/ivacaftor-treated patients compared with matched controls (-1·33, -1·80 to -0·85 vs -2·29, -2·56 to -2·03). The efficacy and safety profile of the lumacaftor 600 mg once daily/ivacaftor 250 mg every 12 h groups was generally similar to that of the lumacaftor 400 mg every 12 h/ivacaftor 250 mg every 12 h groups. The long-term safety profile of lumacaftor/ivacaftor combination therapy was consistent with previous RCTs. Benefits continued to be observed with longer-term treatment, and lumacaftor/ivacaftor was associated with a 42% slower rate of ppFEV 1 decline than in matched registry controls. Vertex Pharmaceuticals Incorporated. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of the CFTR gene in Venezuelan cystic fibrosis patients, identification of six novel cystic fibrosis-causing genetic variants.

PubMed

Sánchez, Karen; de Mendonca, Elizabeth; Matute, Xiorama; Chaustre, Ismenia; Villalón, Marlene; Takiff, Howard

2016-01-01

The mutations in the CFTR gene found in patients with cystic fibrosis (CF) have geographic differences, but there are scant data on their prevalence in Venezuelan patients. This study determined the frequency of common CFTR gene mutations in a group of Venezuelan patients with CF. The 27 exons of the CFTR gene from 110 Venezuelan patients in the National CF Program were amplified and sequenced. A total of 36 different mutations were identified, seven with frequencies greater than 1%: p.Phe508del (27.27%), p.Gly542* (3.18%), c.2988+1G>A (3.18%), p.Arg334Trp (1.36%), p.Arg1162* (1.36%), c.1-8G>C (1.36%), and p.[Gly628Arg;Ser1235Arg](1.36). In 40% of patients, all with a clinical diagnosis of CF, no mutations were found. This report represents the largest cohort of Venezuelan patients with CF ever examined, and includes a wider mutation panel than has been previously studied in this population. Mutations common in Southern European populations predominate, and several new mutations were discovered, but no mutations were found in 40% of the cohort.

Analysis of the CFTR gene in Venezuelan cystic fibrosis patients, identification of six novel cystic fibrosis-causing genetic variants

PubMed Central

Sánchez, Karen; de Mendonca, Elizabeth; Matute, Xiorama; Chaustre, Ismenia; Villalón, Marlene; Takiff, Howard

2016-01-01

The mutations in the CFTR gene found in patients with cystic fibrosis (CF) have geographic differences, but there are scant data on their prevalence in Venezuelan patients. This study determined the frequency of common CFTR gene mutations in a group of Venezuelan patients with CF. The 27 exons of the CFTR gene from 110 Venezuelan patients in the National CF Program were amplified and sequenced. A total of 36 different mutations were identified, seven with frequencies greater than 1%: p.Phe508del (27.27%), p.Gly542* (3.18%), c.2988+1G>A (3.18%), p.Arg334Trp (1.36%), p.Arg1162* (1.36%), c.1-8G>C (1.36%), and p.[Gly628Arg;Ser1235Arg](1.36). In 40% of patients, all with a clinical diagnosis of CF, no mutations were found. This report represents the largest cohort of Venezuelan patients with CF ever examined, and includes a wider mutation panel than has been previously studied in this population. Mutations common in Southern European populations predominate, and several new mutations were discovered, but no mutations were found in 40% of the cohort. PMID:27022295

Remarkable stabilization of a psychrotrophic RNase HI by a combination of thermostabilizing mutations identified by the suppressor mutation method.

PubMed

Tadokoro, Takashi; Matsushita, Kyoko; Abe, Yumi; Rohman, Muhammad Saifur; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

2008-08-05

Ribonuclease HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is much less stable than Escherichia coli RNase HI (Ec-RNase HI) by 22.4 degrees C in T m and 12.5 kJ mol (-1) in Delta G(H 2O), despite their high degrees of structural and functional similarity. To examine whether the stability of So-RNase HI increases to a level similar to that of Ec-RNase HI via introduction of several mutations, the mutations that stabilize So-RNase HI were identified by the suppressor mutation method and combined. So-RNase HI and its variant with a C-terminal four-residue truncation (154-RNase HI) complemented the RNase H-dependent temperature-sensitive (ts) growth phenotype of E. coli strain MIC3001, while 153-RNase HI with a five-residue truncation could not. Analyses of the activity and stability of these truncated proteins suggest that 153-RNase HI is nonfunctional in vivo because of a great decrease in stability. Random mutagenesis of 153-RNase HI using error-prone PCR, followed by screening for the revertants, allowed us to identify six single suppressor mutations that make 153-RNase HI functional in vivo. Four of them markedly increased the stability of the wild-type protein by 3.6-6.7 degrees C in T m and 1.7-5.2 kJ mol (-1) in Delta G(H 2O). The effects of these mutations were nearly additive, and combination of these mutations increased protein stability by 18.7 degrees C in T m and 12.2 kJ mol (-1) in Delta G(H 2O). These results suggest that several residues are not optimal for the stability of So-RNase HI, and their replacement with other residues strikingly increases it to a level similar to that of the mesophilic counterpart.

CFTR RECRUITMENT TO PHAGOSOMES IN NEUTROPHILS

PubMed Central

Zhou, Yun; Song, Kejing; Painter, Richard G.; Aiken, Martha; Reiser, Jakob; Stanton, Bruce A.; Nauseef, William M.; Wang, Guoshun

2013-01-01

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that ~95–99% of LAMP-1 positive mature phagosomes were CFTR-positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed EGFP alone, EGFP-wt-CFTR and EGFP-ΔF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and co-localize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-ΔF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, CFTR corrector compound VRT-325 facilitated the recruitment of ΔF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function. PMID:23486169

A false positive newborn screening result due to a complex allele carrying two frequent CF-causing variants.

PubMed

Bergougnoux, Anne; Boureau-Wirth, Amandine; Rouzier, Cécile; Altieri, Jean-Pierre; Verneau, Fanny; Larrieu, Lise; Koenig, Michel; Claustres, Mireille; Raynal, Caroline

2016-05-01

The detection of two frequent CFTR disease-causing variations in the context of a newborn screening program (NBS) usually leads to the diagnosis of cystic fibrosis (CF) and a relevant genetic counseling in the family. In the present study, CF-causing variants p.Phe508del (F508del) and c.3140-26A>G (3272-26A>G) were identified on a neonate with positive ImmunoReactive Trypsinogen test by the Elucigene™ CF30 kit. The CF diagnosis initially suggested, despite three inconclusive Sweat Chloride Tests (SCT), was finally ruled out after the familial segregation study combined with a negative SCT. Haplotype studies, based on the comparison of 80 p.Phe508del haplotypes, suggested a probable de novo occurrence of c.3140-26A>G on the p.Phe508del ancestral allele in this family. This false positive case emphasizes the importance of SCT in the NBS strategy. Moreover, it raises the need for familial segregation studies in CF and in overall molecular diagnosis strategy of autosomal recessive diseases. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Microwave Spectrum, Structure, and Nuclear Quadrupole Coupling Constants of 1-Bromo-1-fluoroethane.

PubMed

Tatamitani; Kuwano; Fuchigami; Oe; Ogata

1999-08-01

The microwave spectrum of 1-bromo-1-fluoroethane, CHBrF-CH(3) and CHBrF-CH(2)D ((79/81)Br), has been studied for the first time from 8 to 41 GHz. A least-squares analysis of the observed a- and b-type transition frequencies gave rotational and centrifugal distortion constants and components of the bromine nuclear quadrupole coupling constant tensor in the principal axes system as follows: A = 8979.428(5) MHz, B = 2883.898(3) MHz, C = 2310.535(3) MHz, Delta(J) = 0.74(2) kHz, Delta(JK) = 2.49(3) kHz, Delta(K) = 5.3(5) kHz, delta(J) = 0.146(1) kHz, delta(K) = 2.75(4) kHz, chi(aa) = 493.49(29) MHz, chi(bb) - chi(cc) = -38.89(11) MHz, and ||chi(ab) || = 161.8(28) MHz for the CH(79)BrF-CH(3) species; A = 8979.257(5) MHz, B = 2859.072(3) MHz, C = 2294.572(3), Delta(J) = 0.76(2) kHz, Delta(JK) = 2.51(3) kHz, Delta(K) = 4.5(4) kHz, delta(J) = 0.145(1) kHz, delta(K) = 2.70(4) kHz, chi(aa) = 412.42(27) MHz, chi(bb) - chi(cc) = -32.56 (11) MHz, and ||chi(ab) || = 133.3(3) MHz for the CH(81)BrF-CH(3) species. The structural parameters are calculated from the 24 observed rotational constants, and electronic properties of the carbon-bromine bond in 1-bromo-1-fluoroethane are evaluated from the observed nuclear quadrupole coupling constants. These molecular properties are compared with those of other related molecules. The molecular structure of 1-bromo-1-fluoroethane is found to be very close to that of 1,1-difluoroethane except for the C-Br bond. Copyright 1999 Academic Press.

Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations

PubMed Central

1992-01-01

We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222. PMID:1431803

Dynamics of δ-dopant redistribution during heterostructure growth

NASA Astrophysics Data System (ADS)

Pankratov, E. L.

2007-06-01

It has recently been shown that growth of a multilayer structure with one or more delta-layers at high temperature leads to spreading and asymmetrization of the dopant distribution [see, for example, E.F.J. Schubert, Vac. Sci. Technol. A. 8, 2980 (1990), A.M. Nazmul, S. Sugahara, M. Tanaka, J. Crystal Growth 251, 303 (2003); R.C. Newman, M.J. Ashwin, M.R. Fahy, L. Hart, S.N. Holmes, C. Roberts, X. Zhang, Phys. Rev. B 54, 8769 (1996); E.F. Schubert, J.M. Kuo, R.F. Kopf, H.S. Luftman, L.C. Hopkins, N.J. Sauer, J. Appl. Phys. 67, 1969 (1990); P.M. Zagwijn, J.F. van der Veen, E. Vlieg, A.H. Reader, D.J. Gravesteijn, J. Appl. Phys. 78, 4933 (1995); W.S. Hobson, S.J. Pearton, E.F. Schubert, G. Cabaniss, Appl. Phys. Lett. 55, 1546 (1989); Delta Doping of Semiconductors, edited by E.F. Schubert (Cambridge University Press, Cambridge, 1996); Yu.N. Drozdov, N.B. Baidus', B.N. Zvonkov, M.N. Drozdov, O.I. Khrykin, V.I. Shashkin, Semiconductors 37, 194 (2003); E. Skuras, A.R. Long, B. Vogele, M.C. Holland, C.R. Stanley, E.A. Johnson, M. van der Burgt, H. Yaguchi, J. Singleton, Phys. Rev. B 59, 10712 (1999); G. Li, C. Jagadish, Solid-State Electronics 41, 1207 (1997)]. In this work analytical and numerical analysis of dopant dynamics in a delta-doped area of a multilayer structure has been accomplished using Fick's second law. Some reasons for asymmetrization of a delta-dopant distribution are illustrated. The spreading of a delta-layer has been estimated using example materials of a multilayer structure, a delta-layer and an overlayer.

JAK2 V617F, MPL W515L and JAK2 Exon 12 Mutations in Chinese Patients with Primary Myelofibrosis.

PubMed

Xia, Jun; Lu, Mi-Ze; Jiang, Yuan-Qiang; Yang, Guo-Hua; Zhuang, Yun; Sun, Hong-Li; Shen, Yun-Feng

2012-03-01

JAK2 V617F, MPL W515L and JAK2 exon 12 mutations are novel acquired mutations that induce constitutive cytokine-independent activation of the JAK-STAT pathway in myeloproliferative disorders (MPD). The discovery of these mutations provides novel mechanism for activation of signal transduction in hematopoietic malignancies. This research was to investigate their prevalence in Chinese patients with primary myelofibrosis (PMF). We introduced allele-specific PCR (AS-PCR) combined with sequence analysis to simultaneously screen JAK2 V617F, MPL W515L and JAK2 exon 12 mutations in 30 patients with PMF. Fifteen PMF patients (50.0%) carried JAK2 V617F mutation, and only two JAK2 V617F-negative patients (6.7%) harbored MPL W515L mutation. None had JAK2 exon 12 mutations. Furthermore, these three mutations were not detected in 50 healthy controls. MPL W515L and JAK2 V617F mutations existed in PMF patients but JAK2 exon 12 mutations not. JAK2 V617F and MPL W515L and mutations might contribute to the primary molecular pathogenesis in patients with PMF.

Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.

PubMed Central

Kögel, D; Aboud, M; Flügel, R M

1995-01-01

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. Images PMID:7544460

Breed distribution and history of canine mdr1-1Delta, a pharmacogenetic mutation that marks the emergence of breeds from the collie lineage.

PubMed

Neff, Mark W; Robertson, Kathryn R; Wong, Aaron K; Safra, Noa; Broman, Karl W; Slatkin, Montgomery; Mealey, Katrina L; Pedersen, Niels C

2004-08-10

A mutation in the canine multidrug resistance gene, MDR1, has previously been associated with drug sensitivities in two breeds from the collie lineage. We exploited breed phylogeny and reports of drug sensitivity to survey other purebred populations that might be genetically at risk. We found that the same allele, mdr1-1Delta, segregated in seven additional breeds, including two sighthounds that were not expected to share collie ancestry. A mutant haplotype that was conserved among affected breeds indicated that the allele was identical by descent. Based on breed histories and the extent of linkage disequilibrium, we conclude that all dogs carrying mdr1-1Delta are descendants of a dog that lived in Great Britain before the genetic isolation of breeds by registry (ca. 1873). The breed distribution and frequency of mdr1-1Delta have applications in veterinary medicine and selective breeding, whereas the allele's history recounts the emergence of formally recognized breeds from an admixed population of working sheepdogs.

Optimal least-squares finite element method for elliptic problems

NASA Technical Reports Server (NTRS)

Jiang, Bo-Nan; Povinelli, Louis A.

1991-01-01

An optimal least squares finite element method is proposed for two dimensional and three dimensional elliptic problems and its advantages are discussed over the mixed Galerkin method and the usual least squares finite element method. In the usual least squares finite element method, the second order equation (-Delta x (Delta u) + u = f) is recast as a first order system (-Delta x p + u = f, Delta u - p = 0). The error analysis and numerical experiment show that, in this usual least squares finite element method, the rate of convergence for flux p is one order lower than optimal. In order to get an optimal least squares method, the irrotationality Delta x p = 0 should be included in the first order system.

Phenotypic behavior of C2C12 myoblasts upon expression of the dystrophy-related caveolin-3 P104L and TFT mutants.

PubMed

Fanzani, Alessandro; Stoppani, Elena; Gualandi, Laura; Giuliani, Roberta; Galbiati, Ferruccio; Rossi, Stefania; Fra, Anna; Preti, Augusto; Marchesini, Sergio

2007-10-30

Caveolin-3 (Cav-3) is the main scaffolding protein present in myofiber caveolae. We transfected C2C12 myoblasts with dominant negative forms of Cav-3, P104L or DeltaTFT, respectively, which cause the limb-girdle muscular dystrophy 1-C. Both these forms triggered Cav-3 loss during C2C12 cell differentiation. The P104L mutation reduced myofiber formation by impaired AKT signalling, accompanied by dramatic expression of the E3 ubiquitin ligase Atrogin. On the other hand, the DeltaTFT mutation triggered hypertrophic myotubes sustained by prolonged AKT activation, but independent of increased levels of follistatin and interleukin 4 expression. These data suggest that separated mutations within the same dystrophy-related gene may cause muscle degeneration through different mechanisms.

The lipid A of Burkholderia multivorans C1576 smooth-type lipopolysaccharide and its pro-inflammatory activity in a cystic fibrosis airways model.

PubMed

Ieranò, Teresa; Cescutti, Paola; Leone, Maria Rosaria; Luciani, Alessandro; Rizzo, Roberto; Raia, Valeria; Lanzetta, Rosa; Parrilli, Michelangelo; Maiuri, Luigi; Silipo, Alba; Molinaro, Antonio

2010-12-01

Cystic fibrosis is an autosomal recessive disorder and it is characterised by chronic bacterial airway infection which leads to progressive lung deterioration, sometimes with fatal outcome. Burkholderia multivorans and Burkholderia cenocepacia are the species responsible for most of the infections of cystic fibrosis patients. Lipopolysaccharide endotoxins (LPSs) are among the foremost factors of pathogenesis of Gram-negative infection and, in particular, lipid A is the endotoxic portion of LPS responsible for eliciting host innate immune response. In this work, the complete primary structure of the lipid A from B. multivorans C1576 has been defined and, further, its pro-inflammatory activity in a cystic fibrosis airways model is shown. The structure of B. multivorans lipid A was attained by chemical, mass spectrometry and nuclear magnetic resonance analyses whereas its biological activity was assessed on the intestinal epithelial cell line CACO-2 cells, on the airway epithelial IB3-1 cells, carrying the ΔF508/W1282X CFTR mutation and on an ex vivo model of culture explants of nasal polyps.

Relationship between JAK2V617F mutation, allele burden and coagulation function in Ph-negative myeloproliferative neoplasms.

PubMed

Hu, Linhui; Pu, Lianfang; Ding, Yangyang; Li, Manman; Cabanero, Michael; Xie, Jingxin; Zhou, Dejun; Yang, Dongdong; Zhang, Cui; Wang, Huiping; Zhai, Zhimin; Ru, Xiang; Li, Jingrong; Xiong, Shudao

2017-07-01

Our aim was to explore the relationship between JAK2V617F mutation allele burden and hematological parameters especially in coagulation function in Chinese population. This study included 133 Ph-negative myeloproliferative neoplasms (MPNs) patients between 2013 and 2016. All the clinical and experimental data of patients were collected at the time of the diagnosis without any prior treatment, including blood parameters, coagulation function, splenomegaly, vascular events and chromosome karyotype. PCR and qPCR were used to detect JAK2V617F mutation and JAK2V617F mutation allele burden. In polycythemia vera patients, a positive correlation between the allele burden of JAK2V617F mutation and PLT counts was found; in essential thrombocythemia (ET) patients, WBC counts, RBC counts, HB, and HCT were higher in mutated patients than in wild-type patients. Furthermore, PT-INR was higher in ET and PMF mutated patients. In addition, a positive correlation between the allele burden of JAK2V617F mutation and activated partial thromboplastin time (APTT) was observed in JAK2V617F mutated ET patients. Higher hematologic parameters including counts of WBC, RBC, and PLT are closely associated with JAK2V617F mutation and its burden in Ph-negative MPNs; importantly, PT-INR, APTT are also related to JAK2V617F mutation and allele burden. Thus, our data indicate that JAK2V617F mutation allele burden might not only represent the burden of MPN but also alter the coagulation function.

Interactions between nested sunspots. 1: The formation and breakup of a delta-type sunspot

NASA Astrophysics Data System (ADS)

Gaizauskas, V.; Harvey, K. L.; Proulx, M.

1994-02-01

We investigate a nest of sunspots in which three ordinary bipolar pairs of sunspots are aligned collinearly. The usual spreading action of the growing regions brings two spots of leading polarity together (p-p collision) and forces the leading and trailing spots of the two interior regions to overlap into a single penumbra (p-f collision), thus forming a delta-spot. We examine digitally processed images from the Ottawa River Solar Observatory of two related events inside the delta-spot 5 days after the p-f collision begins: the violent disruption of the f-umbra, and the formation in less than a day of an hydrogen-alpha filament. The evolutionary changes in shape, area, relative motions, and brightness that we measure for each spot in the elongated nest are more compatible with Parker's (1979a) hypothesis of a sunspot as a cluster of flux tubes held together by downdrafts than with the notion of a sunspot as a monolithic plug of magnetic flux. From chromospheric developments over the delta-spot, we show that a shearing motion along a polarity inversion is more effective than convergence for creating a chromospheric filament. We invoke the release of an instability, triggered by a sequence of processes lasting 1 day or more, to explain the disruption of the f-umbra in this delta-spot. We show that the sequence is initiated when the colliding p-f umbrae reach a critical separation around 3200 +/- 200 km. We present a descriptive model in which the reconnected magnetic fields block vertical transport of convective heat flux just beneath the photosphere. We observe the formation of an unusual type of penumbra adjacent to the f-polarity portion of this delta-spot just before its disruption. A tangential penumbral band grows out of disordered matter connected to the f-umbra. We present this as evidence for the extrusion of umbral magnetic flux by thermal plumes rising through a loosely bound umbra.

Interactions between nested sunspots. 1: The formation and breakup of a delta-type sunspot

NASA Technical Reports Server (NTRS)

Gaizauskas, V.; Harvey, K. L.; Proulx, M.

1994-01-01

We investigate a nest of sunspots in which three ordinary bipolar pairs of sunspots are aligned collinearly. The usual spreading action of the growing regions brings two spots of leading polarity together (p-p collision) and forces the leading and trailing spots of the two interior regions to overlap inot a single penumbra (p-f collision), thus forming a delta-spot. We examine digitally processed images from the Ottawa River Solar Observatory of two related events inside the delta-spot 5 days after the p-f collision begins: the violent disruption of the f-umbra, and the formation in less than a day of an hydrogen-alpha filament. The evolutionary changes in shape, area, relative motions, and brightness that we measure for each spot in the elongated nest are more compatible with Parker's (1979a) hypothesis of a sunspot as a cluster of flux tubes held together by downdrafts than with the notion of a sunspot as a monolithic plug of magnetic flux. From chromospheric developments over the delta-spot, we show that a shearing motion along a polarity inversion is more effective than convergence for creating a chromospheric filament. We invoke the release of an instability, triggered by a sequence of processes lasting 1 day or more, to explain the disruption of the f-umbra in this delta-spot. We show that the sequence is initiated when the colliding p-f umbrae reach a critical separation around 3200 +/- 200 km. We present a descriptive model in which the reconnected magnetic fields block vertical transport of convective heat flux just beneath the photosphere. We observe the formation of an unusual type of penumbra adjacent to the f-polarity portion of this delta-spot just before its disruption. A tangential penumbral band grows out of disordered matter connected to the f-umbra. We present this as evidence for the extrusion of umbral magnetic flux by thermal plumes rising through a loosely bound umbra.

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

Awata, T.; Matsumoto, C.; Iwamoto, Y.

We studied a woman with acanthosis nigricans and insulin resistance. The patient`s Epstein-Barr virus-transformed lymphocytes revealed slightly decreased insulin binding and markedly decreased insulin-stimulated autophosphorylation of the insulin receptor. The nucleotide sequence analysis of the patient`s genomic DNA revealed a 3-basepair in-frame deletion of one allele, resulting in the loss of leucine at position 999 of the insulin receptor ({Delta}Leu{sup 999}). The messenger ribonucleic acid transcripts from the mutant allele in the patient`s lymphocytes were not decreased. Insulin-stimulated autophosphorylation of the insulin receptor from cells expressing {Delta}Leu{sup 999} mutant insulin receptor complementary DNA was markedly decreased. The proband, her mother,more » elder brother, and younger brother, who were heterozygous for this mutation, showed moderate or marked hyperinsulinemia during oral glucose tolerance tests. Although fasting glucose levels were normal and fasting insulin values were preserved in all subjects with the mutation for the 8-yr period of observation, a tendancy of progressive increase in postload glucose levels were observed. These results suggest that the {Delta}Leu{sup 999} mutation, which reduces tyrosine kinase activity, was responsible for insulin resistance and contributed to postload hyperglycemia. 27 refs., 3 figs., 1 tab.« less

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner* ♦

PubMed Central

Eckford, Paul D. W.; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E.

2012-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe. PMID:22942289

Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner.

PubMed

Eckford, Paul D W; Li, Canhui; Ramjeesingh, Mohabir; Bear, Christine E

2012-10-26

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe.

[Measurement of periapical pressure created by occlusal loading].

PubMed

Dobó, Nagy Csaba; Fejérdy, Pál; Angyal, János; Harasztosi, Lajos; Daróczi, Lajos; Beke, Dezsó; Wesselink, Paul R

2004-04-01

The aim of this study was to develop an in vitro model in which the pressure in the periapical tissues can be measured during loading. Extracted human maxillary central incisors were embedded into resin blocks that had physical characteristics similar to bone and periodontal ligament. Each tooth was loaded with 20, 40, 50, 60, 75, 85, 100, 200, 300 and 450 N vertical forces from the incisal edge of the crown; this procedure was carried out three consecutive times. A minute resistor embedded in the periapical space was used to detect apical pressure changes during occlusal loading. The ratio of apical pressure changes (delta P) to the loading force changes (delta F) was calculated. The periapical pressure detected was in direct proportion to the loading force. The mean value of delta P/delta F was 5.994 kPa/N (SD = 2.04). Direct proportionality was found between the coronal loading and the apical hydrostatic pressure. The (delta P)/(delta F) ratio determined in this study makes it easier to estimate the apical hydrostatic pressure values during occlusal loading of single rooted teeth. In this study the apical pressure generated under occlusal loading was of the same magnitude as that estimated with the finite element method.

Measurement of periapical pressure created by occlusal loading.

PubMed

Dobó-Nagy, C; Fejérdy, P; Angyal, J; Harasztosi, L; Daróczi, L; Beke, D; Wesselink, P R

2003-10-01

To develop an in vitro model in which the pressure in the periapical tissues can be measured during loading. Extracted human maxillary central incisors were embedded in resin blocks that had physical characteristics similar to those of bone and periodontal ligament. Each tooth was loaded with 20, 40, 50, 60, 75, 85, 100, 200, 300 and 450 N vertical forces from the incisal edge of the crown on three consecutive occasions. A minute resistor embedded in the periapical space was used to detect apical pressure changes during occlusal loading. The ratio of apical pressure changes (DeltaP) to the loading force changes (DeltaF) was calculated. The periapical pressure detected was in direct proportion to the loading force. The mean value of DeltaP/DeltaF was 5.994 kPa N-1 (SD = 2.04). Direct proportionality was found between the coronal loading and the apical hydrostatic pressure. The DeltaP/DeltaF ratio determined in this study makes it easier to estimate the apical hydrostatic pressure values during occlusal loading of single-rooted teeth. In this study, the apical pressure generated under occlusal loading was the same magnitude as that estimated with the finite element method.

Robust Inversion and Data Compression in Control Allocation

NASA Technical Reports Server (NTRS)

Hodel, A. Scottedward

2000-01-01

We present an off-line computational method for control allocation design. The control allocation function delta = F(z)tau = delta (sub 0) (z) mapping commanded body-frame torques to actuator commands is implicitly specified by trim condition delta (sub 0) (z) and by a robust pseudo-inverse problem double vertical line I - G(z) F(z) double vertical line less than epsilon (z) where G(z) is a system Jacobian evaluated at operating point z, z circumflex is an estimate of z, and epsilon (z) less than 1 is a specified error tolerance. The allocation function F(z) = sigma (sub i) psi (z) F (sub i) is computed using a heuristic technique for selecting wavelet basis functions psi and a constrained least-squares criterion for selecting the allocation matrices F (sub i). The method is applied to entry trajectory control allocation for a reusable launch vehicle (X-33).

Cardiac autonomic impairment and chronotropic incompetence in fibromyalgia.

PubMed

da Cunha Ribeiro, Roberta Potenza; Roschel, Hamilton; Artioli, Guilherme Gianini; Dassouki, Thalita; Perandini, Luiz Augusto; Calich, Ana Luisa; de Sá Pinto, Ana Lúcia; Lima, Fernanda Rodrigues; Bonfá, Eloísa; Gualano, Bruno

2011-01-01

We aimed to gather knowledge on the cardiac autonomic modulation in patients with fibromyalgia (FM) in response to exercise and to investigate whether this population suffers from chronotropic incompetence (CI). Fourteen women with FM (age: 46 ± 3 years; body mass index (BMI): 26.6 ± 1.4 kg/m2) and 14 gender-, BMI- (25.4 ± 1.3 kg/m2), and age-matched (age: 41 ± 4 years) healthy individuals (CTRL) took part in this cross-sectional study. A treadmill cardiorespiratory test was performed and heart-rate (HR) response during exercise was evaluated by the chronotropic reserve. HR recovery (deltaHRR) was defined as the difference between HR at peak exercise and at both first (deltaHRR1) and second (deltaHRR2) minutes after the exercise test. FM patients presented lower maximal oxygen consumption (VO2 max) when compared with healthy subjects (22 ± 1 versus CTRL: 32 ± 2 mL/kg/minute, respectively; P < 0.001). Additionally, FM patients presented lower chronotropic reserve (72.5 ± 5 versus CTRL: 106.1 ± 6, P < 0.001), deltaHRR1 (24.5 ± 3 versus CTRL: 32.6 ± 2, P = 0.059) and deltaHRR2 (34.3 ± 4 versus CTRL: 50.8 ± 3, P = 0.002) than their healthy peers. The prevalence of CI was 57.1% among patients with FM. Patients with FM who undertook a graded exercise test may present CI and delayed HR recovery, both being indicative of cardiac autonomic impairment and higher risk of cardiovascular events and mortality.

45 CFR 73.735-508 - Other prohibitions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Government duties; (e) Making a Government decision outside official channels; or (f) Affecting adversely the... Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION STANDARDS OF CONDUCT Gifts... office for private gain; (b) Giving preferential treatment to any person; (c) Impeding Government...

45 CFR 73.735-508 - Other prohibitions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Government duties; (e) Making a Government decision outside official channels; or (f) Affecting adversely the... Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION STANDARDS OF CONDUCT Gifts... office for private gain; (b) Giving preferential treatment to any person; (c) Impeding Government...

Lava delta deformation as a proxy for submarine slope instability

NASA Astrophysics Data System (ADS)

Di Traglia, Federico; Nolesini, Teresa; Solari, Lorenzo; Ciampalini, Andrea; Frodella, William; Steri, Damiano; Allotta, Benedetto; Rindi, Andrea; Marini, Lorenzo; Monni, Niccolò; Galardi, Emanuele; Casagli, Nicola

2018-04-01

The instability of lava deltas is a recurrent phenomenon affecting volcanic islands, which can potentially cause secondary events such as littoral explosions (due to interactions between hot lava and seawater) and tsunamis. It has been shown that Interferometric Synthetic Aperture Radar (InSAR) is a powerful technique to forecast the collapse of newly emplaced lava deltas. This work goes further, demonstrating that the monitoring of lava deltas is a successful strategy by which to observe the long-term deformation of subaerial-submarine landslide systems on unstable volcanic flanks. In this paper, displacement measurements derived from Synthetic Aperture Radar (SAR) imagery were used to detect lava delta instability at Stromboli volcano (Italy). Recent flank eruptions (2002-2003, 2007 and 2014) affected the Sciara del Fuoco (SdF) depression, created a "stacked" lava delta, which overlies a pre-existing scar produced by a submarine-subaerial tsunamigenic landslide that occurred on 30 December 2002. Space-borne X-band COSMO-SkyMED (CSK) and C-band SENTINEL-1A (SNT) SAR data collected between February 2010 and October 2016 were processed using the SqueeSAR algorithm. The obtained ground displacement maps revealed the differential ground motion of the lava delta in both CSK and SNT datasets, identifying a stable area (characterized by less than 2 mm/y in both datasets) within the northern sector of the SdF and an unstable area (characterized by velocity fields on the order of 30 mm/y and 160 mm/y in the CSK and SNT datasets, respectively) in the central sector of the SdF. The slope stability of the offshore part of the SdF, as reconstructed based on a recently performed multibeam bathymetric survey, was evaluated using a 3D Limit Equilibrium Method (LEM). In all the simulations, Factor of Safety (F) values between 0.9 and 1.1 always characterized the submarine slope between the coastline and -250 m a.s.l. The critical surfaces for all the search volumes corresponded to the 30 December 2002 landslide, which involved the lava delta and its surrounding areas. InSAR data provided the post-effusive deformation field after the 2007 and 2014 flank eruptions, whereas LEM results highlighted that the accumulation of lava flows on the prone-to-failure SdF submarine slope is the main cause of the detected lava delta deformation. Lava delta instability, measured also at Pico Island (Azores) and Kilauea volcano (Hawaii), is evidence of the broader spectrum of instability phenomena that take place in the coastal or submarine area of the flanks of the volcanoes. At Kilauea, past lava deltas have moved faster than the surrounding slope and the recorded movements relate only to the collapses of the deltas themselves, producing rapid mass wasting near the coasts. In contrast, at Stromboli and Pico, lava deltas move at the same velocity as the surrounding slope. In these cases, the displacement at lava deltas can be considered as a proxy for the deformation of submarine slides. There are very few studies dealing with lava delta deformation, thus, the analysis presented in this work will benefit the monitoring of submarine slopes in other prone-to-failure coastal or island volcanic systems which have the potential to generate tsunamis.

Frequency and clinical features of the JAK2 V617F mutation in pediatric patients with sporadic essential thrombocythemia.

PubMed

Nakatani, Takuya; Imamura, Toshihiko; Ishida, Hiroyuki; Wakaizumi, Katsuji; Yamamoto, Tohru; Otabe, Osamu; Ishigami, Tsuyoshi; Adachi, Souichi; Morimoto, Akira

2008-12-01

Pediatric essential thrombocythemia (ET) is a rare and heterogenous disease entity. While several recent studies have focused on the role of the JAK2 V617F mutation in pediatric ET, the frequency of pediatric ET cases with this mutation and the associated clinical features remain unclear. We examined six childhood cases who had been diagnosed with ET according to WHO criteria (onset age: 0.2-14 years) for the presence of the JAK2 V617F mutation, MPLW515L mutation and JAK2 exon 12 mutations. Two sensitive PCR-based methods were used for the JAK2 V617F genotyping. We also examined the expression of polycythemia rubra vera-1 (PRV-1), which is a diagnostic marker for clonal ET. We found that three of the six cases had the JAK2 V617F mutation and that all six cases expressed PRV-1 in their peripheral granulocytes. Neither MPL W515L mutation nor JAK2 exon 12 mutations was detected in the patients without JAK2 V617F mutation. The two patients who developed thrombocythemia during infancy were JAK2 V617F-negative. These findings suggest that the JAK2 V617F mutation is not rare in childhood sporadic ET cases, and that these cases might be older and myeloproliferative features.

Analysis of a delta spot

NASA Astrophysics Data System (ADS)

Liu, Y.; Zhang, H. Q.

2002-05-01

delta -groups generally develop in three different ways: eruption of a single complex active region, eruption of large satellite spots near a large older spot, or collision of spots of opposite polarity from different dipoles. In this paper, we present a rare observational result in which a delta -spot forms from rapid coalescence of two opposite magnetic features in a pre-existing delta -configuration. The white-light (WL) observations of this delta -spot in active region NOAA 9077 were performed by TRACE. The time-lapse movie of the co-aligned WL images shows that the whole active region was undergoing rapid disintegration and reconstruction on 11-17 July 2000. The preceding ({p}) and following ({f}) components of the delta -spot continue to grow in size, while the active region is in the decaying phase. Their proper motions are determined as a function of time using SOHO/MDI full-disk magnetograms. A major flare (3B/X5.7) erupted in the active region on 14 July. Initially, a relative shearing motion is visible between the {p} and {f} spots on this day. About two hours before the major flare, the shearing motion suddenly stops and the velocities change significantly. The ribbons of the flare are located just outside of the delta -configuration. An interesting finding is the sign reversal of the helicity of the {f} spot also just 2 hours before the flare. The delta -spot obviously separates after the flare. Our results clearly demonstrate that helicity reversal in magnetic features of a delta -configuration is likely to destabilize the compact structure, as well as to re-organize the magnetic field configuration, and, hence, is important for the rapid disintegration of a delta -spot during major flares. A model is presented to explain why a spot can change its chirality.

Delta opioid receptor analgesia: recent contributions from pharmacology and molecular approaches

PubMed Central

Gavériaux-Ruff, Claire; Kieffer, Brigitte Lina

2012-01-01

Delta opioid receptors represent a promising target for the development of novel analgesics. A number of tools have been developed recently that have significantly improved our knowledge of delta receptor function in pain control. These include several novel delta agonists with potent analgesic properties, as well as genetic mouse models with targeted mutations in the delta opioid receptor gene. Also, recent findings have further documented the regulation of delta receptor function at cellular level, which impacts on the pain-reducing activity of the receptor. These regulatory mechanisms occur at transcriptional and post-translational levels, along agonist-induced receptor activation, signaling and trafficking, or in interaction with other receptors and neuromodulatory systems. All these tools for in vivo research, as well as proposed mechanisms at molecular level, have tremendously increased our understanding of delta receptor physiology, and contribute to designing innovative strategies for the treatment of chronic pain and other diseases such as mood disorders. PMID:21836459

A new missense mutation in the BCKDHB gene causes the classic form of maple syrup urine disease (MSUD).

PubMed

Miryounesi, Mohammad; Ghafouri-Fard, Soudeh; Goodarzi, Hamedreza; Fardaei, Majid

2015-05-01

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disease caused by mutations in the BCKDHA, BCKDHB, DBT and DLD genes, which encode the E1α, E1β, E2 and E3 subunits of the branched chain α ketoacid dehydrogenase (BCKD) complex, respectively. This complex is involved in the metabolism of branched-chain amino acids. In this study, we analyzed the DNA sequences of BCKDHA and BCKDHB genes in an infant who suffered from MSUD and died at the age of 6 months. We found a new missense mutation in exon 5 of BCKDHB gene (c.508C>T). The heterozygosity of the parents for the mentioned nucleotide change was confirmed by direct sequence analysis of the corresponding segment. Another missense mutation has been found in the same codon previously and shown by in silico analyses to be deleterious. This report provides further evidence that this amino acid change can cause classic MSUD.

Newly proposed hormonal criteria via genotypic proof for type II 3beta-hydroxysteroid dehydrogenase deficiency.

PubMed

Lutfallah, Chantal; Wang, Weihua; Mason, J Ian; Chang, Ying Tai; Haider, Anzar; Rich, Barry; Castro-Magana, Mariano; Copeland, Kenneth C; David, Raphael; Pang, Songya

2002-06-01

To define the hormonal criteria via genotypic proof for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency in the adrenals and gonads, we investigated the type II 3beta-HSD genotype in 55 patients with clinical and/or hormonal presentation suggesting compromised adrenal with or without gonadal 3beta-HSD activity. Fourteen patients (11 males and 3 females) had ambiguous genitalia with or without salt wasting and with or without premature pubarche. One female neonate had salt wasting only. Twenty-five children (4 males and 21 females) had premature pubarche only. Fifteen adolescent and adult females had hirsutism with or without menstrual disorder. The type II 3beta-HSD gene, including the promoter region up to -1053 base, all exons I, II, III, IV, and exon and intron boundaries, was sequenced in all subjects. Eight patients had a proven or predictably deleterious mutation in both alleles of the type II 3beta-HSD gene, and 47 patients had no apparent mutation in the gene. ACTH-stimulated (1 h post iv bolus of 250 microg Cortrosyn) serum 17-hydroxypregnenolone (Delta5-17P) levels and basal and ACTH-stimulated ratios of Delta5-17P to cortisol (F) in the genotypic proven patients were unequivocally higher than those of age-matched or pubic hair stage matched genotype-normal patients or control subjects (n = 7-30 for each group). All other baseline and ACTH-stimulated hormone parameters, including dehydroepiandrosterone (DHEA) levels, ratios of Delta5-17P to 17-OHP and DHEA to androstenedione in the genotype-proven patients, overlapped with the genotype-normal patients or control subjects. The hormonal findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3beta-HSD deficiency congenital adrenal hyperplasia (numeric and graphic reference standards from infancy to adulthood are provided): ACTH-stimulated Delta5-17P levels in 1) neonatal infants with ambiguous genitalia at or greater than 378 nmol/liter equivalent to or greater than 5.3 SD above the control mean level [95 +/- 53 (SD) nmol/liter]; 2) Tanner I children with ambiguous genitalia at or greater than 165 nmol/liter equivalent to or greater than 35 SD above the control mean level [12 +/- 4.3 (SD) nmol/liter]; 3) children with premature pubarche at or greater than 294 nmol/liter equivalent to or greater than 54 SD above Tanner II pubic hair stage matched control mean level [17 +/- 5 (SD) nmol/liter]; and 4) adults with at or greater than 289 nmol/liter equivalent to or greater than 21 SD above the normal mean level [25 +/- 12 (SD) nmol/liter]. ACTH-stimulated ratio of Delta5-17P to F in 1) neonatal infants at or greater than 434 equivalent to or greater than 6.4 SD above the control mean ratio [88 +/- 54 (SD)]; 2) Tanner I children at or greater than 216 equivalent to or greater than 23 SD above the control mean ratio [12 +/- 9 (SD)]; 3) children with premature pubarche at or greater than 363 equivalent to or greater than 38 SD above the control mean ratio [20 +/- 9 (SD)]; and 4) adults at or greater than 4010 equivalent to or greater than 221 SD above the normal mean ratio [29 +/- 18 (SD)]. Conversely, the hormonal data in the genotype-normal patients suggest the following hormonal criteria are not consistent with 3beta-HSD deficiency congenital adrenal hyperplasia: ACTH-stimulated Delta5-17P levels in children with premature pubarche up to 72 nmol/liter equivalent to up to 11 SD above the control mean level, and in hirsute females up to 150 nmol/liter equivalent to up to 12 SD above the normal female mean level [28 +/- 10 (SD) nmol/liter]; and ACTH-stimulated Delta5-17P to F ratio in children with premature pubarche up to 67 equivalent to up to 5 SD above the control mean ratio, and in hirsute females up to 151 equivalent to up to 10 SD above the normal mean ratio [32 +/- 12 (SD)]. These findings help define newly proposed hormonal criteria to accurately predict inherited 3beta-HSD deficiency.

Increase of heat-shock protein and induction of gamma/delta T cells in peritoneal exudate of mice after injection of live Fusobacterium nucleatum.

PubMed Central

Saito, K; Katsuragi, H; Mikami, M; Kato, C; Miyamaru, M; Nagaso, K

1997-01-01

Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans are Gram-negative rod periodontal pathogens. The peritoneal cavity of Institute of Cancer Research (ICR) mice was used as the local infection model. In vivo production of heat-shock proteins (hsp) was studied by injection of 1/10 minimum lethal dose (MLD) of each live bacteria into mice. Heat-shock proteins 70 and 60 were examined in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with either F. nucleatum or A. actinomycetemcomitans by using sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting analysis. Although hsp are present in PEC without injection of the bacteria, both hsp increased and reached a peak on day 3 after F. nucleatum injection but not after A. actinomycetemcomitans. Kinetic study of gamma/delta cells in PEC after injection of bacteria showed that the increase of gamma/delta T cells was observed only in the PEC from mice injected with F. nucleatum but not A. actinomycetemcomitans. The gamma/delta T cells in PEC were either CD3+ and CD4+ or CD3+ and CD8+. The differential cell count of PEC suggested that gamma/delta T-cell induction is related to the expansion of the macrophage population. The phagocytic and chemiluminescence responses of macrophages against the same bacteria were compared after intensive immunization with live F. nucleatum and A. actinomycetemcomitans. Elevations of chemiluminescence response and phagocytic function by immunization were observed in the macrophages of mice immunized with F. nucleatum. These results suggest the sequential appearance of hsp, gamma/delta T cells and macrophage activation after fusobacterial infection. Images Figure 2 PMID:9135551

Epidemiology and genetics of cystic fibrosis in Asia: In preparation for the next-generation treatments.

PubMed

Singh, Meenu; Rebordosa, Cristina; Bernholz, Juliane; Sharma, Neeraj

2015-11-01

Cystic fibrosis (CF) in the Asian population is less frequently reported due to under-diagnosis and lack of centralized CF patient registries. Clinical studies on CF cases from Asia have documented a severe course of the disease. The spectrum of the cystic fibrosis transmembrane conductance regulator (CFTR) variants in this population is quite heterogeneous. In total, 166 variants have been reported on approximately 3700 Asian CF chromosomes. The frequency of F508del among Asians is low compared with Caucasians. Recent in vitro studies have shown promise of small molecule correction and potentiation of 45 different CFTR variants. Of these variants, 16 (including G551D and F508del) have also been observed among Asian CF individuals. We suggest undertaking molecular studies extensively to annotate CFTR variants that will help Asian CF individuals to benefit from the precision medicine gaining momentum in the Western countries. © 2015 Asian Pacific Society of Respirology.

Modulations of neural activity in auditory streaming caused by spectral and temporal alternation in subsequent stimuli: a magnetoencephalographic study.

PubMed

Chakalov, Ivan; Draganova, Rossitza; Wollbrink, Andreas; Preissl, Hubert; Pantev, Christo

2012-06-20

The aim of the present study was to identify a specific neuronal correlate underlying the pre-attentive auditory stream segregation of subsequent sound patterns alternating in spectral or temporal cues. Fifteen participants with normal hearing were presented with series' of two consecutive ABA auditory tone-triplet sequences, the initial triplets being the Adaptation sequence and the subsequent triplets being the Test sequence. In the first experiment, the frequency separation (delta-f) between A and B tones in the sequences was varied by 2, 4 and 10 semitones. In the second experiment, a constant delta-f of 6 semitones was maintained but the Inter-Stimulus Intervals (ISIs) between A and B tones were varied. Auditory evoked magnetic fields (AEFs) were recorded using magnetoencephalography (MEG). Participants watched a muted video of their choice and ignored the auditory stimuli. In a subsequent behavioral study both MEG experiments were replicated to provide information about the participants' perceptual state. MEG measurements showed a significant increase in the amplitude of the B-tone related P1 component of the AEFs as delta-f increased. This effect was seen predominantly in the left hemisphere. A significant increase in the amplitude of the N1 component was only obtained for a Test sequence delta-f of 10 semitones with a prior Adaptation sequence of 2 semitones. This effect was more pronounced in the right hemisphere. The additional behavioral data indicated an increased probability of two-stream perception for delta-f = 4 and delta-f = 10 semitones with a preceding Adaptation sequence of 2 semitones. However, neither the neural activity nor the perception of the successive streaming sequences were modulated when the ISIs were alternated. Our MEG experiment demonstrated differences in the behavior of P1 and N1 components during the automatic segregation of sounds when induced by an initial Adaptation sequence. The P1 component appeared enhanced in all Test-conditions and thus demonstrates the preceding context effect, whereas N1 was specifically modulated only by large delta-f Test sequences induced by a preceding small delta-f Adaptation sequence. These results suggest that P1 and N1 components represent at least partially-different systems that underlie the neural representation of auditory streaming.

Method for ambiguity resolution in range-Doppler measurements

NASA Technical Reports Server (NTRS)

Heymsfield, Gerald M. (Inventor); Miller, Lee S. (Inventor)

1994-01-01

A method for resolving range and Doppler target ambiguities when the target has substantial range or has a high relative velocity in which a first signal is generated and a second signal is also generated which is coherent with the first signal but at a slightly different frequency such that there exists a difference in frequency between these two signals of Delta f(sub t). The first and second signals are converted into a dual-frequency pulsed signal, amplified, and the dual-frequency pulsed signal is transmitted towards a target. A reflected dual-frequency signal is received from the target, amplified, and changed to an intermediate dual-frequency signal. The intermediate dual-frequency signal is amplified, with extracting of a shifted difference frequency Delta f(sub r) from the amplified intermediate dual-frequency signal done by a nonlinear detector. The final step is generating two quadrature signals from the difference frequency Delta f(sub t) and the shifted difference frequency Delta f(sub r) and processing the two quadrature signals to determine range and Doppler information of the target.

Frequency modulation drive for a piezoelectric motor

DOEpatents

Mittas, Anthony

2001-01-01

A piezoelectric motor has peak performance at a specific frequency f.sub.1 that may vary over a range of frequencies. A drive system is disclosed for operating such a motor at peak performance without feedback. The drive system consists of the motor and an ac source connected to power the motor, the ac source repeatedly generating a frequency over a range from f.sub.1 -.DELTA.x to f.sub.1 +.DELTA.y.

The association of JAK2V617F mutation and leukocytosis with thrombotic events in essential thrombocythemia.

PubMed

Hsiao, Hui-Hua; Yang, Ming-Yu; Liu, Yi-Chang; Lee, Ching-Ping; Yang, Wen-Chi; Liu, Ta-Chih; Chang, Chao-Sung; Lin, Sheng-Fung

2007-11-01

The Janus kinase 2 mutation, JAK2 (V617F), and megakaryocytic mutations, MPL (W515L/K), have been identified and correlated with a subtype of essential thrombocythemia (ET) patients. We investigated the frequency of mutations in ET patients and analyzed the relationship with their clinical features. Fifty-three ET patients were enrolled in the study. The amplification refractory mutation system was applied for the mutation survey of the JAK2V617F, while the polymerase chain reaction with sequencing was used for the mutation survey of MPLW515L/K. Thirty-five (66%) patients harboring the JAK2 (V617F) mutation, including 3 homozygous and 32 heterozygous changes, but no MPLW515L/K mutation, were found. During follow-up, 17 (32.1%) patients suffered from documented thrombotic events, with 15 having JAK2V617F mutations. Statistical analysis showed that patients with the JAK2 mutation had significantly higher leukocytes, hemoglobin level, and thrombotic event (p = 0.043, p = 0.001, and p = 0.029, respectively). Thrombotic events were also significantly correlated with leukocytosis and older age. The JAK2V617F mutation was noted in a certain population of ET patients and correlated with leukocytosis, high hemoglobin level, and thrombosis. Therefore, detection of the JAK2V617F mutation can affect not only the diagnosis, but also the management of ET patients.

Bone disease in cystic fibrosis: new pathogenic insights opening novel therapies.

PubMed

Jacquot, J; Delion, M; Gangloff, S; Braux, J; Velard, F

2016-04-01

Mutations within the gene encoding for the chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) results in cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease that causes a number of long-term health problems, as the bone disease. Osteoporosis and increased vertebral fracture risk associated with CF disease are becoming more important as the life expectancy of patients continues to improve. The etiology of low bone density is multifactorial, most probably a combination of inadequate peak bone mass during puberty and increased bone losses in adults. Body mass index, male sex, advanced pulmonary disease, malnutrition and chronic therapies are established additional risk factors for CF-related bone disease (CFBD). Consistently, recent evidence has confirmed that CFTR plays a major role in the osteoprotegerin (OPG) and COX-2 metabolite prostaglandin E2 (PGE2) production, two key regulators in the bone formation and regeneration. Several others mechanisms were also recognized from animal and cell models contributing to malfunctions of osteoblast (cell that form bone) and indirectly of bone-resorpting osteoclasts. Understanding such mechanisms is crucial for the development of therapies in CFBD. Innovative therapeutic approaches using CFTR modulators such as C18 have recently shown in vitro capacity to enhance PGE2 production and normalized the RANKL-to-OPG ratio in human osteoblasts bearing the mutation F508del-CFTR and therefore potential clinical utility in CFBD. This review focuses on the recently identified pathogenic mechanisms leading to CFBD and potential future therapies for treating CFBD.

Assessing the improvements in the newborn screening strategy for cystic fibrosis in the Balearic Islands.

PubMed

Bauça, Josep Miquel; Morell-Garcia, Daniel; Vila, Magdalena; Pérez, Gerardo; Heine-Suñer, Damián; Figuerola, Joan

2015-04-01

Newborn screening strategies for cystic fibrosis (CF) are run worldwide, and aim at the early detection of the disorder to significantly improve the quality of life. Elevated levels of immunoreactive trypsinogen (IRT) represent a high likelihood for the screened child to be affected with CF. However, the specificity of IRT is low. The objective of this study was to assess the screening program in the Balearic Islands during the past 14 years. We evaluated all results of the screening program after 14 years, by considering all changes in the protocol and assessing the number of positive samples, the mutations detected, the number of sweat tests performed, the incidence of CF and the presence of false-negative cases. Despite a great variability among the different Balearic Islands, the global incidence of CF was 1:6059 for the 14 years assessed. The incidence in the smaller islands is about 5 times higher than in Majorca (1:2376 versus 1:10,613). After different changes in the protocol, an IRT cut-off value of 60 ng/mL was established. The two most common mutations are ΔF508 and G542X, in accordance with other geographical regions. The changes in the protocol helped reduce the number of sweat tests performed without any increase in the false-negative rate. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Evaluation of eddy-current proximity devices for measuring thin potassium film thicknesses

NASA Technical Reports Server (NTRS)

Asadourian, A. S.

1972-01-01

Two eddy current proximity probe systems were tested over a range of 0 to 508 micrometers (0 to 20 mils) of simulated potassium film thicknesses for simulated temperatures of 66 C (150 F), 232 C (450 F), and 666 C (1230 F). The results of short time calibration tests are presented. Instrument drift was a problem throughout the testing and, without correction, may limit the use of such systems to short periods of time. Additional development will be required prior to their being usable as practical instrumentation systems.

Acousto-Optic Applications for Multichannel Adaptive Optical Processor

DTIC Science & Technology

1992-06-01

AO cell and the two- channel line-scan camera system described in Subsection 4.1. The AO material for this IntraAction AOD-70 device was flint glass (n...Single-Channel 1.68 (flint glass ) 60,.0 AO Cell Multichannel 2.26 (TeO 2) 20.0 AO Cell Beam splitter 1.515 ( glass ) 50.8 Multichannel correlation was...Tone Intermodulation Dynamic Ranges of Longitudinal TeO2 Bragg Cells for Several Acoustic Power Densities 4-92 f f2 f 3 1 t SOURCE: Reference 21 TR-92

Calreticulin mutation analysis in non-mutated Janus kinase 2 essential thrombocythemia patients in Chiang Mai University: analysis of three methods and clinical correlations.

PubMed

Rattarittamrong, Ekarat; Tantiworawit, Adisak; Kumpunya, Noppamas; Wongtagan, Ornkamon; Tongphung, Ratchanoo; Phusua, Arunee; Chai-Adisaksopha, Chatree; Hantrakool, Sasinee; Rattanathammethee, Thanawat; Norasetthada, Lalita; Charoenkwan, Pimlak; Lekawanvijit, Suree

2018-03-09

The primary objective was to determine the prevalence of calreticulin (CALR) mutation in patients with non-JAK2V617F mutated essential thrombocythemia (ET). The secondary objectives were to evaluate the accuracy of CALR mutation analysis by high-resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) compared with DNA sequencing and to compare clinical characteristics of CALR mutated and JAK2V617F mutated ET. This was a prospective cohort study involving ET patients registered at Chiang Mai University in the period September 2015-September 2017 who were aged more than 2 years, and did not harbor JAK2V617F mutation. The presence of CALR mutation was established by DNA sequencing, HRM, and real-time PCR for type 1 and type 2 mutation. Clinical data were compared with that from ET patients with mutated JAK2V617F. Twenty-eight patients were enrolled onto the study. CALR mutations were found in 10 patients (35.7%). Three patients had type 1 mutation, 5 patients had type 2 mutation, 1 patient had type 18 mutation, and 1 patients had novel mutations (c.1093 C-G, c.1098_1131 del, c.1135 G-A). HRM could differentiate between the types of mutation in complete agreement with DNA sequencing. Patients with a CALR mutation showed a significantly greater male predominance and had a higher platelet count when compared with 42 JAK2V617F patients. The prevalence of CALR mutation in JAK2V617F-negative ET in this study is 35.7%. HRM is an effective method of detecting CALR mutation and is a more advantageous method of screening for CALR mutation.

A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants.

PubMed

DebRoy, Sruti; Thilmony, Roger; Kwack, Yong-Bum; Nomura, Kinya; He, Sheng Yang

2004-06-29

Salicylic acid (SA)-mediated host immunity plays a central role in combating microbial pathogens in plants. Inactivation of SA-mediated immunity, therefore, would be a critical step in the evolution of a successful plant pathogen. It is known that mutations in conserved effector loci (CEL) in the plant pathogens Pseudomonas syringae (the Delta CEL mutation), Erwinia amylovora (the dspA/E mutation), and Pantoea stewartii subsp. stewartii (the wtsE mutation) exert particularly strong negative effects on bacterial virulence in their host plants by unknown mechanisms. We found that the loss of virulence in Delta CEL and dspA/E mutants was linked to their inability to suppress cell wall-based defenses and to cause normal disease necrosis in Arabidopsis and apple host plants. The Delta CEL mutant activated SA-dependent callose deposition in wild-type Arabidopsis but failed to elicit high levels of callose-associated defense in Arabidopsis plants blocked in SA accumulation or synthesis. This mutant also multiplied more aggressively in SA-deficient plants than in wild-type plants. The hopPtoM and avrE genes in the CEL of P. syringae were found to encode suppressors of this SA-dependent basal defense. The widespread conservation of the HopPtoM and AvrE families of effectors in various bacteria suggests that suppression of SA-dependent basal immunity and promotion of host cell death are important virulence strategies for bacterial infection of plants.

PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

PubMed

Suzuki, Motoshi; Niimi, Atsuko; Limsirichaikul, Siripan; Tomida, Shuta; Miao Huang, Qin; Izuta, Shunji; Usukura, Jiro; Itoh, Yasutomo; Hishida, Takashi; Akashi, Tomohiro; Nakagawa, Yoshiyuki; Kikuchi, Akihiko; Pavlov, Youri; Murate, Takashi; Takahashi, Takashi

2009-07-01

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

Optical transmembrane potential measurements during defibrillation-strength shocks in perfused rabbit hearts.

PubMed

Zhou, X; Ideker, R E; Blitchington, T F; Smith, W M; Knisley, S B

1995-09-01

To study the optical transmembrane potential change (delta F) induced during shocks, optical recordings were obtained in 15 isolated perfused rabbit hearts treated with the potentiometric dye di-4-ANEPPS and diacetyl monoxime. Shock electrodes were sutured on the right and left ventricles. A laser beam 30 microns in diameter was used to optically excite di-4-ANEPPS. Fluorescence from a region 150 microns in diameter was recorded during a shock. In the macroscopic study (six animals), there were nine recording spots that were 3 mm apart between the two shock electrodes. In the microscopic study, there were three recording regions that were 3 mm away from either shock electrode and midway between them, with nine recording spots that were 30 microns (three animals), 100 microns (three animals), and 300 microns (three animals) apart in each region. After 20 S1 stimuli, a 10-ms truncated exponential S2 shock of defibrillation-threshold strength was given during the plateau of the last S1 action potential. In the microscopic study, shocks were also given during diastole, with delta F recordings at the three recording regions. Shocks of both polarities were tested. delta F during the shock was expressed as a percentage of the fluorescence change during the S1 upstroke action potential amplitude (the S1 Fapa), ie, delta F/Fapa%. In the macroscopic study, the magnitudes of delta F/Fapa% from recording spots 1 to 9, numbered from the left to the right ventricular electrodes, were 77 +/- 41%, 46 +/- 32%, 32 +/- 27%, 28 +/- 20%, 37 +/- 25%, 24 +/- 20%, 33 +/- 22%, 37 +/- 25%, and 59 +/- 29%, respectively (P < .05 among the nine spots). Depolarization or hyperpolarization could occur near either shock electrode with both shock polarities, but the magnitude of hyperpolarization was 1.8 +/- 0.9 times that of depolarization at the same recording spot when the shock polarity was reversed (P < .01). In the microscopic study, the change in delta F/Fapa% varied significantly over the microscopic regions examined. The maximum values of delta F/Fapa% for hyperpolarizing shocks during diastole reached only 7 +/- 10% of those for shocks during the plateau (P < .01). During diastole, the time until a new action potential occurred after the beginning of the shock was shorter when the membrane was depolarized (1.1 +/- 0.5 ms) than when it was hyperpolarized (12.8 +/- 9.1 ms, P < .01). Conclusions are as follows: (1) A shock can induce either hyperpolarization or depolarization. (2) Hyperpolarization or depolarization during a shock can occur near either the anodal or cathodal shock electrode. (3) Variation of delta F/Fapa% exists within a microscopic region.(ABSTRACT TRUNCATED AT 400 WORDS)

Evasion of adaptive immunity by HIV through the action of host APOBEC3G/F enzymes.

PubMed

Grant, Michael; Larijani, Mani

2017-09-12

APOBEC3G (A3G) and APOBEC3F (A3F) are DNA-mutating enzymes expressed in T cells, dendritic cells and macrophages. A3G/F have been considered innate immune host factors, based on reports that they lethally mutate the HIV genome in vitro. In vivo, A3G/F effectiveness is limited by viral proteins, entrapment in inactive complexes and filtration of mutations during viral life cycle. We hypothesized that the impact of sub-lethal A3G/F action could extend beyond the realm of innate immunity confined to the cytoplasm of infected cells. We measured recognition of wild type and A3G/F-mutated epitopes by cytotoxic T lymphocytes (CTL) from HIV-infected individuals and found that A3G/F-induced mutations overwhelmingly diminished CTL recognition of HIV peptides, in a human histocompatibility-linked leukocyte antigen (HLA)-dependent manner. Furthermore, we found corresponding enrichment of A3G/F-favored motifs in CTL epitope-encoding sequences within the HIV genome. These findings illustrate that A3G/F-mediated mutations mediate immune evasion by HIV in vivo. Therefore, we suggest that vaccine strategies target T cell or antibody epitopes that are not poised for mutation into escape variants by A3G/F action.

Early-onset lymphoma and extensive embryonic apoptosis in two domain-specific Fen1 mice mutants.

PubMed

Larsen, Elisabeth; Kleppa, Liv; Meza, Trine J; Meza-Zepeda, Leonardo A; Rada, Christina; Castellanos, Cesilie G; Lien, Guro F; Nesse, Gaute J; Neuberger, Michael S; Laerdahl, Jon K; William Doughty, Richard; Klungland, Arne

2008-06-15

Flap endonuclease 1 (FEN1) processes Okazaki fragments in lagging strand DNA synthesis, and FEN1 is involved in several DNA repair pathways. The interaction of FEN1 with the proliferating cell nuclear antigen (PCNA) processivity factor is central to the function of FEN1 in both DNA replication and repair. Here we present two gene-targeted mice with mutations in FEN1. The first mutant mouse carries a single amino acid point mutation in the active site of the nuclease domain of FEN1 (Fen1(E160D/E160D)), and the second mutant mouse contains two amino acid substitutions in the highly conserved PCNA interaction domain of FEN1 (Fen1(DeltaPCNA/DeltaPCNA)). Fen1(E160D/E160D) mice develop a considerably elevated incidence of B-cell lymphomas beginning at 6 months of age, particularly in females. By 16 months of age, more than 90% of the Fen1(E160D/E160D) females have tumors, primarily lymphomas. By contrast, Fen1(DeltaPCNA/DeltaPCNA) mouse embryos show extensive apoptosis in the forebrain and vertebrae area and die around stage E9.5 to E11.5.

Increased frequency of co-existing JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden.

PubMed

Nussenzveig, Roberto H; Pham, Ha T; Perkins, Sherrie L; Prchal, Josef T; Agarwal, Archana M; Salama, Mohamed E

2016-01-01

The frequency of co-existing JAK2(V617F)/MPL and JAK2(V617F)/JAK2 exon-12 mutations has not been previously investigated in MPNs. Poor survival was reported in primary myelofibrosis with low JAK2(V617F) allelic burden. However, mutational status of JAK2 exon-12 or MPL were not reported in these patients. This study developed a cost-effective multiplex high resolution melt assay that screens for mutations in JAK2 gene exons-12 and -14 ((V617F)) and MPL gene exon-10. Co-existing mutations with JAK2(V617F) were detected in 2.9% (6/208; two JAK2 exon-12 and four MPL exon-10) patient specimens with known JAK2(V617F) (allelic-burden range: 0.1-96.8%). Co-existing mutations were detected in specimens with < 12% JAK2(V617F) allelic burden. Current WHO guidelines do not recommend further testing once JAK2(V617F) mutation is detected in MPNs. The findings, however, indicate that quantification of JAK2(V617F) allele burden may be clinically relevant in MPNs and in those with low allelic burden additional testing for JAK2 exon-12 and MPL exon-10 mutation should be pursued.

Technical Objective Document for Food and Food Service Systems

DTIC Science & Technology

1990-10-01

full ntission posture for exlended duration . 3. POCGRESS AND ACCa -fPLISHMENI’S Natick is responsible for many Research, Development, Test , and...Dr. David L. Kaplan Telephone (508) 651-5525 (Biotechnology) b. Technology Program (1) FY91 Planned Programs Complete Joint Services Front End

Control of adaptive optic element displacement with the help of a magnetic rheology drive

NASA Astrophysics Data System (ADS)

Deulin, Eugeni A.; Mikhailov, Valeri P.; Sytchev, Victor V.

2000-10-01

The control system of adaptive optic of a large astronomical segmentated telescope was designed and tested. The dynamic model and the amplitude-frequency analysis of the new magnetic rheology (MR) drive are presented. The loop controlled drive consists of hydrostatic carrier, MR hydraulic loop controlling system, elastic thin wall seal, stainless seal which are united in a single three coordinate manipulator. This combination ensures short positioning error (delta) (phi)

CCAAT/enhancer-binding protein alpha (CEBPA) polymorphisms and mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia.

PubMed

Fuchs, O; Kostecka, A; Provazníková, D; Krásná, B; Kotlín, R; Stanková, M; Kobylka, P; Dostálová, G; Zeman, M; Chochola, M

2010-01-01

The CCAAT/enhancer-binding protein alpha, encoded by the intronless CEBPA gene, is a transcription factor that induces expression of genes involved in differentiation of granulocytes, monocytes, adipocytes and hepatocytes. Both mono- and bi-allelic CEBPA mutations were detected in acute myeloid leukaemia and myelodysplastic syndrome. In this study we also identified CEBPA mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia. We found 16 various deletions with the presence of two direct repeats in CEBPA by analysis of 431 individuals. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493- 498_865-870), GG (486-487_885-886), and GCCAAGCAGC (508-517_907-916), all according to GenBank Accession No. NM_004364.2. In one case we identified that a father with ischaemic heart disease and his healthy son had two identical deletions (493_864del and 508_906del, both according to GenBank Accession No. NM_004364.2) in CEBPA. The occurrence of deletions between two repetitive sequences may be caused by recombination events in the repair process. A double-stranded cut in DNA may initiate these recombination events in adjacent DNA sequences. Four types of polymorphisms in the CEBPA gene were also detected in the screened individuals. Polymorphism in CEBPA gene 690 G>T according to GenBank Accession No. NM_004364.2 is the most frequent type in our analysis. Statistical analysis did not find significant differences in the frequency of polymorphisms in CEBPA in patients and in healthy individuals with the exception of P4 polymorphism (580_585dup according to GenBank Accesion No. NM_004364.2). P4 polymorphism was significantly increased in ischaemic heart disease patients.

Nuclear accumulation of SHIP1 mutants derived from AML patients leads to increased proliferation of leukemic cells.

PubMed

Nalaskowski, Marcus M; Ehm, Patrick; Rehbach, Christoph; Nelson, Nina; Täger, Maike; Modest, Kathrin; Jücker, Manfred

2018-05-28

The inositol 5-phosphatase SHIP1 acts as negative regulator of intracellular signaling in myeloid cells and is a tumor suppressor in myeloid leukemogenesis. After relocalization from the cytoplasm to the plasma membrane SHIP1 terminates PI3-kinase mediated signaling processes. Furthermore, SHIP1 is also found in distinct puncta in the cell nucleus and nuclear SHIP1 has a pro-proliferative function. Here we report the identification of five nuclear export signals (NESs) which regulate together with the two known nuclear localization signals (NLSs) the nucleocytoplasmic shuttling of SHIP1. Mutation of NLSs reduced the nuclear import and mutation of NESs decreased the nuclear export of SHIP1 in the acute myeloid leukemia (AML) cell line UKE-1. Interestingly, four SHIP1 mutants (K210R, N508D, V684E, Q1153L) derived from AML patients showed a nuclear accumulation after expression in UKE-1 cells. In addition, overexpression of the AML patient-derived mutation N508D caused an increased proliferation rate of UKE-1 cells in comparison to wild type SHIP1. Furthermore, we identified serine and tyrosine phosphorylation as a molecular mechanism for the regulation of nucleocytoplasmic shuttling of SHIP1 where tyrosine phosphorylation of distinct residues i.e. Y864, Y914, Y1021 reduces nuclear localization, whereas serine phosphorylation at S933 enhances nuclear localization of SHIP1. In summary, our data further implicate nuclear SHIP1 in cellular signaling and suggest that enhanced accumulation of SHIP1 mutants in the nucleus may be a contributory factor of abnormally high proliferation of AML cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X-ray crystallographic analyses.

PubMed

Rohman, Muhammad S; Tadokoro, Takashi; Angkawidjaja, Clement; Abe, Yumi; Matsumura, Hiroyoshi; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

2009-01-01

The Arg97 --> Gly and Asp136 --> His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7 degrees C, respectively, in T(m), and 3.5 and 6.1 kJ x mol(-1), respectively, in DeltaG(H2O). These mutations also stabilized the So-RNase HI derivative (4x-RNase HI) with quadruple thermostabilizing mutations in an additive manner. As a result, the resultant sextuple mutant protein (6x-RNase HI) was more stable than the wild-type protein by 28.8 degrees C in T(m) and 27.0 kJ x mol(-1) in DeltaG(H2O). To analyse the effects of the mutations on the protein structure, the crystal structure of the 6x-RNase HI protein was determined at 2.5 A resolution. The main chain fold and interactions of the side-chains of the 6x-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites. These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive. The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6x-RNase HI protein. We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability.

Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations.

PubMed

Hoyt, M A; He, L; Totis, L; Saunders, W S

1993-09-01

The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae

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

Saparbaev, M.; Prakash, L.; Prakash, S.

1996-03-01

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3-A mutation has an effect on recombination similar to that of the rad1{Delta} and rad10{Delta} mutations. The msh2{Delta} mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integrationmore » of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAM-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process. 59 refs., 2 figs., 7 tabs.« less

The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome

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

Engel, Amber R., E-mail: engelam@mail.nih.go; Rumyantsev, Alexander A., E-mail: alexander.rumyantsev@sanofipasteur.co; Maximova, Olga A., E-mail: maximovao@mail.nih.go

Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E{sub 315}) and NS5 (NS5{sub 654,655}) proteins, and into the 3' non-coding region ({Delta}30) of TBEV/DEN4.more » The variant that contained all three mutations (v{Delta}30/E{sub 315}/NS5{sub 654,655}) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that v{Delta}30/E{sub 315}/NS5{sub 654,655} should be further evaluated as a TBEV vaccine.« less

Enhanced toxicity of Bacillus thuringiensis Cry3A 8-endotoxin in coleopterans by mutagenesis in recetor binding loop

Treesearch

Sheng-Jiun Wu; Noah C. Koller; Deborah L. Miller; Leah S. Bauer; Donald H. Dean

2000-01-01

We used site-directed mutagenesis to modify the Bacillus cry3A gene in amino acid residues 350-354. Two mutant toxins, A1 (R345A, Y350F, Y351F) and A2 (R345A,DeltaY350, DeltaY351, showed significantly improved...

Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

PubMed Central

Huyet, Jessica; Naylor, Claire E.; Savva, Christos G.; Gibert, Maryse; Popoff, Michel R.; Basak, Ajit K.

2013-01-01

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins. PMID:23805259

Elastic scattering and particle production in two-prong. pi. /sup -/p interactions at 8 GeV/c

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

Kitagaki, T.; Tanaka, S.; Yuta, H.

1982-10-01

Results of a high-statistics study of elastic scattering and meson resonances produced by ..pi../sup -/p interactions at 8 GeV/c are presented. Large statistics and small systematic errors permit examination of the complete kinematic region. Total differential cross sections are given for rho/sup 0,-/, f/sup 0/, g/sup 0,-/, ..delta../sup + -/, ..delta../sup 0/, and N* resonances. Spin-density matrix elements and Legendre-polynomial moments are given for rho, f, and ..delta.. resonances. The results for rho/sup 0/ and f/sup 0/ resonances are compared with the predictions of a Regge-pole-exchange model. Properties of the above resonances are compared and discussed. In particular, we presentmore » evidence that the rho/sup 0/ and f/sup 0/ production mechanisms are similar. The similarity of the g/sup 0/ t distribution to that of the rho/sup 0/ and f/sup 0/ suggests a common production mechanism for all three resonances.« less

Microstructural examination of fatigue crack tip in high strength steel

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

Fukuoka, C.; Yoshizawa, H.; Nakagawa, Y.G.

1993-10-01

Fatigue tests were performed to examine how microstructural conditioning influences crack initiation and propagation in SA508 class 3 low-carbon steel. A 3-mm-long crack was introduced in compact tension (CT) fatigue test specimens under four different loads in order to obtain crack tip plastic zones at different stress intensity factor ranges, [Delta]K = 18, 36, 54, and 72 MPa[radical]m. The microstructure of the plastic zones around the crack tip were examined by transmission electron microscopy (TEM) and selected area electron diffraction (SAD). Micro-orientation of the dislocation cells in the plastic zones of all of the CT samples increased to 4 degmore » from the level of an as-received sample. Four-point bending fatigue tests were performed for plate shape samples with a large cyclic strain range. The SAD value of the bending samples was also 4 deg in the damaged area where cracks already initiated at an early stage of the fatigue process. These test results indicate that the microstructural conditioning is a prerequisite for the fatigue crack initiation and propagation in SA508. These observations may lead to better understanding of how fatigue initiation processes transit to cracks.« less

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.

F1174V mutation alters the ALK active conformation in response to Crizotinib in NSCLC: Insight from molecular simulations.

PubMed

Dehghanian, Fariba; Kay, Maryam; Vallian, Sadeq

2017-08-01

Crizotinib is an efficient antineoplastic drug for treatment of non-small cell lung carcinoma (NSCLC), which is identified as an anaplastic lymphoma kinase (ALK) inhibitor. F1174V is a recently identified acquired point mutation relating to the Crizotinib resistance in NSCLC patients. The mechanism of Crizotinib resistance relating to F1174V mutation as a non-active site mutation remains unclear. In this study, the molecular dynamic simulation was used to investigate the possible mechanisms by which F1174V mutation may affect the structure and activity of ALK kinase domain. The results suggested that F1174V mutation could cause two important secondary structure alterations, which led to the local conformational change in ALK kinase domain. This causes more positive free energy in the mutant complex in comparison with the wild-type one. In addition, our structural analyses illustrated that F1174V mutation could result in some important interactions, which represent the key characteristics of the ALK active conformation. This study provided a molecular mechanism for ALK Crizotinib resistance caused by F1174V mutation,which could facilitate designing more efficient drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

50 CFR 660.508 - Annual specifications.

Code of Federal Regulations, 2013 CFR

2013-10-01

..., quota, Annual Catch Limit (ACL) (defined at § 600.310(f)(2)) or Annual Catch Target (ACT) (defined at... guideline, quota, ACL, or ACT, including any apportionment between the directed fishery and set-aside for..., quota, ACL or ACT will contain the following information if available or applicable: (1) The estimated...

50 CFR 660.508 - Annual specifications.

Code of Federal Regulations, 2014 CFR

2014-10-01

..., quota, Annual Catch Limit (ACL) (defined at § 600.310(f)(2)) or Annual Catch Target (ACT) (defined at... guideline, quota, ACL, or ACT, including any apportionment between the directed fishery and set-aside for..., quota, ACL or ACT will contain the following information if available or applicable: (1) The estimated...

50 CFR 660.508 - Annual specifications.

Code of Federal Regulations, 2012 CFR

2012-10-01

..., quota, Annual Catch Limit (ACL) (defined at § 600.310(f)(2)) or Annual Catch Target (ACT) (defined at... guideline, quota, ACL, or ACT, including any apportionment between the directed fishery and set-aside for..., quota, ACL or ACT will contain the following information if available or applicable: (1) The estimated...

78 FR 42514 - City of Clarksville, Tennessee; Notice of Application

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-16

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. CP13-508-000] City of Clarksville, Tennessee; Notice of Application Take notice that on June 26, the City of Clarksville, Tennessee (Clarksville) filed an application pursuant to section 7(f) of the Natural Gas Act (NGA) requesting the...

8 CFR 247.11 - Notice.

Code of Federal Regulations, 2012 CFR

2012-01-01

... (Waiver of Rights, Privileges, Exemptions and Immunities) and, if a French national receiving salary from the French Republic, Form I-508F (election as to tax exemption under the Convention between the United States and the French Republic), within 10 days after service of the notice, or the alien, within such 10...

8 CFR 247.11 - Notice.

Code of Federal Regulations, 2014 CFR

2014-01-01

... (Waiver of Rights, Privileges, Exemptions and Immunities) and, if a French national receiving salary from the French Republic, Form I-508F (election as to tax exemption under the Convention between the United States and the French Republic), within 10 days after service of the notice, or the alien, within such 10...

8 CFR 247.11 - Notice.

Code of Federal Regulations, 2013 CFR

2013-01-01

... (Waiver of Rights, Privileges, Exemptions and Immunities) and, if a French national receiving salary from the French Republic, Form I-508F (election as to tax exemption under the Convention between the United States and the French Republic), within 10 days after service of the notice, or the alien, within such 10...

8 CFR 247.11 - Notice.

Code of Federal Regulations, 2011 CFR

2011-01-01

... (Waiver of Rights, Privileges, Exemptions and Immunities) and, if a French national receiving salary from the French Republic, Form I-508F (election as to tax exemption under the Convention between the United States and the French Republic), within 10 days after service of the notice, or the alien, within such 10...

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan.

PubMed

Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph (+)) CML patients in Pakistan. The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region - Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. It is concluded that the co-existence of Ph (+)CML and JAK2 V617F mutation is possible.

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders.

PubMed

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, S H; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients.

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders

PubMed Central

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, SH; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Background Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Material and Methods Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Results Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. Conclusions The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients. PMID:25914801

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families.

PubMed

Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

2018-01-01

Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients' families. Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients' F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson's correlation coefficient and the nonparametric Mann-Whitney test. Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity.

High-level ab initio predictions for the ionization energy, bond dissociation energies, and heats of formations of iron carbide (FeC) and its cation (FeC+).

PubMed

Lau, Kai-Chung; Chang, Yih-Chung; Lam, Chow-Shing; Ng, C Y

2009-12-31

The ionization energy (IE) of FeC and the 0 K bond dissociation energies (D(0)) and the heats of formation at 0 K (DeltaH(o)(f0)) and 298 K (DeltaH(o)(f298)) for FeC and FeC(+) are predicted by the single-reference wave function based CCSDTQ(Full)/CBS approach, which involves the approximation to the complete basis set (CBS) limit at the coupled cluster level up to full quadruple excitations. The zero-point vibrational energy (ZPVE) correction, the core-valence electronic corrections (up to CCSDT level), spin-orbit couplings, and relativistic effects (up to CCSDTQ level) are included in the calculations. The present calculations provide the correct symmetry predictions for the ground states of FeC and FeC(+) to be (3)Delta and (2)Delta, respectively. We have also examined the theoretical harmonic vibrational frequencies of FeC/FeC(+) at the ROHF-UCCSD(T) and UHF-UCCSD(T) levels. While the UHF-UCCSD(T) harmonic frequencies are in good agreement with the experimental measurements, the ROHF-UCCSD(T) yields significantly higher harmonic frequency predictions for FeC/FeC(+). The CCSDTQ(Full)/CBS IE(FeC) = 7.565 eV is found to compare favorably with the experimental IE value of 7.59318 +/- 0.00006 eV, suggesting that the single-reference-based coupled cluster theory is capable of providing reliable IE prediction for FeC, despite its multireference character. The CCSDTQ(Full)/CBS D(0)(Fe(+)-C) and D(0)(Fe-C) give the prediction of D(0)(Fe(+)-C) - D(0)(Fe-C) = 0.334 eV, which is consistent with the experimental determination of 0.3094 +/- 0.0001 eV. The D(0) calculations also support the experimental D(0)(Fe(+)-C) = 4.1 +/- 0.3 eV and D(0)(Fe-C) = 3.8 +/- 0.3 eV determined by the previous ion photodissociation study. The present calculations also provide the DeltaH(o)(f0)(DeltaH(o)(f298)) predictions for FeC/FeC(+). The analysis of the correction terms in these calculations shows that the core-valence and valence-valence electronic correlations beyond CCSD(T) wave function and the relativistic effects make significant contributions to the calculated thermochemical properties of FeC/FeC(+). For the experimental D(0) and DeltaH(o)(f0) values of FeC/FeC(+), which are not known to high precision, we recommend the CCSDTQ(Full)/CBS predictions [D(0)(Fe-C) = 3.778 eV, D(0)(Fe(+)-C) = 4.112 eV, DeltaH(o)(f0)(FeC) = 760.8 kJ/mol and DeltaH(o)(f0)(FeC(+)) = 1490.6 kJ/mol] based on the ZPVE corrections using the experimental vibrational frequencies of FeC and FeC(+).

Selfish prion of Rnq1 mutant in yeast.

PubMed

Kurahashi, Hiroshi; Shibata, Shoichiro; Ishiwata, Masao; Nakamura, Yoshikazu

2009-05-01

[PIN(+)] is a prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI(+)]. We previously isolated a truncated form of Rnq1, named Rnq1Delta100, as a [PSI(+)]-eliminating factor in S. cerevisiae. Rnq1Delta100 deletes the N-terminal non-prion domain of Rnq1, and eliminates [PSI(+)] in [PIN(+)] yeast. Here we found that [PIN(+)] is transmissible to Rnq1Delta100 in the absence of full-length Rnq1, forming a novel prion variant [RNQ1Delta100(+)]. [RNQ1Delta100(+)] has similar [PIN(+)] properties as it stimulates the de novo induction of [PSI(+)] and is eliminated by the null hsp104Delta mutation, but not by Hsp104 overproduction. In contrast, [RNQ1Delta100(+)] inherits the inhibitory activity and hampers the maintenance of [PSI(+)] though less efficiently than [PIN(+)] made of Rnq1-Rnq1Delta100 co-aggregates. Interestingly, [RNQ1Delta100(+)] prion was eliminated by de novo [PSI(+)] induction. Thus, the [RNQ1Delta100(+)] prion demonstrates selfish activity to eliminate a heterologous prion in S. cerevisiae, showing the first instance of a selfish prion variant in living organisms.

Enhanced sensitivity to the time variation of the fine-structure constant and m{p}/m{e} in diatomic molecules.

PubMed

Flambaum, V V; Kozlov, M G

2007-10-12

Sensitivity to temporal variation of the fundamental constants may be strongly enhanced in transitions between narrow close levels of different nature. This enhancement may be realized in a large number of molecules due to cancellation between the ground state fine-structure omega{f} and vibrational interval omega{v} [omega=omega{f}-nomega{v} approximately 0, delta omega/omega=K(2delta alpha/alpha+0.5 delta mu/mu), K>1, mu=m{p}/m{e}]. The intervals between the levels are conveniently located in microwave frequency range and the level widths are very small. Required accuracy of the shift measurements is about 0.01-1 Hz. As examples, we consider molecules Cl(+)(2), CuS, IrC, SiBr, and HfF(+).

Immersion angle dependence of the resonant-frequency shift of the quartz crystal microbalance in a liquid: effects of longitudinal wave.

PubMed

Yoshimoto, Minoru; Kobirata, Satoshi; Aizawa, Hideo; Kurosawa, Shigeru

2007-06-19

We investigated the effects of the longitudinal wave on the immersion angle dependence of the resonant-frequency shift, deltaF, of the quartz crystal microbalance, QCM. In order to study exactly the effects, we employed the three types of cells: normal cell, cell with the glass beads and cell with sponge. The longitudinal wave exists in the normal cell. On the other hand, both the cell with the glass beads and the cell with sponge eliminate the longitudinal wave. As results, we have found that the tendencies of deltaF are the same in the three types of cells. That is, we conclude that the longitudinal wave does not have effects on the immersion angle dependence of deltaF.

[Frequency of the most common mutations of the CFTR gene in peruvian patients with cystic fibrosis using the ARMS-PCR technique].

PubMed

Aquino, Ruth; Protzel, Ana; Rivera, Juan; Abarca, Hugo; Dueñas, Milagros; Nestarez, Cecilia; Purizaga, Nestor; Diringer, Benoit

2017-01-01

To determine the frequency of the ten most common mutations of the CFTR gene reported in Latin Americausing amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) in patients with cystic fibrosis (CF) in two referral hospitals in Peru during the year 2014. The frequency of the ten most common mutations of the CFTR gene was assessed in patients of the Hospital Nacional Edgardo Rebagliati Martins and the Instituto Nacional de Salud del Niño, both located in Lima, Peru. Blood samples were collected from 36 patients with CF, and the ARMS-PCR technique was used to determine the presence of these mutations. The study group included 73.5% of patients with a known diagnosis of CF in the country when the study was carried out. ARMS-PCR allowed three of the mutations to be identified in a combined 30.6% of the alleles from patients with CF, and 64.9% of the mutated alleles were not identified. The mutations found were p.Phe508del (22,2%), p.Gly542* (6,9%), and p.Arg1162* (1,4%). There is significant variability in both the frequency and type of mutations present in our study population and in what has been reported in other Latin American countries. It is necessary to perform studies that use complete sequencing technology for the CFTR gene to identify other mutations present in our population.

Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function.

PubMed Central

Poon, P P; Cassel, D; Spang, A; Rotman, M; Pick, E; Singer, R A; Johnston, G C

1999-01-01

ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the actions of GTPase-activating proteins (GAPs) for their function. The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport. However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist. We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)-Golgi stage of vesicular transport. Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi. The glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER-Golgi alleviates the effects of a glo3Delta mutation. An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins. These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER. PMID:9927415

Roles of germline JAK2 activation mutation JAK2 V625F in the pathology of myeloproliferative neoplasms.

PubMed

Wu, Qing-Yun; Ma, Meng-Meng; Fu, Lin; Zhu, Yuan-Yuan; Liu, Yang; Cao, Jiang; Zhou, Ping; Li, Zhen-Yu; Zeng, Ling-Yu; Li, Feng; Wang, Xiao-Yun; Xu, Kai-Lin

2018-05-18

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

Dissipation of the Proton Electrochemical Potential in Intact Chloroplasts (II. The pH Gradient Monitored by Cytochrome f Reduction Kinetics).

PubMed Central

Nishio, J. N.; Whitmarsh, J.

1993-01-01

The potency of various uncouplers for collapsing the light-induced pH gradient across thylakoid membranes in intact chloroplasts was investigated by time-resolved optical spectroscopy. The thylakoid transmembrane pH gradient ([delta]pH) was monitored indirectly by measuring the rate of cytochrome (Cyt) f reduction following a light flash of sufficient duration to create a sizable [delta]pH. The results show that the rate of Cyt f reduction is controlled in part by the internal pH of the thylakoid inner aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was maximal, whereas at lower pH values from 6.5 to 5.5 the reduction rate decreased to 25% of the maximal rate. The ability of three uncouplers, nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to accelerate the rate of Cyt f reduction was determined for intact chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the uncouplers for collapsing the [delta]pH was determined using the empirical relationship between the [delta]pH and the Cyt f reduction rate. For intact chloroplasts, nigericin was the most effective uncoupler, followed by carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with bovine serum albumin. Gramicidin D, even at high gramicidin:chlorophyll ratios, did not completely collapse the pH gradient, probably because it partitions in the envelope membranes and does not enter the intact chloroplast. PMID:12231669

Krüppel-like factor 1 mutations and expression of hemoglobins F and A2 in homozygous hemoglobin E syndrome.

PubMed

Tepakhan, Wanicha; Yamsri, Supawadee; Fucharoen, Goonnapa; Sanchaisuriya, Kanokwan; Fucharoen, Supan

2015-07-01

The basis for variability of hemoglobin (Hb) F in homozygous Hb E disease is not well understood. We have examined multiple mutations of the Krüppel-like factor 1 (KLF1) gene; an erythroid specific transcription factor and determined their associations with Hbs F and A2 expression in homozygous Hb E. Four KLF1 mutations including G176AfsX179, T334R, R238H, and -154 (C-T) were screened using specific PCR assays on 461 subjects with homozygous Hb E and 100 normal controls. None of these four mutations were observed in 100 normal controls. Among 461 subjects with homozygous Hb E, 306 had high (≥5 %) and 155 had low (<5 %) Hb F. DNA analysis identified the KLF1 mutations in 35 cases of the former group with high Hb F, including the G176AfsX179 mutation (17/306 = 5.6 %), T334R mutation (9/306 = 2.9 %), -154 (C-T) mutation (7/306 = 2.3 %), and R328H mutation (2/306 = 0.7 %). Only two subjects in the latter group with low Hb F carried the G176AfsX179 and -154 (C-T) mutations. Significant higher Hb A2 level was observed in those of homozygous Hb E with the G176AfsX179 mutation as compared to those without KLF1 mutations. These results indicate that KLF1 is among the genetic factors associated with increased Hbs F and A2, and in combination with other factors could explain the variabilities of these Hb expression in Hb E syndrome.

The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase.

PubMed

Nolan, D P; Voorheis, H P

1992-10-01

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

PubMed

Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Novel FAM20A mutation causes autosomal recessive amelogenesis imperfecta.

PubMed

Volodarsky, Michael; Zilberman, Uri; Birk, Ohad S

2015-06-01

To relate the peculiar phenotype of amelogenesis imperfecta in a large Bedouin family to the genotype determined by whole genome linkage analysis. Amelogenesis imperfecta (AI) is a broad group of inherited pathologies affecting enamel formation, characterized by variability in phenotypes, causing mutations and modes of inheritance. Autosomal recessive or compound heterozygous mutations in FAM20A, encoding sequence similarity 20, member A, have been shown to cause several AI phenotypes. Five members from a large consanguineous Bedouin family presented with hypoplastic amelogenesis imperfecta with unerupted and resorbed permanent molars. Following Soroka Medical Center IRB approval and informed consent, blood samples were obtained from six affected offspring, five obligatory carriers and two unaffected siblings. Whole genome linkage analysis was performed followed by Sanger sequencing of FAM20A. The sequencing unravelled a novel homozygous deletion mutation in exon 11 (c.1523delC), predicted to insert a premature stop codon (p.Thr508Lysfs*6). We provide an interesting case of novel mutation in this rare disorder, in which the affected kindred is unique in the large number of family members sharing a similar phenotype. Copyright © 2015 Elsevier Ltd. All rights reserved.

An accurate, simple prognostic model consisting of age, JAK2, CALR, and MPL mutation status for patients with primary myelofibrosis.

PubMed

Rozovski, Uri; Verstovsek, Srdan; Manshouri, Taghi; Dembitz, Vilma; Bozinovic, Ksenija; Newberry, Kate; Zhang, Ying; Bove, Joseph E; Pierce, Sherry; Kantarjian, Hagop; Estrov, Zeev

2017-01-01

In most patients with primary myelofibrosis, one of three mutually exclusive somatic mutations is detected. In approximately 60% of patients, the Janus kinase 2 gene is mutated, in 20%, the calreticulin gene is mutated, and in 5%, the myeloproliferative leukemia virus gene is mutated. Although patients with mutated calreticulin or myeloproliferative leukemia genes have a favorable outcome, and those with none of these mutations have an unfavorable outcome, prognostication based on mutation status is challenging due to the heterogeneous survival of patients with mutated Janus kinase 2. To develop a prognostic model based on mutation status, we screened primary myelofibrosis patients seen at the MD Anderson Cancer Center, Houston, USA, between 2000 and 2013 for the presence of Janus kinase 2, calreticulin, and myeloproliferative leukemia mutations. Of 344 primary myelofibrosis patients, Janus kinase 2 V617F was detected in 226 (66%), calreticulin mutation in 43 (12%), and myeloproliferative leukemia mutation in 16 (5%); 59 patients (17%) were triple-negatives. A 50% cut-off dichotomized Janus kinase 2-mutated patients into those with high Janus kinase 2 V617F allele burden and favorable survival and those with low Janus kinase 2 V617F allele burden and unfavorable survival. Patients with a favorable mutation status (high Janus kinase 2 V617F allele burden/myeloproliferative leukemia/calreticulin mutation) and aged 65 years or under had a median survival of 126 months. Patients with one risk factor (low Janus kinase 2 V617F allele burden/triple-negative or age >65 years) had an intermediate survival duration, and patients aged over 65 years with an adverse mutation status (low Janus kinase 2 V617F allele burden or triple-negative) had a median survival of only 35 months. Our simple and easily applied age- and mutation status-based scoring system accurately predicted the survival of patients with primary myelofibrosis. Copyright© Ferrata Storti Foundation.

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan

PubMed Central

Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Background and Objective: Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph +) CML patients in Pakistan. Methods: The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region – Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. Results: All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. Conclusion: It is concluded that the co-existence of Ph +CML and JAK2 V617F mutation is possible. PMID:24639858

Heterozygous TGFBR2 mutations in Marfan syndrome

PubMed Central

Mizuguchi, Takeshi; Collod-Beroud, Gwenaëlle; Akiyama, Takushi; Abifadel, Marianne; Harada, Naoki; Morisaki, Takayuki; Allard, Delphine; Varret, Mathilde; Claustres, Mireille; Morisaki, Hiroko; Ihara, Makoto; Kinoshita, Akira; Yoshiura, Koh-ichiro; Junien, Claudine; Kajii, Tadashi; Jondeau, Guillaume; Ohta, Tohru; Kishino, Tatsuya; Furukawa, Yoichi; Nakamura, Yusuke; Niikawa, Norio; Boileau, Catherine; Matsumoto, Naomichi

2004-01-01

Marfan syndrome (MFS) is an extracellular matrix disorder with cardinal manifestations in the eye, skeleton, and cardiovascular systems and associated with defects in the fibrillin gene (FBN1) at 15q21.1 1. We previously mapped the second locus for MFS (MFS type 2, MFS2, OMIM *154705), at 3p24.2-p25 in a large French family (MS1)2. Identification of a 3p24.1 chromosomal breakpoint disrupting the TGF-beta receptor 2 gene (TGFBR2) in a Japanese MFS patient led us to consider TGFBR2 as the MSF2 gene. We found a Q508Q mutation of TGFBR2 that resulted in abnormal splicing and segregated with MFS2 in MS1. Three other missense mutations were found in four unrelated probands and were shown by luciferase-assays to lead to loss of function of the TGF-β signaling activity on extracellular matrix formation. These results show that heterozygous mutations in TGFBR2, a putative tumor suppressor gene implicated in several malignancies, are also associated with inherited connective-tissue disorders. PMID:15235604

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.

PubMed

Xia, Zhen-Wei; Zhou, Wen-Pu; Cui, Wen-Jun; Zhang, Xue-Hong; Shen, Qing-Xiang; Li, Yun-Zhu; Yu, Shan-Chang

2004-08-15

To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities. Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1. Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

Thermodynamical interpretation of an adaptive walk on a Mt. Fuji-type fitness landscape: Einstein relation-like formula holds in a stochastic evolution.

PubMed

Aita, Takuyo; Husimi, Yuzuru

2003-11-21

We have theoretically studied the statistical properties of adaptive walks (or hill-climbing) on a Mt. Fuji-type fitness landscape in the multi-dimensional sequence space through mathematical analysis and computer simulation. The adaptive walk is characterized by the "mutation distance" d as the step-width of the walker and the "population size" N as the number of randomly generated d-fold point mutants to be screened. In addition to the fitness W, we introduced the following quantities analogous to thermodynamical concepts: "free fitness" G(W) is identical with W+T x S(W), where T is the "evolutionary temperature" T infinity square root of d/lnN and S(W) is the entropy as a function of W, and the "evolutionary force" X is identical with d(G(W)/T)/dW, that is caused by the mutation and selection pressure. It is known that a single adaptive walker rapidly climbs on the fitness landscape up to the stationary state where a "mutation-selection-random drift balance" is kept. In our interpretation, the walker tends to the maximal free fitness state, driven by the evolutionary force X. Our major findings are as follows: First, near the stationary point W*, the "climbing rate" J as the expected fitness change per generation is described by J approximately L x X with L approximately V/2, where V is the variance of fitness distribution on a local landscape. This simple relationship is analogous to the well-known Einstein relation in Brownian motion. Second, the "biological information gain" (DeltaG/T) through adaptive walk can be described by combining the Shannon's information gain (DeltaS) and the "fitness information gain" (DeltaW/T).

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.

X-ray crystallographic studies of RNase A variants engineered at the most destabilizing positions of the main hydrophobic core: further insight into protein stability.

PubMed

Kurpiewska, Katarzyna; Font, Josep; Ribó, Marc; Vilanova, Maria; Lewiński, Krzysztof

2009-11-15

To investigate the structural origin of decreased pressure and temperature stability, the crystal structure of bovine pancreatic ribonuclease A variants V47A, V54A, V57A, I81A, I106A, and V108A was solved at 1.4-2.0 A resolution and compared with the structure of wild-type protein. The introduced mutations had only minor influence on the global structure of ribonuclease A. The structural changes had individual character that depends on the localization of mutated residue, however, they seemed to expand from mutation site to the rest of the structure. Several different parameters have been evaluated to find correlation with decrease of free energy of unfolding DeltaDeltaG(T), and the most significant correlation was found for main cavity volume change. Analysis of the difference distance matrices revealed that the ribonuclease A molecule is organized into five relatively rigid subdomains with individual response to mutation. This behavior consistent with results of unfolding experiments is an intrinsic feature of ribonuclease A that might be surviving remnants of folding intermediates and reflects the dynamic nature of the molecule. 2009 Wiley-Liss, Inc.

Treatment burden in patients with at least one class IV or V CFTR mutation.

PubMed

Dewulf, Jonas; Vermeulen, François; Wanyama, Simeon; Thomas, Muriel; Proesmans, Marijke; Dupont, Lieven; De Boeck, Kris

2015-12-01

CFTR mutations are grouped according to disease-causing mechanism. Several studies demonstrated that patients having at least one mutation of class IV/V, present with a milder phenotype, but little is known about their relative treatment burden. We compared treatment burden between patients with two class I, II, or III mutations and patients with at least one mutation of class IV/V in the 2010 database of the Belgian CF Registry. We calculated a "Treatment Burden Index" (TBI) by assigning long term therapies to categories low, medium and high intensity, for differential weighing in the total score. There were 779 patients with two known class I/II/III mutations and 94 patients with at least one class IV/V mutation. Compared to class I/II/III, class IV/V patients had a lower median number of clinic visits (4 vs. 5; P < 0.001), a lower risk of hospitalization (24.7% vs. 50.8%; P < 0.001) and intravenous antibiotic treatment (23.5% vs. 46.0%; P < 0.001) and a lower median TBI (6 vs. 9; P < 0.001). These differences remained significant when only class IV/V patients with pancreatic insufficiency (n = 31) were considered. This study clearly demonstrates the significantly lower treatment burden in patients with CF and at least one class IV/V mutation compared to patients with two class I/II/III mutations and contributes to providing better individual counseling at time of diagnosis. © 2015 Wiley Periodicals, Inc.

The complete set of spin observables for the (13)C(polarized proton, polarized neutron)(13)N and (15)N(polarized proton, polarized neutron)(15)O reactions

NASA Astrophysics Data System (ADS)

Du, Qun Qun

1998-12-01

The 13C(p,n)13N and 15N(p,n)15O reactions have been a puzzle for more than ten years. The ground state transitions are Jπ=1/2- to Jπ=1/2-. These are 'mixed' transitions because they can involve quantum number changes either (/Delta T=1,/ /Delta J=0,/ /Delta/pi=0,/ /Delta S=0), or (/Delta T=1,/ /Delta J=1,/ /Delta/pi=0,/ /Delta S=1); these quantum number changes are refered to as 'Fermi' and 'Gamow-Teller' respectively. Because the quantum number changes are the same as for Fermi and Gamow-Teller beta decay. From the systematics of (p,n) and (n,p) reactions on pure Fermi transitions (e.g. 0 + to 0+) and pure Gamow-Teller transitions (e.g. 0+ to 1+), calibrations have been established of cross section per unit B(F) or unit B(GT), where 'B' refers to doubly reduced matrix elements extracted from beta decay. However, cross sections for the 13C(p,n)13N(g.s.) and 15N(p,n)15O(g.s.) reactions are substantially larger than one would then predict from the known B(F)s and B(GT)s for these transitions. To explore this anomaly, spin observables were used to extract separately the Fermi and Gamow-Teller cross sections for these reactions. To acquire the complete sets of polarization- transfer observables, a new neutron polarimeter was designed, built, commissioned and calibrated. This polarimeter, call the '2π polarimeter' because of its complete azimuthal coverage for scattered neutrons, has very good position and timing resolution (354 ps). The complete sets of spin-transfer coefficients Dij for 13C(p,n)13N (at 0o , 5.5o , and 11o ) and 15N(p,n)15O (at 0o ) at 135 MeV were measured. Following the formalism of Ichimura and Kawahigashi, we extracted the spin-longitudinal, and spin-transverse and spin-independent responses D0,/ Dq,/ Dn and Dp from the measured Dijs. The F and GT fractions of the (p,n) cross sections are then extracted as f F=D0 and fGT=Dn+Dp+Dq=1- d0. Values of Dk for both the 13C(p,n)13N(g.s) and 15N(p,n)15O(g.s.) were extracted. From these responses, we extracted. From these responses, we extracted fGT and fF, the GT and Fermi fractions for these transitions in a model-independent fashion. Values for FGT extracted by another group at other beam energies in a model-dependent manner from DNN/sp' alone, are in good agreement with our model-independent values. These results were also compared with the empirical energy-dependence of ratio of σtau to τ strength for the (p,n) reaction extracted primarily from even-A targets. Our measured values of fGT are typically 10% larger than predicted from β-decay matrix elements and even-A (p,n) systematics. Since the cross sections for these transitions are anomalously large, this suggests that the GT strength in these transitions is ~50% larger than expected from β-decay and even-A (p,n) systematics. The measured values of Dk and cross sections for the 13C(p,n)13N(g.s) transition were compared with the DWIA calculations, where modest agreement was achieved.

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families

PubMed Central

Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

2018-01-01

Background Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients’ families. Material and methods Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients’ F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson’s correlation coefficient and the nonparametric Mann-Whitney test. Results Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Discussion Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity. PMID:27723456

A novel heterozygous intronic mutation in POU1F1 is associated with combined pituitary hormone deficiency.

PubMed

Takagi, Masaki; Kamasaki, Hotaka; Yagi, Hiroko; Fukuzawa, Ryuji; Narumi, Satoshi; Hasegawa, Tomonobu

2017-02-27

POU class 1 homeobox 1 (POU1F1) regulates pituitary cell-specific gene expression of somatotropes, lactotropes, and thyrotropes. In humans, two POU1F1 isoforms (long and short isoform), which are generated by the alternative use of the splice acceptor site for exon 2, have been identified. To date, more than 30 POU1F1 mutations in patients with combined pituitary hormone deficiency (CPHD) have been described. All POU1F1 variants reported to date affect both the short and long isoforms of the POU1F1 protein; therefore, it is unclear at present whether a decrease in the function of only one of these two isoforms is sufficient for disease onset in humans. Here, we described a sibling case of CPHD carrying a heterozygous mutation in intron 1 of POU1F1. In vitro experiments showed that this mutation resulted in exon 2-skipping of only in the short isoform of POU1F1, while the long isoform remained intact. This result strongly suggests the possibility, for the first time, that isolated mutations in the short isoform of POU1F1 could be sufficient for induction of POU1F1-related CPHD. This finding improves our understanding of the molecular mechanisms, and developmental course associated with mutations in POU1F1.

Biomechanical analysis of cervical distraction.

PubMed

Miller, L S; Cotler, H B; De Lucia, F A; Cotler, J M; Hume, E L

1987-11-01

A biomechanical analysis of cervical distraction is presented, and a model comparing closed reduction of cervical spine dislocations to spring mechanics is developed. Behavior of a spring may be described as F = k delta x where F = distraction force; delta x = elongation of the spring; and k = spring constant. The records and roentgenograms of 24 cervical spine dislocations were reviewed retrospectively. Evaluation of cervical distraction vs traction weight indicates that Ftraction = kneck delta x; where F = traction weight and x = distraction at the injured level. The constant, kneck, is different for bilateral and unilateral dislocations (P less than .001) and is a function of magnitude of injury and neck morphology. As determined in this study, traction weight needed for reduction of facet dislocations may be estimated using the formulae: Ftx = 107.1 lbs/cm (x) unilateral, and Ftx = 76.4 lbs/cm (x) bilateral.

F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

PubMed

Goetstouwers, T; Van Poucke, M; Nguyen, V U; Melkebeek, V; Coddens, A; Deforce, D; Cox, E; Peelman, L J

2014-05-01

Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

Calorimetric determination of the enthalpy of 1-butyl-3-methylimidazolium bromide synthesis: a key quantity in thermodynamics of ionic liquids.

PubMed

Paulechka, Yauheni U; Kabo, Andrey G; Blokhin, Andrey V

2009-11-05

The enthalpy of the 1-butyl-3-methylimidazolium bromide [C(4)mim]Br ionic liquid synthesis reaction 1-methylimidazole (liq) + 1-bromobutane (liq) --> [C(4)mim]Br (liq) was determined in a homemade small-volume isoperibol calorimeter to be Delta(r)H degrees (298) = -87.7 +/- 1.6 kJ x mol(-1). The activation energy for this reaction in a homogeneous system E(A) = 73 +/- 4 kJ x mol(-1) was found from the results of calorimetric measurements. The formation enthalpies for the crystalline and liquid [C(4)mim]Br were determined from the calorimetric data: Delta(f)H degrees (298)(cr) = -178 +/- 5 kJ x mol(-1) and Delta(f)H degrees (298)(liq) = -158 +/- 5 kJ x mol(-1). The ideal-gas formation enthalpy of this compound Delta(f)H degrees (298)(g) = 16 +/- 7 kJ x mol(-1) was calculated using the methods of quantum chemistry and statistical thermodynamics. The vaporization enthalpy of [C(4)mim]Br, Delta(vap)H degrees (298) = 174 +/- 9 kJ x mol(-1), was estimated from the experimental and calculated formation enthalpies. It was demonstrated that vapor pressure of this ionic liquid cannot be experimentally determined.

The Astronomical Almanac Online - Glossary

Science.gov Websites

Astronomical Almanac. Δ A B C D E F G H I J K L M N O P Q R S T U V W X Y Z $\\boldmath{\\Delta {\\rm T}}$: the difference between Terrestrial Time (TT) and Universal Time (UT): $\\Delta {\\rm T} = {\\rm TT} - {\\rm UT}1 $. $\\boldmath{\\Delta {\\rm UT1}}$ (or $\\boldmath{\\Delta {\\rm UT}}$): the value of the difference between

JAK2 Exon 12 Mutations in Polycythemia Vera and Idiopathic Erythrocytosis

PubMed Central

Scott, Linda M.; Tong, Wei; Levine, Ross L.; Scott, Mike A.; Beer, Philip A.; Stratton, Michael R.; Futreal, P. Andrew; Erber, Wendy N.; McMullin, Mary Frances; Harrison, Claire N.; Warren, Alan J.; Gilliland, D. Gary; Lodish, Harvey F.; Green, Anthony R.

2010-01-01

BACKGROUND The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. METHODS We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. RESULTS We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. CONCLUSIONS JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis. PMID:17267906

MPL W515L/K Mutations in Chronic Myeloproliferative Neoplasms.

PubMed

Akpınar, Timur Selçuk; Hançer, Veysel Sabri; Nalçacı, Meliha; Diz-Küçükkaya, Reyhan

2013-03-01

The MPL gene encodes the thrombopoietin receptor. Recently MPL mutations (MPL W515L or MPL W515K) were described in patients with essential thrombocythemia (ET) and primary (idiopathic) myelofibrosis (PMF). The prevalence and the clinical importance of these mutations are not clear. In the present study, we aimed to investigate the frequency and clinical significance of MPL W515L/K mutations in our patients with ET and PMF. A total of 77 patients (66 were diagnosed with ET and 11 with PMF) and 42 healthy controls were included in the study. Using peripheral blood samples, the presence of MPL W515L/K mutations and JAK-2 V617F mutation were analyzed by real-time polymerase chain reaction. In our study, MPL W515L/K or JAK-2 V617F mutations were not observed in healthy controls. JAK-2 V617F mutation was present in 35 patients, of whom 29 had ET (43.9%, 29/66) and 6 had PMF (54.5%, 6/11). In the patient group, MPL W515L/K mutations were found in only 2 PMF cases, and these cases were negative for JAK-2 V617F mutation. The prevalence of MPL W515L/K mutations in the patient group was 2.6%, and the prevalence of MPL W515L/K mutations among the cases negative for the JAK-2 V617F mutation was found to be 4.8%. The 2 cases with MPL W515L/K mutations had long follow-up times (124 months and 71 months, respectively), had no thrombotic or hemorrhagic complications, and had no additional cytogenetic anomalies. MPL W515L/K mutations may be helpful for identifying clonal disease in MPN patients with no established Ph chromosome or JAK-2 V617F mutation. None declared.

EFFECT OF RAPAMYCIN ON THE FATE OF P23H OPSIN ASSOCIATED WITH RETINITS PIGMENTOSA (AN AMERICAN OPHTHALMOLOGICAL SOCIETY THESIS)

PubMed Central

Kaushal, Shalesh

2006-01-01

Purpose To determine the effect of rapamycin on the fate of misfolded opsin associated with retinitis pigmentosa. Methods Stable cell lines separately expressing WT and P23H opsins and WT and ΔF508 CFTR were used. Cells were incubated with complete media or amino acid–depleted medium or in the presence of rapamycin. At various time points thereafter, quantitative opsin and CFTR immunoblotting was performed. Immunofluorescence and electron microscopy were also performed to observe the expression and colocalization of autophagy specific marker proteins with opsin or CFTR. Results Upon incubation with rapamycin, the levels of P23H opsin and ΔF508 CFTR were reduced more rapidly than in untreated controls while no observable changes in the amounts of WT opsin was seen. The autophagy specific marker proteins, Atg7, Atg8 (LC3), and LAMP-1, which associate with autophagic vacuoles, colocalized with P23H opsin. A dramatic increase in the immunofluorescence signals of Atg7, LC3, and LAMP-1 was observed. All three of these proteins were found to decorate P23H opsin, suggesting that autophagy may be directly responsible for the clearance of this protein. Also, it was determined that neither the unfolded protein response nor the heat shock response was induced upon rapamycin-associated degradation of P23H opsin. Conclusions These data suggest that rapamycin induces the loss of P23H opsin and ΔF508 CFTR from the cell under the experimental conditions described. Concomitantly, there is increased expression and colocalization of autophagy marker proteins with P23H opsin. Immunogold electron microscopic studies demonstrate autophagic vacuoles clustered in physical proximity to the aggregates of P23H opsin, suggesting that some of the loss of P23H is related to the induction of autophagy. Thus, rapamycin may be useful to clear misfolded proteins associated with retinal degeneration. PMID:17471359

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.

Detection of MPL exon10 mutations in 103 Chinese patients with JAK2V617F-negative myeloproliferative neoplasms.

PubMed

Chen, Xiuhua; Qi, Xiling; Tan, Yanhong; Xu, Zhifang; Xu, Aining; Zhang, Linlin; Wang, Hongwei

2011-06-15

JAK2V617F mutation has been reported in 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thromobocythemia (ET) and primary myelofibrosis (PMF). Recently, acquired mutations in the transmembrane-juxtamembrane region of MPL (MPLW515 mutations) have been reported in approximately 5% of JAK2V617F-negative PMF and about 1% of all cases of ET. MPL is the receptor for thrombopoietin that regulates the production of platelets by bone marrow. It is likely that some mutations more closely related to ET in MPL exon10 may have been missed by current assays. We inferred that there might be other mutations in MPL exon10 for MPN patients in addition to MPLW515 mutations. To investigate its mutation types and prevalence in Chinese patients with myeloproliferative neoplasms (MPN), we performed mutation detection on MPL exon10 in 103 JAK2V617F-negative MPN patients by single strand conformation polymorphism (SSCP) and allele-specific PCR (AS-PCR) combined with sequencing. As a result, one previously unrecognized MPL mutation (12-bp in-frame insertion) was identified in one patient with ET in addition to an MPLW515K mutation identified in one PMF patient. This confirms our hypothesis that BCR/ABL negative and JAK2V617F-negative MPN patients have other mutations besides W515 mutation in MPL exon10 and mutations other than single nucleotide exchange also exist. In addition, MPL mutation was associated with Chinese MPN patients. Copyright © 2011 Elsevier Inc. All rights reserved.

Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

PubMed

Hannan, Nicholas R F; Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A; Vallier, Ludovic

2015-07-15

Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.

Powerful tools for genetic modification: Advances in gene editing.

PubMed

Roesch, Erica A; Drumm, Mitchell L

2017-11-01

Recent discoveries and technical advances in genetic engineering, methods called gene or genome editing, provide hope for repairing genes that cause diseases like cystic fibrosis (CF) or otherwise altering a gene for therapeutic benefit. There are both hopes and hurdles with these technologies, with new ideas emerging almost daily. Initial studies using intestinal organoid cultures carrying the common, F508del mutation have shown that gene editing by CRISPR/Cas9 can convert cells lacking CFTR function to cells with normal channel function, providing a precedent that this technology can be harnessed for CF. While this is an important precedent, the challenges that remain are not trivial. A logistical issue for this and many other genetic diseases is genetic heterogeneity. Approximately, 2000 mutations associated with CF have been found in CFTR, the gene responsible for CF, and thus a feasible strategy that would encompass all individuals affected by the disease is particularly difficult to envision. However, single strategies that would be applicable to all subjects affected by CF have been conceived and are being investigated. With all of these approaches, efficiency (the proportion of cells edited), accuracy (how often other sites in the genome are affected), and delivery of the gene editing components to the desired cells are perhaps the most significant, impending hurdles. Our understanding of each of these areas is increasing rapidly, and while it is impossible to predict when a successful strategy will reach the clinic, there is every reason to believe it is a question of "when" and not "if." © 2017 Wiley Periodicals, Inc.

Mutational subtypes of JAK2 and CALR correlate with different clinical features in Japanese patients with myeloproliferative neoplasms.

PubMed

Misawa, Kyohei; Yasuda, Hajime; Araki, Marito; Ochiai, Tomonori; Morishita, Soji; Shirane, Shuichi; Edahiro, Yoko; Gotoh, Akihiko; Ohsaka, Akimichi; Komatsu, Norio

2018-06-01

The majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) harbor JAK2, CALR, or MPL mutations. We compared clinical manifestations of different subtypes of JAK2 and CALR mutations in Japanese patients with MPNs. Within our cohort, we diagnosed 166 patients as polycythemia vera (PV), 212 patients as essential thrombocythemia (ET), 23 patients as pre-primary myelofibrosis (PMF), 65 patients as overt PMF, and 27 patients as secondary myelofibrosis following the 2016 WHO criteria. Compared to patients with JAK2V617F-mutated PV, JAK2 exon 12-mutated PV patients were younger, showed lower white blood cell (WBC) counts, lower platelet counts, higher red blood cell counts, and higher frequency of thrombotic events. Compared to JAK2-mutated ET patients, CALR-mutated ET patients were younger, showed lower WBC counts, lower hemoglobin levels, higher platelet counts, and fewer thrombotic events. CALR type 1-like mutation was the dominant subtype in CALR-mutated overt PMF patients. Compared with JAK2V617F-mutated ET patients, JAK2V617F-mutated pre-PMF patients showed higher LDH levels, lower hemoglobin levels, higher JAK2V617F allele burden, and higher frequency of splenomegaly. In conclusion, Japanese patients with MPNs grouped by different mutation subtypes exhibit characteristics similar to those of their Western counterparts. In addition, ET and pre-PMF patients show different characteristics, even when restricted to JAK2V617F-mutated patients.

In situ impact of multiple pulses of metal and herbicide on the seagrass, Zostera capricorni.

PubMed

Macinnis-Ng, Catriona M O; Ralph, Peter J

2004-04-28

Tides and freshwater inflow which influence water movement in estuarine areas govern the exposure-regime of pollutants. In this experiment, we examined the in situ impact of double pulses of copper and the herbicide Irgarol 1051 on the photosynthesis of the seagrass, Zostera capricorni. Despite a 4-day recovery period between the two 10h pulses of toxicant, the effective quantum yield of photosystem II (DeltaF/Fm') and total chlorophyll concentrations indicated that multiple-pulses had a greater impact than a single pulse. During the first exposure period, samples exposed to Irgarol 1051 had DeltaF/Fm' values as low as zero while controls remained around 0.6 relative units. After the second exposure period, treated samples recovered to only 0.4 relative units. Samples exposed to copper had DeltaF/Fm' values around 0.3 relative units during the first exposure period and while these samples recovered before the second dose, they remained below 0.2 relative units after the second exposure period. Alternate samples were also exposed to one toxicant, allowed to recover and then exposed to the other toxicant. DeltaF/Fm' values indicated that copper exposure followed by Irgarol 1051 exposure was more toxic than Irgarol 1051 exposure followed by copper exposure.

Influence of ligand polarizability on the reversible binding of O2 by trans-[Rh(X)(XNC)(PPh3)2] (X = Cl, Br, SC6F5, C2Ph; XNC = xylyl isocyanide). Structures and a kinetic study.

PubMed

Carlton, Laurence; Mokoena, Lebohang V; Fernandes, Manuel A

2008-10-06

The complexes trans-[Rh(X)(XNC)(PPh 3) 2] (X = Cl, 1; Br, 2; SC 6F 5, 3; C 2Ph, 4; XNC = xylyl isocyanide) combine reversibly with molecular oxygen to give [Rh(X)(O 2)(XNC)(PPh 3) 2] of which [Rh(SC 6F 5)(O 2)(XNC)(PPh 3) 2] ( 7) and [Rh(C 2Ph)(O 2)(XNC)(PPh 3) 2] ( 8) are sufficiently stable to be isolated in crystalline form. Complexes 2, 3, 4, and 7 have been structurally characterized. Kinetic data for the dissociation of O 2 from the dioxygen adducts of 1- 4 were obtained using (31)P NMR to monitor changes in the concentration of [Rh(X)(O 2)(XNC)(PPh 3) 2] (X = Cl, Br, SC 6F 5, C 2Ph) resulting from the bubbling of argon through the respective warmed solutions (solvent chlorobenzene). From data recorded at temperatures in the range 30-70 degrees C, activation parameters were obtained as follows: Delta H (++) (kJ mol (-1)): 31.7 +/- 1.6 (X = Cl), 52.1 +/- 4.3 (X = Br), 66.0 +/- 5.8 (X = SC 6F 5), 101.3 +/- 1.8 (X = C 2Ph); Delta S (++) (J K (-1) mol (-1)): -170.3 +/- 5.0 (X = Cl), -120 +/- 13.6 (X = Br), -89 +/- 18.2 (X = SC 6F 5), -6.4 +/- 5.4 (X = C 2Ph). The values of Delta H (++) and Delta S (++) are closely correlated (R (2) = 0.9997), consistent with a common dissociation pathway along which the rate-determining step occurs at a different position for each X. Relative magnitudes of Delta H (++) are interpreted in terms of differing polarizabilities of ligands X.

Infertility due to congenital absence of vas deferens in mainly caused by variable exon 9 skipping of the CFTR gene in heterozygous males for cystic fibrosis mutations

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

Chillon, M.; Casals, T.; Nunes, V.

1994-09-01

About 65% or the individuals with congenital bilateral absence of the vas deferens (CBAVD) have mutations in at least one of the CFTR alleles. We have studied the phenotypic effects of the CFTR gene intron 8 polyT tract 5T allele in 90 CBAVD subjects and in parents of CF patients. This group was compared with normal individuals, and with fathers and mothers of CF patients. Allele 5T was significantly associated with CBAVD (19.6%) when compared to the general population (5.2%) ({chi}{sup 2} = 33.3%; p<<0.0001). It was represented poorly in fathers of CF patients (1.3%). Mutations were identified in onemore » (60%) or both CFTR alleles (8.9%) of CBAVD patients. Heterozygosity for the 5T allele was strongly associated with heterozygosity for CF mutations ({chi}{sup 2} = 10.9; p<0.0004). The strong correlation between allele 5T and CBAVD, together with the low frequency of this allele in fathers of CF patients, demonstrates that variable {Delta}exon 9 produces infertility in males if associated with a CF mutation on the other chromosome. The 30% of CBAVD cases with only one CFTR mutation and without a 5T-allele may be due to other molecular mechanisms involving CFTR, distinct from {Delta}exon 9. Since there is a relatively high proportion of CBAVD without CF mutations (25%), other gene(s), distinct from CFTR, may have a role in the CBAVD phenotype.« less

Improved phase-ellipse method for in-situ geophone calibration.

USGS Publications Warehouse

Liu, Huaibao P.; Peselnick, L.

1986-01-01

For amplitude and phase response calibration of moving-coil electromagnetic geophones 2 parameters are needed, namely the geophone natural frequency, fo, and the geophone upper resonance frequency fu. The phase-ellipse method is commonly used for the in situ determination of these parameters. For a given signal-to-noise ratio, the precision of the measurement of fo and fu depends on the phase sensitivity, f(delta PHI/delta PHIf). For some commercial geophones (f(delta PHI/delta PHI) at fu can be an order of magnitude less than the sensitivity at fo. Presents an improved phase-ellipse method with increased precision. Compared to measurements made with the existing phase-ellipse methods, the method shows a 6- and 3-fold improvement in the precision, respectively, on measurements of fo and fu on a commercial geophone.-from Authors

JAK2V617F somatic mutation in the general population: myeloproliferative neoplasm development and progression rate

PubMed Central

Nielsen, Camilla; Bojesen, Stig E.; Nordestgaard, Børge G.; Kofoed, Klaus F.; Birgens, Henrik S.

2014-01-01

Clinical significance of the JAK2V617F mutation in patients with a myeloproliferative neoplasm has been the target of intensive research in recent years. However, there is considerably uncertainty about prognosis in JAK2V617F positive individuals without overt signs of myeloproliferative disease. In this study, we tested the hypothesis that increased JAK2V617F somatic mutation burden is associated with myeloproliferative neoplasm progression rate in the general population. Among 49,488 individuals from the Copenhagen General Population Study, 63 (0.1%) tested positive for the JAK2V617F mutation in the time period 2003–2008. Of these, 48 were available for re-examination in 2012. Level of JAK2V617F mutation burden was associated with myeloproliferative neoplasm progression rate, consistent with a biological continuum of increasing JAK2V617F mutation burden across increasing severity of myeloproliferative neoplasm from no disease (n=8 at re-examination) through essential thrombocythemia (n=20) and polycythemia vera (n=13) to primary myelofibrosis (n=7). Among those diagnosed with a myeloproliferative neoplasm only at re-examination in 2012, in the preceding years JAK2V617F mutation burden increased by 0.55% per year, erythrocyte volume fraction increased by 1.19% per year, and erythrocyte mean corpuscular volume increased by 1.25% per year, while there was no change in platelet count or erythropoietin levels. Furthermore, we established a JAK2V617F mutation burden cut-off point of 2% indicative of disease versus no disease; however, individuals with a mutation burden below 2% may suffer from a latent form of myeloproliferative disease revealed by a slightly larger spleen and/or slightly higher lactic acid dehydrogenase concentration compared to controls. Of all 63 JAK2V617F positive individuals, 48 were eventually diagnosed with a myeloproliferative neoplasm. PMID:24907356

124I-HuCC49deltaCH2 for TAG-72 antigen-directed positron emission tomography (PET) imaging of LS174T colon adenocarcinoma tumor implants in xenograft mice: preliminary results

PubMed Central

2010-01-01

Background 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) is widely used in diagnostic cancer imaging. However, the use of 18F-FDG in PET-based imaging is limited by its specificity and sensitivity. In contrast, anti-TAG (tumor associated glycoprotein)-72 monoclonal antibodies are highly specific for binding to a variety of adenocarcinomas, including colorectal cancer. The aim of this preliminary study was to evaluate a complimentary determining region (CDR)-grafted humanized CH2-domain-deleted anti-TAG-72 monoclonal antibody (HuCC49deltaCH2), radiolabeled with iodine-124 (124I), as an antigen-directed and cancer-specific targeting agent for PET-based imaging. Methods HuCC49deltaCH2 was radiolabeled with 124I. Subcutaneous tumor implants of LS174T colon adenocarcinoma cells, which express TAG-72 antigen, were grown on athymic Nu/Nu nude mice as the xenograft model. Intravascular (i.v.) and intraperitoneal (i.p.) administration of 124I-HuCC49deltaCH2 was then evaluated in this xenograft mouse model at various time points from approximately 1 hour to 24 hours after injection using microPET imaging. This was compared to i.v. injection of 18F-FDG in the same xenograft mouse model using microPET imaging at 50 minutes after injection. Results At approximately 1 hour after i.v. injection, 124I-HuCC49deltaCH2 was distributed within the systemic circulation, while at approximately 1 hour after i.p. injection, 124I-HuCC49deltaCH2 was distributed within the peritoneal cavity. At time points from 18 hours to 24 hours after i.v. and i.p. injection, 124I-HuCC49deltaCH2 demonstrated a significantly increased level of specific localization to LS174T tumor implants (p = 0.001) when compared to the 1 hour images. In contrast, approximately 50 minutes after i.v. injection, 18F-FDG failed to demonstrate any increased level of specific localization to a LS174T tumor implant, but showed the propensity toward more nonspecific uptake within the heart, Harderian glands of the bony orbits of the eyes, brown fat of the posterior neck, kidneys, and bladder. Conclusions On microPET imaging, 124I-HuCC49deltaCH2 demonstrates an increased level of specific localization to tumor implants of LS174T colon adenocarcinoma cells in the xenograft mouse model on delayed imaging, while 18F-FDG failed to demonstrate this. The antigen-directed and cancer-specific 124I-radiolabled anti-TAG-72 monoclonal antibody conjugate, 124I-HuCC49deltaCH2, holds future potential for use in human clinical trials for preoperative, intraoperative, and postoperative PET-based imaging strategies, including fused-modality PET-based imaging platforms. PMID:20691066

A statistical model to predict one-year risk of death in patients with cystic fibrosis.

PubMed

Aaron, Shawn D; Stephenson, Anne L; Cameron, Donald W; Whitmore, George A

2015-11-01

We constructed a statistical model to assess the risk of death for cystic fibrosis (CF) patients between scheduled annual clinical visits. Our model includes a CF health index that shows the influence of risk factors on CF chronic health and on the severity and frequency of CF exacerbations. Our study used Canadian CF registry data for 3,794 CF patients born after 1970. Data up to 2010 were analyzed, yielding 44,390 annual visit records. Our stochastic process model postulates that CF health between annual clinical visits is a superposition of chronic disease progression and an exacerbation shock stream. Death occurs when an exacerbation carries CF health across a critical threshold. The data constitute censored survival data, and hence, threshold regression was used to connect CF death to study covariates. Maximum likelihood estimates were used to determine which clinical covariates were included within the regression functions for both CF chronic health and CF exacerbations. Lung function, Pseudomonas aeruginosa infection, CF-related diabetes, weight deficiency, pancreatic insufficiency, and the deltaF508 homozygous mutation were significantly associated with CF chronic health status. Lung function, age, gender, age at CF diagnosis, P aeruginosa infection, body mass index <18.5, number of previous hospitalizations for CF exacerbations in the preceding year, and decline in forced expiratory volume in 1 second in the preceding year were significantly associated with CF exacerbations. When combined in one summative model, the regression functions for CF chronic health and CF exacerbation risk provided a simple clinical scoring tool for assessing 1-year risk of death for an individual CF patient. Goodness-of-fit tests of the model showed very encouraging results. We confirmed predictive validity of the model by comparing actual and estimated deaths in repeated hold-out samples from the data set and showed excellent agreement between estimated and actual mortality. Our threshold regression model incorporates a composite CF chronic health status index and an exacerbation risk index to produce an accurate clinical scoring tool for prediction of 1-year survival of CF patients. Our tool can be used by clinicians to decide on optimal timing for lung transplant referral. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular dynamics simulations reveal the allosteric effect of F1174C resistance mutation to ceritinib in ALK-associated lung cancer.

PubMed

Ni, Zhong; Wang, Xiting; Zhang, Tianchen; Jin, Rong Zhong

2016-12-01

Anaplastic lymphoma kinase (ALK) has become as an important target for the treatment of various human cancers, especially non-small-cell lung cancer. A mutation, F1174C, suited in the C-terminal helix αC of ALK and distal from the small-molecule inhibitor ceritinib bound to the ATP-binding site, causes the emergence of drug resistance to ceritinib. However, the detailed mechanism for the allosteric effect of F1174C resistance mutation to ceritinib remains unclear. Here, molecular dynamics (MD) simulations and binding free energy calculations [Molecular Mechanics/Generalized Born Surface Area (MM/GBSA)] were carried out to explore the advent of drug resistance mutation in ALK. MD simulations observed that the exquisite aromatic-aromatic network formed by residues F1098, F1174, F1245, and F1271 in the wild-type ALK-ceritinib complex was disrupted by the F1174C mutation. The resulting mutation allosterically affected the conformational dynamic of P-loop and caused the upward movement of the P-loop from the ATP-binding site, thereby weakening the interaction between ceritinib and the P-loop. The subsequent MM/GBSA binding free energy calculations and decomposition analysis of binding free energy validated this prediction. This study provides mechanistic insight into the allosteric effect of F1174C resistance mutation to ceritinib in ALK and is expected to contribute to design the next-generation of ALK inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of CALR and MPL Mutations in Low Allelic Burden JAK2 V617F Essential Thrombocythemia.

PubMed

Usseglio, Fabrice; Beaufils, Nathalie; Calleja, Anne; Raynaud, Sophie; Gabert, Jean

2017-01-01

Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by aberrant proliferation and an increased tendency toward leukemic transformation. The genes JAK2, MPL, and CALR are frequently altered in these syndromes, and their mutations are often a strong argument for diagnosis. We analyzed the mutational profiles of these three genes in a cohort of 164 suspected myeloproliferative neoplasms. JAK2 V617F mutation was detected by real-time PCR, whereas high-resolution melting analysis followed by Sanger sequencing were used for searching for mutations in JAK2 exon 12, CALR, and MPL. JAK2 V617F mutation was associated with CALR (n = 4) and MPL (n = 4) mutations in 8 of 103 essential thrombocytosis patients. These cases were harboring a JAK2 V617F allelic burden of <4% and a significantly higher platelet count compared with JAK2 V617F (P < 0.001) and CALR (P = 0.001) single-mutation patients. The findings from this study support the possibility of coexisting mutations of the JAK2, CALR, and MPL genes in myeloproliferative neoplasms and suggest that CALR and MPL should be analyzed not only in JAK2-negative patients but also in low V617F mutation patients. Follow-up of these double-mutation cases will be important for determining whether this group of patients presents particular evolution or complications. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Impact of JAK2V617F Mutation Burden on Disease Phenotype in Chinese Patients with JAK2V617F-positive Polycythemia Vera (PV) and Essential thrombocythemia (ET).

PubMed

Zhao, Shixiang; Zhang, Xiang; Xu, Yang; Feng, Yufeng; Sheng, Wenhong; Cen, Jiannong; Wu, Depei; Han, Yue

2016-01-01

Most patients with polycythemia vera (PV) and half of essential thrombocythemia (ET) possess an activating JAK2V617F mutation. The objective of this study was to better define the effect of JAK2V617F mutant allele burden on clinical phenotypes in Chinese patients, especially thrombosis. By real-time polymerase chain reaction (RT-PCR), the JAK2V617F mutation burden was detected in 170 JAK2V617F-positive patients, including 54 PV and 116 ET. The results showed that JAK2V617F allele burden was higher in PV than in ET (P< 0.001). Higher percentage of patients had JAK2V617F allele burden over 20% in PV than in ET (68.5% VS 26.7%) (P< 0.001). In PV patients, higher JAK2V617F allele burden was observed in female (P< 0.05) and leukocytosis patients (WBC above 10 × 10(9)/L) (P< 0.001). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher hemoglobin (HGB above 150 g/L) (P< 0.05), leukocytosis (WBC above 10 × 10(9)/L) (P< 0.001), splenomegaly (P< 0.05) and thrombosis (P< 0.05). In conclusion, the JAK2V617F mutation allele burden is higher in Chinese patients with PV than ET. In PV patients, JAK2V617F mutation burden had influence on WBC counts. And the clinical characteristics of ET patients, such as WBC counts, hemoglobin level, splenomegaly and thrombosis, were influenced by JAK2V617F mutation burden. Male, high hemoglobin (HGB above 150 g/L), and increased JAK2V617F mutation burden (JAK2V617F allele burden ≥ 16.5%) were risks of thrombosis (P< 0.05) for ET patients by Logistic Regression.

Establishment and rapid detection of a heterozygous missense mutation in the CACNA1F gene by ARMS technique with double-base mismatched primers.

PubMed

Yang, W C; Zhu, L; Zhou, B X; Tania, S; Zhou, Q; Khan, M A; Fu, X L; Cheng, J L; Lv, H B; Fu, J J

2015-09-25

Retinitis pigmentosa (RP) is a retinal degenerative disorder that often causes complete blindness. Mutations of more than 50 genes have been identified as associated with RP, including the CACNA1F gene. In a recent study, by employing next-generation sequencing, we identified a novel mutation in the CACNA1F gene. In this study, we used the amplification refractory mutation system (ARMS) and identified a single nucleotide change c.1555C>T in exon 13 of the CACNA1F gene, leading to the substitution of arginine by tryptophan (p.R519W) in a Chinese individual affected by RP. This study actually confirms this novel mutation, and establishes the ARMS technique for the detection of mutations in RP.

Roles for the yeast RAD18 and RAD52 DNA repair genes in UV mutagenesis.

PubMed

Armstrong, J D; Chadee, D N; Kunz, B A

1994-11-01

Experimental evidence indicates that although the Saccharomyces cerevisiae RAD18 and RAD52 genes are not required for nucleotide excision repair, they function in the processing of UV-induced DNA damage in yeast. Conflicting statements regarding the UV mutability of strains deleted for RAD18 prompted us to re-examine the influence of RAD18, and RAD52, on UV mutagenesis. To do so, we characterized mutations induced by UV in SUP4-o, a yeast suppressor tRNA gene. SUP4-o was maintained on a plasmid in isogenic strains that either carried one of two different rad18 deletions (rad18 delta) or had RAD52 disrupted. Both rad18 deletions decreased the frequency of UV-induced SUP4-o mutations to levels close to those for spontaneous mutagenesis in the rad18 delta backgrounds, and prevented a net increase in mutant yield. A detailed analysis of mutations isolated after UV irradiation of one of the rad18 delta strains uncovered little evidence of the specificity features typical for UV mutagenesis in the isogenic repair-proficient (RAD) parent (e.g., predominance of G.C-->A.T transitions). Evidently, UV induction of SUP4-o mutations is highly dependent on the RAD18 gene. Compared to the RAD strain, disruption of RAD52 reduced the frequency and yield of UV mutagenesis by about two-thirds. Closer inspection revealed that 80% of this reduction was due to a decrease in the frequency of G.C-->A.T transitions. In addition, there were differences in the distributions and site specificities of single base-pair substitutions. Thus, RAD52 also participates in UV mutagenesis of a plasmid-borne gene in yeast, but to a lesser extent than RAD18.

[Expression of JAK2V617F and MPLW515L/K mutation in 30 suspected cases of early myeloproliferative disorders].

PubMed

Fan, Zheng; Zhang, Ri; Shen, Yi-Min; Fei, Hai-Rong; Zhu, Zi-Ling; Cen, Jian-Nong

2008-09-01

To investigate the prevalence of JAK2V617F and MPLW515L/K mutation in patients with slightly elevated platelets (BPC) or hemoglobin (Hb) not meeting the criteria of polycythemia vera (PV) or essential thrombocythemia (ET). Genomic DNA from bone marrow or blood mononuclear cells was screened with allele specific polymerase chain reaction (AS-PCR) for JAK2V617F and MPLW515L/K mutation. The history of thrombosis was assessed retrospectively by patients files. Of 30 patients, 14 (46.7%) were positive for the JAK2V617F mutation, none of them had the MPLW515L/ K. Five of these 14 patients had a history of thrombosis. Follow-up results were available in 22 patients. Among them, 12 patients with JAK2V617F mutation turned out to be MPD in 6-24 months; only 2 out of 10 patients without this mutation evolved to MPD. JAK2V617F mutation could be one of the diagnosis criteria of early MPD. No MPLW515L/K expression was found in early MPD.

Atoms-in-molecules study of the genetically encoded amino acids. III. Bond and atomic properties and their correlations with experiment including mutation-induced changes in protein stability and genetic coding.

PubMed

Matta, Chérif F; Bader, Richard F W

2003-08-15

This article presents a study of the molecular charge distributions of the genetically encoded amino acids (AA), one that builds on the previous determination of their equilibrium geometries and the demonstrated transferability of their common geometrical parameters. The properties of the charge distributions are characterized and given quantitative expression in terms of the bond and atomic properties determined within the quantum theory of atoms-in-molecules (QTAIM) that defines atoms and bonds in terms of the observable charge density. The properties so defined are demonstrated to be remarkably transferable, a reflection of the underlying transferability of the charge distributions of the main chain and other groups common to the AA. The use of the atomic properties in obtaining an understanding of the biological functions of the AA, whether free or bound in a polypeptide, is demonstrated by the excellent statistical correlations they yield with experimental physicochemical properties. A property of the AA side chains of particular importance is the charge separation index (CSI), a quantity previously defined as the sum of the magnitudes of the atomic charges and which measures the degree of separation of positive and negative charges in the side chain of interest. The CSI values provide a correlation with the measured free energies of transfer of capped side chain analogues, from the vapor phase to aqueous solution, yielding a linear regression equation with r2 = 0.94. The atomic volume is defined by the van der Waals isodensity surface and it, together with the CSI, which accounts for the electrostriction of the solvent, yield a linear regression (r2 = 0.98) with the measured partial molar volumes of the AAs. The changes in free energies of transfer from octanol to water upon interchanging 153 pairs of AAs and from cyclohexane to water upon interchanging 190 pairs of AAs, were modeled using only three calculated parameters (representing electrostatic and volume contributions) yielding linear regressions with r2 values of 0.78 and 0.89, respectively. These results are a prelude to the single-site mutation-induced changes in the stabilities of two typical proteins: ubiquitin and staphylococcal nuclease. Strong quadratic correlations (r2 approximately 0.9) were obtained between DeltaCSI upon mutation and each of the two terms DeltaDeltaH and TDeltaDeltaS taken from recent and accurate differential scanning calorimetry experiments on ubiquitin. When the two terms are summed to yield DeltaDeltaG, the quadratic terms nearly cancel, and the result is a simple linear fit between DeltaDeltaG and DeltaCSI with r2 = 0.88. As another example, the change in the stability of staphylococcal nuclease upon mutation has been fitted linearly (r2 = 0.83) to the sum of a DeltaCSI term and a term representing the change in the van der Waals volume of the side chains upon mutation. The suggested correlation of the polarity of the side chain with the second letter of the AA triplet genetic codon is given concrete expression in a classification of the side chains in terms of their CSI values and their group dipole moments. For example, all amino acids with a pyrimidine base as their second letter in mRNA possess side-chain CSI < or = 2.8 (with the exception of Cys), whereas all those with CSI > 2.8 possess an purine base. The article concludes with two proposals for measuring and predicting molecular complementarity: van der Waals complementarity expressed in terms of the van der Waals isodensity surface and Lewis complementarity expressed in terms of the local charge concentrations and depletions defined by the topology of the Laplacian of the electron density. A display of the experimentally accessible Laplacian distribution for a folded protein would offer a clear picture of the operation of the "stereochemical code" proposed as the determinant in the folding process. Copyright 2003 Wiley-Liss, Inc.

JAK2 (V617F) mutation is not associated with thrombosis in Behcet syndrome.

PubMed

Ar, M Cem; Hatemi, Gülen; Ekizoğlu, Seda; Bilgen, Hülya; Saçli, Sevgi; Buyru, A Nur; Soysal, Teoman; Ülkü, Birsen; Yazici, Hasan

2012-07-01

The Janus kinase 2(V617F) (JAK2 (V617F)) mutation is an acquired genetic defect that is considered to enhance thrombosis in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Thrombosis is also a well-defined component of Behcet syndrome (BS). The aim of this study was to determine the frequency of JAK2 ( V617F ) mutation in BS-associated thrombosis. A total of 152 patients with BS (62 with thrombosis and 90 without thrombosis) were enrolled. An additional 186 patients with MPNs and 107 healthy blood donors were included to serve as diseased and healthy controls, respectively. None of the patients with BS and healthy controls carried the JAK2 (V617F) mutation, whereas 67% of patients with MPNs were positive for JAK2 ( V617F ). The frequency of thrombosis in patients with MPNs was not statistically different between carriers and non-carriers of JAK2 ( V617F ) mutation. Our data suggest that JAK2 (V617F) is not directly related to thrombosis in MPNs and in other thrombotic entities, such as BS.

Increased fMRI signal with age in familial Alzheimer’s disease mutation carriers

PubMed Central

Braskie, Meredith N.; Medina, Luis D.; Rodriguez-Agudelo, Yaneth; Geschwind, Daniel H.; Macias-Islas, Miguel Angel; Cummings, Jeffrey L.; Bookheimer, Susan Y.; Ringman, John M.

2010-01-01

Although many Alzheimer’s disease (AD) patients have a family history of the disease, it is rarely inherited in a predictable way. Functional magnetic resonance imaging (fMRI) studies of non-demented adults carrying familial AD mutations provide an opportunity to prospectively identify brain differences associated with early AD-related changes. We compared fMRI activity of 18 non-demented autosomal dominant AD mutation carriers with fMRI activity in 8 of their non-carrier relatives as they performed a novelty encoding task in which they viewed novel and repeated images. Because age of disease onset is relatively consistent within families, we also correlated fMRI activity with subjects’ distance from the median age of diagnosis for their family. Mutation carriers did not show significantly different voxelwise fMRI activity from non-carriers as a group. However, as they approached their family age of disease diagnosis, only mutation carriers showed increased fMRI activity in the fusiform and middle temporal gyri. This suggests that during novelty encoding, increased fMRI activity in the temporal lobe may relate to incipient AD processes. PMID:21129823

Identification of a Gypsy SHOX mutation (p.A170P) in Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia

PubMed Central

Barca-Tierno, Verónica; Aza-Carmona, Miriam; Barroso, Eva; Heine-Suner, Damia; Azmanov, Dimitar; Rosell, Jordi; Ezquieta, Begoña; Montané, Lucia Sentchordi; Vendrell, Teresa; Cruz, Jaime; Santos, Fernando; Rodríguez, José Ignacio; Pozo, Jesús; Argente, Jesús; Kalaydjieva, Luba; Gracía, Ricardo; Campos-Barros, Ángel; Benito-Sanz, Sara; Heath, Karen E

2011-01-01

We report the clinical and molecular characteristics of 12 Spanish families with multiple members affected with Léri-Weill dyschondrosteosis (LWD) or Langer mesomelic dysplasia (LMD), who present the SHOX (short stature homeobox gene) mutation p.A170P (c.508G>C) in heterozygosity or homozygosity, respectively. In all studied families, the A170P mutation co-segregated with the fully penetrant phenotype of mesomelic limb shortening and Madelung deformity. A shared haplotype around SHOX was observed by microsatellite analysis, confirming the presence of a common ancestor, probably of Gypsy origin, as 11 of the families were of this ethnic group. Mutation screening in 359 Eastern-European Gypsies failed to identify any carriers. For the first time, we have shown SHOX expression in the human growth plate of a 22-week LMD fetus, homozygous for the A170P mutation. Although the mutant SHOX protein was expressed in all zones of the growth plate, the chondrocyte columns in the proliferative zone were disorganized with the chondrocytes occurring in smaller columnal clusters. We have also identified a novel mutation at the same residue, c. 509C>A (p.A170D), in two unrelated Spanish LWD families, which similar to A170P mutation impedes nuclear localization of SHOX. In conclusion, we have identified A170P as the first frequent SHOX mutation in Gypsy LWD and LMD individuals. PMID:21712857

Identification of a Gypsy SHOX mutation (p.A170P) in Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia.

PubMed

Barca-Tierno, Verónica; Aza-Carmona, Miriam; Barroso, Eva; Heine-Suner, Damia; Azmanov, Dimitar; Rosell, Jordi; Ezquieta, Begoña; Montané, Lucia Sentchordi; Vendrell, Teresa; Cruz, Jaime; Santos, Fernando; Rodríguez, José Ignacio; Pozo, Jesús; Argente, Jesús; Kalaydjieva, Luba; Gracía, Ricardo; Campos-Barros, Angel; Benito-Sanz, Sara; Heath, Karen E

2011-12-01

We report the clinical and molecular characteristics of 12 Spanish families with multiple members affected with Léri-Weill dyschondrosteosis (LWD) or Langer mesomelic dysplasia (LMD), who present the SHOX (short stature homeobox gene) mutation p.A170P (c.508G>C) in heterozygosity or homozygosity, respectively. In all studied families, the A170P mutation co-segregated with the fully penetrant phenotype of mesomelic limb shortening and Madelung deformity. A shared haplotype around SHOX was observed by microsatellite analysis, confirming the presence of a common ancestor, probably of Gypsy origin, as 11 of the families were of this ethnic group. Mutation screening in 359 Eastern-European Gypsies failed to identify any carriers. For the first time, we have shown SHOX expression in the human growth plate of a 22-week LMD fetus, homozygous for the A170P mutation. Although the mutant SHOX protein was expressed in all zones of the growth plate, the chondrocyte columns in the proliferative zone were disorganized with the chondrocytes occurring in smaller columnal clusters. We have also identified a novel mutation at the same residue, c. 509C>A (p.A170D), in two unrelated Spanish LWD families, which similar to A170P mutation impedes nuclear localization of SHOX. In conclusion, we have identified A170P as the first frequent SHOX mutation in Gypsy LWD and LMD individuals.

Characterization of Nucleoside Reverse Transcriptase Inhibitor-Associated Mutations in the RNase H Region of HIV-1 Subtype C Infected Individuals.

PubMed

Ngcapu, Sinaye; Theys, Kristof; Libin, Pieter; Marconi, Vincent C; Sunpath, Henry; Ndung'u, Thumbi; Gordon, Michelle L

2017-11-08

The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher's exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation.

Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum

PubMed Central

Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

2014-01-01

Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

Conditioning monitoring by microstructural evaluation of cumulative fatigue damage

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

Fukuoka, C.; Nakagawa, Y.G.; Lance, J.J.

1996-12-01

The objective of this work is to evaluate the damage induced below and above the fatigue limit ({Delta}{sigma}{sub t} = 360 MPa) in pressure vessel steels, such as SA508. Fatigue damage was induced in samples taken from an SA508 steel plate by various loading histories in order to examine the influence of prior cyclic loading below the fatigue limit. Cell-to-cell misorientation differences were measured by the selected area diffraction (SAD) method. Surface cracking was also studied by the replication method. Small cracks were observed after precycling both below and above the fatigue limit. It was, however, found that fatigue testmore » bars had a longer lifetime after precycling below the fatigue limit, while precycling above the fatigue limit caused other specimens to fail even when subsequently cycled below the fatigue limit. Cell-to-cell misorientation usually increases with accumulation of fatigue damage, but it was found that the misorientations measured after precycling below the fatigue limit decreased again at the beginning of the subsequent cycling above the fatigue limit. It should be noted that the misorientation at failure was always about 4 to 5 deg, regardless of loading histories. Misorientation showed good correlation with the fatigue lifetime of the samples.« less

Extending Jak2V617F and MplW515 mutation analysis to single hematopoietic colonies and B and T lymphocytes.

PubMed

Pardanani, Animesh; Lasho, Terra L; Finke, Christy; Mesa, Ruben A; Hogan, William J; Ketterling, Rhett P; Gilliland, Dwight Gary; Tefferi, Ayalew

2007-09-01

JAK2V617F and MPLW515L/K are myeloproliferative disorder (MPD)-associated mutations. We genotyped 552 individual hematopoietic colonies obtained by CD34+ cell culture from 16 affected patients (13 JAK2V617F and 3 MPLW515L/K) to determine (a) the proportion of colonies harboring a particular mutation in the presence or absence of cytokines, (b) the lineage distribution of endogenous colonies for each mutation, and (c) the differences (if any) in the pattern of mutation among the various MPDs, as established by genotyping of individual colonies. Genotyping analysis revealed cohabitation of mutation-negative and mutation-positive endogenous colonies in polycythemia vera as well as other MPDs. Culture of progenitor cells harboring MPLW515L/K yielded virtually no endogenous erythroid colonies in contrast to JAK2V617F-harboring progenitor cells. The mutation pattern (i.e., relative distribution of homozygous, heterozygous, or wild-type colonies) was not a distinguishing feature among the MPDs, and MPLW515 mutations were detected in B and/or T lymphocytes in all three patients tested. These observations suggest that clonal myelopoiesis antedates acquisition of JAK2V617F or MPLW515L/K mutations and that the latter is acquired in a lympho-myeloid progenitor cell.

A novel mutation in the alpha-helix 1 of the C subunit of the F(1)/F(0) ATPase responsible for optochin resistance of a Streptococcus pneumoniae clinical isolate.

PubMed

Cogné, N; Claverys, J; Denis, F; Martin, C

2000-10-01

Previously reported mutations involved in optochin resistance of Streptococcus pneumoniae clinical isolates changed residues 48, 49 or 50, in the transmembrane alpha-helix 2 of the F(1)/F(0) ATPase subunit. We report here an unusual mutation which changes the sequence of the transmembrane alpha-helix 1 of the AtpC subunit. This mutation involves a Gly to Ser substitution resulting from a G to A transition at codon 14 of the atpC gene.

Interferometric apparatus for ultra-high precision displacement measurement

NASA Technical Reports Server (NTRS)

Zhao, Feng (Inventor)

2004-01-01

A high-precision heterodyne interferometer measures relative displacement by creating a thermally-insensitive system generally not subject to polarization leakage. By using first and second light beams separated by a small frequency difference (.DELTA.f), beams of light at the first frequency (f.sub.0) are reflected by co-axial mirrors, the first mirror of which has a central aperture through which the light is transmitted to and reflected by the second mirror. Prior to detection, the light beams from the two mirrors are combined with light of the second and slightly different frequency. The combined light beams are separated according to the light from the mirrors. The change in phase (.DELTA..phi.) with respect to the two signals is proportional to the change in distance of Fiducial B by a factor of wavelength (.lambda.) divided by 4.pi. (.DELTA.L=.lambda..DELTA..phi.1/(4.pi.)). In a second embodiment, a polarizing beam splitting system can be used.

Convair F-106B Delta Dart Prepares for a Flight

NASA Image and Video Library

1969-05-21

National Aeronautics and Space Administration (NASA) pilot Cliff Crabbs and the flight operations crew prepare a Convair F-106B Delta Dart for a flight from the Lewis Research Center in Cleveland, Ohio. NASA acquired the aircraft three years earlier to investigate noise-reducing inlet and nozzle designs for the supersonic transport engine program. Two General Electric J85 engines were installed underneath the aircraft’s delta wings to simulate the general shape of the supersonic transport’s engines. One of the engines was modified with experimental inlet or nozzle configurations. The unmodified engine was used for comparison. Most F-106B flights were flown in a 200-mile path over the lake between Buffalo and Sandusky, known as the Lake Erie Corridor. The 1100-miles per hour flight took only 11 minutes at an altitude of 30,000 feet. The aircraft almost always returned with a depleted fuel supply so a Visual Flight Rules operation was required. Following the crash of another jet fighter at Lewis in July 1969, the F-106s were stationed at Selfridge Air Force Base in Michigan. NASA pilots flew transport planes each morning to the base before commencing the F-106B missions.

Association between Abdominal Fat (DXA) and Its Subcomponents (CT Scan) before and after Weight Loss in Obese Postmenopausal Women: A MONET Study.

PubMed

Doyon, Caroline Y; Brochu, Martin; Messier, Virginie; Lavoie, Marie-Ève; Faraj, May; Doucet, Eric; Rabasa-Lhoret, Rémi; Dionne, Isabelle J

2011-01-01

Introduction. Subcutaneous fat (ScF) and visceral fat (VF) measurements using CT scan are expensive and may imply significant radiation doses. Cross-sectional studies using CT scan showed that ScF and VF are significantly correlated with abdominal fat measured by DXA (AF-DXA). The association has not been studied after a weight loss. Objective. To determine (1) the associations between AF-DXA and ScF and VF before and after weight loss and (2) the associations between their changes. Methods. 137 overweight/obese postmenopausal women were divided in two groups (1-caloric restriction or 2-caloric restriction + resistance training). AF was assessed using DXA and CT scan. Results. Correlations between AF-DXA and ScF (before: r = 0.87, after; r = 0.87; P < .01) and, AF-DXA and VF (before: r = 0.61, after; r = 0.69; P < .01) are not different before and after the weight loss. Correlations between delta AF-DXA and delta ScF (r = 0.72; P < .01) or delta VF (r = 0.51; P < .01) were found. Conclusion. The use of AF-DXA as a surrogate for VF after weight loss is questionable, but may be interesting for ScF.

Association between Abdominal Fat (DXA) and Its Subcomponents (CT Scan) before and after Weight Loss in Obese Postmenopausal Women: A MONET Study

PubMed Central

Doyon, Caroline Y.; Brochu, Martin; Messier, Virginie; Lavoie, Marie-Ève; Faraj, May; Doucet, Éric; Rabasa-Lhoret, Rémi; Dionne, Isabelle J.

2011-01-01

Introduction. Subcutaneous fat (ScF) and visceral fat (VF) measurements using CT scan are expensive and may imply significant radiation doses. Cross-sectional studies using CT scan showed that ScF and VF are significantly correlated with abdominal fat measured by DXA (AF-DXA). The association has not been studied after a weight loss. Objective. To determine (1) the associations between AF-DXA and ScF and VF before and after weight loss and (2) the associations between their changes. Methods. 137 overweight/obese postmenopausal women were divided in two groups (1-caloric restriction or 2-caloric restriction + resistance training). AF was assessed using DXA and CT scan. Results. Correlations between AF-DXA and ScF (before: r = 0.87, after; r = 0.87; P < .01) and, AF-DXA and VF (before: r = 0.61, after; r = 0.69; P < .01) are not different before and after the weight loss. Correlations between delta AF-DXA and delta ScF (r = 0.72; P < .01) or delta VF (r = 0.51; P < .01) were found. Conclusion. The use of AF-DXA as a surrogate for VF after weight loss is questionable, but may be interesting for ScF. PMID:21603261

From Wine to Pineapples: Delta Company Takes on New Satellite Role

DTIC Science & Technology

2011-01-01

2011 Spring / Summer edition army Space Journal 2F Delta Company Takes on New Satellite Role FROM Wine TO PineAPPLeS WAHIAWA, Hawaii – 2011 has yet...next to pineapple fields on the island of Oahu, Hawaii. Delta Company has provided network and payload control for the Defense Satellite...1. REPORT DATE 2011 2. REPORT TYPE 3. DATES COVERED 00-00-2011 to 00-00-2011 4. TITLE AND SUBTITLE From Wine to Pineapples : Delta Company

Crystal Structure of Human Liver [delta][superscript 4]-3-Ketosteroid 5[beta]-Reductase (AKR1D1) and Implications for Substrate Binding and Catalysis

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

Di Costanzo, Luigi; Drury, Jason E.; Penning, Trevor M.

2008-07-15

AKR1D1 (steroid 5{beta}-reductase) reduces all {Delta}{sup 4}-3-ketosteroids to form 5{beta}-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an {alpha}{beta}-unsaturated ketone by 5{beta}-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the {beta}-face of a {Delta}{sup 4}-3-ketosteroid yields a cis-A/B-ring configuration with an {approx}90{sup o} bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human {Delta}{sup 4}-3-ketosteroid 5{beta}-reductase (AKR1D1), and its complexes withmore » intact substrates. We have determined the structures of AKR1D1 complexes with NADP{sup +} at 1.79- and 1.35-{angstrom} resolution (HEPES bound in the active site), NADP{sup +} and cortisone at 1.90-{angstrom} resolution, NADP{sup +} and progesterone at 2.03-{angstrom} resolution, and NADP{sup +} and testosterone at 1.62-{angstrom} resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP{sup +}. This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr{sup 58} and Glu{sup 120}. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.« less

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

PubMed

Huang, Yuehua; Tai, Andrew W; Tong, Shuping; Lok, Anna S F

2013-06-01

Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed

Singdong, Roongrudee; Siriboonpiputtana, Teerapong; Chareonsirisuthigul, Takol; Kongruang, Adcharee; Limsuwanachot, Nittaya; Sirirat, Tanasan; Chuncharunee, Suporn; Rerkamnuaychoke, Budsaba

2016-10-01

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome. Material and Methods: Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9.0 mutations in 100.0 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U). Results: The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load > 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9.0 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF. Conclusions: The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET. Creative Commons Attribution License

Characterization and Prognosis Significance of JAK2 (V617F), MPL, and CALR Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed Central

Singdong, Roongrudee; Siriboonpiputtana, Teerapong; Chareonsirisuthigul, Takol; Kongruang, Adcharee; Limsuwanachot, Nittaya; Sirirat, Tanasan; Chuncharunee, Suporn; Rerkamnuaychoke, Budsaba

2016-01-01

Background: The discovery of somatic acquired mutations of JAK2 (V617F) in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) has not only improved rational disease classification and prognostication but also brings new understanding insight into the pathogenesis of diseases. Dosage effects of the JAK2 (V617F) allelic burden in Ph-negative MPNs may partially influence clinical presentation, disease progression, and treatment outcome. Material and Methods: Pyrosequencing was performed to detect JAK2 (V617F) and MPL (W515K/L) and capillary electrophoresis to identify CALR exon 9 mutations in 100 samples of Ph-negative MPNs (38.0 PV, 55 ET, 4 PMF, and 3 MPN-U). Results: The results showed somatic mutations of JAK2 (V617F) in 94.7% of PV, 74.5% of ET, 25.0% of PMF, and all MPN-U. A high proportion of JAK2 (V617F) mutant allele burden (mutational load > 50.0%) was predominantly observed in PV when compared with ET. Although a high level of JAK2 (V617F) allele burden was strongly associated with high WBC counts in both PV and ET, several hematological parameters (hemoglobin, hematocrit, and platelet count) were independent of JAK2 (V617F) mutational load. MPL (W515K/L) mutations could not be detected whereas CALR exon 9 mutations were identified in 35.7% of patients with JAK2 negative ET and 33.3% with JAK2 negative PMF. Conclusions: The JAK2 (V617F) allele burden may be involved in progression of MPNs. Furthermore, a high level of JAK2 (V617F) mutant allele appears strongly associated with leukocytosis in both PV and ET. PMID:27892678

A complex of serine protease genes expressed preferentially in cytotoxic T-lymphocytes is closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14.

PubMed

Crosby, J L; Bleackley, R C; Nadeau, J H

1990-02-01

A complex of genes encoding serine proteases that are preferentially expressed in cytotoxic T-cells was shown to be closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14. A striking difference in recombination frequencies among linkage crosses was reported. Two genes, Np-1 and Tcra, which fail to recombine in crosses involving conventional strains of mice, were shown to recombine readily in interspecific crosses involving Mus spretus. This difference in recombination frequency suggests chromosomal rearrangements that suppress recombination in conventional crosses, recombination hot spots in interspecific crosses, or selection against recombinant haplotypes during development of recombinant inbred strains. Finally, a mutation called disorganization, which is located near the serine protease complex, is of considerable interest because it causes an extraordinarily wide variety of congenital defects. Because of the involvement of serine protease loci in several homeotic mutations in Drosophila, disorganization must be considered a candidate for a mutation in a serine protease-encoding gene.

Memory Performance and fMRI Signal in Presymptomatic Familial Alzheimer’s Disease

PubMed Central

Braskie, Meredith N.; Medina, Luis D.; Rodriguez-Agudelo, Yaneth; Geschwind, Daniel H.; Macias-Islas, Miguel Angel; Thompson, Paul M.; Cummings, Jeffrey L.; Bookheimer, Susan Y.; Ringman, John M.

2013-01-01

Rare autosomal dominant mutations result in familial Alzheimer’s disease (FAD) with a relatively consistent age of onset within families. This provides an estimate of years until disease onset (relative age) in mutation carriers. Increased AD risk has been associated with differences in functional magnetic resonance imaging (fMRI) activity during memory tasks, but most of these studies have focused on possession of apolipoprotein E allele 4 (APOE4), a risk factor, but not causative variant, of late-onset AD. Evaluation of fMRI activity in presymptomatic FAD mutation carriers versus noncarriers provides insight into preclinical changes in those who will certainly develop AD in a prescribed period of time. Adults from FAD mutation-carrying families (nine mutation carriers, eight noncarriers) underwent fMRI scanning while performing a memory task. We examined fMRI signal differences between carriers and noncarriers, and how signal related to fMRI task performance within mutation status group, controlling for relative age and education. Mutation noncarriers had greater retrieval period activity than carriers in several AD-relevant regions, including the left hippocampus. Better performing noncarriers showed greater encoding period activity including in the parahippocampal gyrus. Poorer performing carriers showed greater retrieval period signal, including in the frontal and temporal lobes, suggesting underlying pathological processes. PMID:22806961

Effect of different high-palladium metal-ceramic alloys on the color of opaque and dentin porcelain.

PubMed

Stavridakis, Minos M; Papazoglou, Efstratios; Seghi, Robert R; Johnston, William M; Brantley, William A

2004-08-01

The color of dental porcelain depends on the type of metal substrate. Little research has been done to document the effects of different types of high-palladium alloys on the color of dental porcelain. The purpose of this in vitro study was to evaluate the effects of different high-palladium alloys on the resulting color of dentin porcelain, as well as on that of opaque porcelain after simulated dentin and glazing firing cycles. Three Pd-Cu-Ga alloys, Spartan Plus (S), Liberty (B), and Freedom Plus (F), and 5 Pd-Ga alloys, Legacy (L), IS 85 (I), Protocol (P), Legacy XT (X), and Jelenko No.1 (N), were examined. A Pd-Ag alloy, Super Star (T), was included for comparison to the high-palladium alloys, and the Au-Pd alloy, Olympia (O), served as the control. Six cast discs (16 x 1 mm) were prepared from each of the alloys. Shade B1 opaque porcelain (Vita-Omega) was applied at a final thickness of 0.1 mm. After 2 opaque porcelain firing cycles, the surfaces were airborne-particle abraded, and the specimens were divided into 2 groups. In the first group, 0.9 mm of B1 dentin porcelain was applied. The other group of specimens with only opaque porcelain underwent the same dentin porcelain and glazing firing cycles. Color differences (DeltaE) were determined with a colorimeter between the control and each experimental group, after the second opaque porcelain, second dentin porcelain, and glazing firing cycles. One-way analysis of variance and Dunnett's multiple range test were performed on the DeltaE data (alpha=.05). After the application of dentin porcelain, the 3 Pd-Cu-Ga alloys showed significantly different (P<.05) DeltaE values (S=2.3 +/- 0.5, B=1.4 +/- 0.3, and F=1.3 +/- 0.7) than the control group. After the glazing cycle of this group, the 3 Pd-Cu-Ga alloys and the Pd-Ag alloy exhibited significantly different (P<.05) DeltaE values (S=2.8 +/- 0.8, B=2.2 +/- 0.3, F=1.9 +/- 1.0, and T=1.4 +/- 0.5) than the control group. After the simulated dentin porcelain firing cycles, the specimens with only opaque porcelain exhibited significantly different (P<.05) DeltaE values (S=5.2 +/- 1.4, B=5.4 +/- 0.6, and F=3.9 +/- 0.2) than the control group. The color difference between the 3 Pd-Cu-Ga alloys with only opaque porcelain and the control group increased more after the simulated glazing cycle (S=6.6 +/- 1.5, B=6.3 +/- 0.5, and F=4.6 +/- 0.1). The observed color differences between the Pd-Ga alloys and the control group were not statistically significant at any point. The Pd-Cu-Ga alloys with only opaque porcelain, after the simulated dentin porcelain and glazing firing cycles, exhibited clinically unacceptable color differences. The application of dentin porcelain to the Pd-Cu-Ga alloys resulted in clinically acceptable color differences. The application of dentin porcelain to the Pd-Ag alloy, after the glazing firing cycle, resulted in clinically acceptable color differences (approximately 2.8 to 3.7 DeltaE CIELAB units). The Pd-Ag alloy specimens with only opaque porcelain did not exhibit significant color differences from the control group, whereas significant color differences from the control group after the dentin porcelain and glazing firing cycles were still clinically acceptable.

Holocarboxylase synthetase deficiency: novel clinical and molecular findings.

PubMed

Tammachote, R; Janklat, S; Tongkobpetch, S; Suphapeetiporn, K; Shotelersuk, V

2010-07-01

Multiple carboxylase deficiency (MCD) is an autosomal recessive metabolic disorder caused by defective activity of biotinidase or holocarboxylase synthetase (HLCS) in the biotin cycle. Clinical symptoms include skin lesions and severe metabolic acidosis. Here, we reported four unrelated Thai patients with MCD, diagnosed by urine organic acid analysis. Unlike Caucasians, which biotinidase deficiency has been found to be more common, all of our four Thai patients were affected by HLCS deficiency. Instead of the generally recommended high dose of biotin, our patients were given biotin at 1.2 mg/day. This low-dose biotin significantly improved their clinical symptoms and stabilized the metabolic state on long-term follow-up. Mutation analysis by polymerase chain reaction-sequencing of the entire coding region of the HLCS gene revealed the c.1522C>T (p.R508W) mutation in six of the eight mutant alleles. This suggests it as the most common mutation in the Thai population, which paves the way for a rapid and unsophisticated diagnostic method for the ethnic Thai. Haplotype analysis revealed that the c.1522C>T was on three different haplotypes suggesting that it was recurrent, not caused by a founder effect. In addition, a novel mutation, c.1513G>C (p.G505R), was identified, expanding the mutational spectrum of this gene.

Investigation of dynamic ground effect

NASA Technical Reports Server (NTRS)

Chang, Ray Chung; Muirhead, Vincent U.

1987-01-01

An experimental investigation of dynamic ground effect was conducted in the Univ. of Kansas wind tunnel using delta wings of 60, 70, 75 deg sweep; the XB-70 wing; and the F-104A wing. Both static and dynamic tests were made. Test data were compared to other test data, including dynamic flight test data of the XB-70 and F-104A. Limited flow visualization test were conducted. A significant dynamic effect was found for highly swept delta wings.

Thermochemistry and gas-phase ion energetics of 2-hydroxy-4-methoxy-benzophenone (oxybenzone).

PubMed

Lago, A F; Jimenez, P; Herrero, R; Dávalos, J Z; Abboud, J-L M

2008-04-10

We have investigated the thermochemistry and ion energetics of the oxybenzone (2-hydroxy-4-methoxy-benzophenone, C14H12O3, 1H) molecule. The following parameters have been determined for this species: gas-phase enthalpy for the of neutral molecule at 298.15K, (Delta(f)H0(m)(g) = -303.5 +/- 5.1 kJ x mol-1), the intrinsic (gas-phase) acidity (GA(1H) = 1402.1 +/- 8.4 kJ x mol-1), enthalpy of formation for the oxybenzone anion (Delta(f)H0(m)(1-,g) = -402.3 +/- 9.8 kJ x mol-1). We also have obtained the enthalpy of formation of, 4-hydroxy-4'-methoxybenzophenone (Delta(f)H0(m)(g) = -275.4 +/- 10 kJ x mol-1) and 3-methoxyphenol anion (Delta(f)H0(m)(C7H7O2-,g) = -317.7 +/- 8.7 kJ x mol-1). A reliable experimental estimation of enthalpy related to intramolecular hydrogen bonding in oxybenzone has also been obtained (30.1 +/- 6.3 kJ x mol-1) and compared with our theoretical calculations at the B3LYP/6-311++G** level of theory, by means of an isodesmic reaction scheme. In addition, heat capacities, temperature, and enthalpy of fusion have been determined for this molecule by differential scanning calorimetry.

Predicting rates of inbreeding in populations undergoing selection.

PubMed Central

Woolliams, J A; Bijma, P

2000-01-01

Tractable forms of predicting rates of inbreeding (DeltaF) in selected populations with general indices, nonrandom mating, and overlapping generations were developed, with the principal results assuming a period of equilibrium in the selection process. An existing theorem concerning the relationship between squared long-term genetic contributions and rates of inbreeding was extended to nonrandom mating and to overlapping generations. DeltaF was shown to be approximately (1)/(4)(1 - omega) times the expected sum of squared lifetime contributions, where omega is the deviation from Hardy-Weinberg proportions. This relationship cannot be used for prediction since it is based upon observed quantities. Therefore, the relationship was further developed to express DeltaF in terms of expected long-term contributions that are conditional on a set of selective advantages that relate the selection processes in two consecutive generations and are predictable quantities. With random mating, if selected family sizes are assumed to be independent Poisson variables then the expected long-term contribution could be substituted for the observed, providing (1)/(4) (since omega = 0) was increased to (1)/(2). Established theory was used to provide a correction term to account for deviations from the Poisson assumptions. The equations were successfully applied, using simple linear models, to the problem of predicting DeltaF with sib indices in discrete generations since previously published solutions had proved complex. PMID:10747074

HOL1 mutations confer novel ion transport in Saccharomyces cerevisiae.

PubMed Central

Gaber, R F; Kielland-Brandt, M C; Fink, G R

1990-01-01

Saccharomyces cerevisiae histidine auxotrophs are unable to use L-histidinol as a source of histidine even when they have a functional histidinol dehydrogenase. Mutations in the hol1 gene permit growth of His- cells on histidinol by enhancing the ability of cells to take up histidinol from the medium. Second-site mutations linked to HOL1-1 further increase histidinol uptake. HOL1 double mutants and, to a lesser extent, HOL1-1 single mutants show hypersensitivity to specific cations added to the growth medium, including Na+, Li+, Cs+, Be2+, guanidinium ion, and histidinol, but not K+, Rb+, Ca2+, or Mg2+. The Na(+)-hypersensitive phenotype is correlated with increased uptake and accumulation of this ion. The HOL1-1-101 gene was cloned and used to generate a viable haploid strain containing a hol1 deletion mutation (hol1 delta). The uptake of cations, the dominance of the mutant alleles, and the relative inability of hol1 delta cells to take up histidinol or Na+ suggest that hol1 encodes an ion transporter. The novel pattern of ion transport conferred by HOL1-1 and HOL1-1-101 mutants may be explained by reduced selectivity for the permeant ions. Images PMID:2405251

Computational design of variants for cephalosporin C acylase from Pseudomonas strain N176 with improved stability and activity.

PubMed

Tian, Ye; Huang, Xiaoqiang; Li, Qing; Zhu, Yushan

2017-01-01

In this report, redesigning cephalosporin C acylase from the Pseudomonas strain N176 revealed that the loss of stability owing to the introduced mutations at the active site can be recovered by repacking the nearby hydrophobic core regions. Starting from a quadruple mutant M31βF/H57βS/V68βA/H70βS, whose decrease in stability is largely owing to the mutation V68βA at the active site, we employed a computational enzyme design strategy that integrated design both at hydrophobic core regions for stability enhancement and at the active site for activity improvement. Single-point mutations L154βF, Y167βF, L180βF and their combinations L154βF/L180βF and L154βF/Y167βF/L180βF were found to display improved stability and activity. The two-point mutant L154βF/L180βF increased the protein melting temperature (T m ) by 11.7 °C and the catalytic efficiency V max /K m by 57 % compared with the values of the starting quadruple mutant. The catalytic efficiency of the resulting sixfold mutant M31βF/H57βS/V68βA/H70βS/L154βF/L180βF is recovered to become comparable to that of the triple mutant M31βF/H57βS/H70βS, but with a higher T m . Further experiments showed that single-point mutations L154βF, L180βF, and their combination contribute no stability enhancement to the triple mutant M31βF/H57βS/H70βS. These results verify that the lost stability because of mutation V68βA at the active site was recovered by introducing mutations L154βF and L180βF at hydrophobic core regions. Importantly, mutation V68βA in the six-residue mutant provides more space to accommodate the bulky side chain of cephalosporin C, which could help in designing cephalosporin C acylase mutants with higher activities and the practical one-step enzymatic route to prepare 7-aminocephalosporanic acid at industrial-scale levels.

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

Robertson, R.M.; Barnes, V.E.; Carmony, D.D.

The multiplicity-cross-section differences ..delta..sigma/sub n/ between p-barp and pp interactions are determined and are compared with measured annihilations and ..delta..sigma/sub n/ at other energies. To the extent that these cross-section differences measure the values of sigma/sup A//sub n/, the topological cross sections for annihilations, we present evidence for a decided break from a single-cluster-model prediction for the parameter f/sup A/--/sub 2/. Alternatively, a picture of precocious Koba-Nielsen-Olesen scaling in p-barp annihilations leads to a reasonably good representation of f/sup A/--/sub 2/ vs over the whole measured range. We find =7.57 +- 0.31, D/sup A/=2.77 +- 0.10, f/sup A/--/sub 2/=-1.86 +-more » 0.20, and /D=2.73 +- 0.15. Finally we observe a remarkable agreement with theoretical prediction for R/sup asterisk//sub n/=..delta..sigma/sub n//sigma/sub n/+2( pp), an experimental ratio based on a strict application of the counting rules for quark duality diagrams, and we thereby find evidence that in the topological-cross-section difference ..delta..sigma/sub n/ the small nonannihilation contribution becomes progressively more negligible as n increases.« less

Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy.

PubMed

Bahrudin, Udin; Morisaki, Hiroko; Morisaki, Takayuki; Ninomiya, Haruaki; Higaki, Katsumi; Nanba, Eiji; Igawa, Osamu; Takashima, Seiji; Mizuta, Einosuke; Miake, Junichiro; Yamamoto, Yasutaka; Shirayoshi, Yasuaki; Kitakaze, Masafumi; Carrier, Lucie; Hisatome, Ichiro

2008-12-26

The ubiquitin-proteasome system is responsible for the disappearance of truncated cardiac myosin-binding protein C, and the suppression of its activity contributes to cardiac dysfunction. This study investigated whether missense cardiac myosin-binding protein C gene (MYBPC3) mutation in hypertrophic cardiomyopathy (HCM) leads to destabilization of its protein, causes UPS impairment, and is associated with cardiac dysfunction. Mutations were identified in Japanese HCM patients using denaturing HPLC and sequencing. Heterologous expression was investigated in COS-7 cells as well as neonatal rat cardiac myocytes to examine protein stability and proteasome activity. The cardiac function was measured using echocardiography. Five novel MYBPC3 mutations -- E344K, DeltaK814, Delta2864-2865GC, Q998E, and T1046M -- were identified in this study. Compared with the wild type and other mutations, the E334K protein level was significantly lower, it was degraded faster, it had a higher level of polyubiquination, and increased in cells pretreated with the proteasome inhibitor MG132 (50 microM, 6 h). The electrical charge of its amino acid at position 334 influenced its stability, but E334K did not affect its phosphorylation. The E334K protein reduced cellular 20 S proteasome activity, increased the proapoptotic/antiapoptotic protein ratio, and enhanced apoptosis in transfected Cos-7 cells and neonatal rat cardiac myocytes. Patients carrying the E334K mutation presented significant left ventricular dysfunction and dilation. The conclusion is the missense MYBPC3 mutation E334K destabilizes its protein through UPS and may contribute to cardiac dysfunction in HCM through impairment of the ubiquitin-proteasome system.

Graphite intercalation with fluoroanions by chemical and electrochemical methods

NASA Astrophysics Data System (ADS)

Ozmen-Monkul, Bahar

New acceptor-type graphite intercalation compounds (GICs) containing perfluoroalkyl anions have been synthesized by using both chemical and electrochemical methods and characterized by elemental and thermogravimetric analyses. Investigation into these graphite intercalation compounds can provide novel materials and a detailed understanding of their properties. GICs of composition Cx[FB(C2F 5)3]·deltaF are prepared for the first time by the intercalation of fluoro-tris(pentafluoroethyl)borate anion, [FB(C2F 5)3]-, under ambient conditions in aqueous (48%) hydrofluoric acid containing the oxidant K2[MnF6]. Powder-XRD data indicate that products are pure stage 2 and physical mixture of stage 2 and stage 3 after 1 h to 20 h reaction times. The calculated basal repeat distance, Ic, is 1.20 nm for stage 2 and 1.54-1.56 nm for stage 3 GICs, corresponding to gallery heights of di = 0.86-0.89 nm. In addition, stage 2 GIC of C x[FB(C2F5)3]·deltaCH 3NO2 having di = 0.84 nm is prepared by electrochemical oxidation of graphite in a nitromethane electrolyte. The elemental analyses of these complex GICs required that a new sample digestion protocol be developed. After digestion, the fluoride amounts in these GIC samples were analyzed by using ion-selective fluoride combination electrode. The method developed is able to provide fluoride anion content in GICs without interference from the decomposition products of [FB(C 2F5)3]- anion. For the boron analyses the same digestion procedure above is used and the B contents were determined by ICP-AES. For Cx[FB(C2F 5)3]·deltaF, both compositional parameters x and delta are obtained from the results of elemental B and F analyses. For the chemically prepared GICs at 1 h to 20 h, calculated x values were in the range of 51-56 and the calculated delta values increased with reaction time from approx. 0-2. Combining B analysis and TGA mass loss gives a composition of x = 44 and delta = 0.37 for the electrochemically prepared GIC of Cx[FB(C2F5)3]·deltaCH 3NO2. Energy minimized structure for the isolated borate anion and powder XRD data show that the borate anions adopt a "lying-down" orientation where the long axes of [FB(C2F5)3] - intercalate anions are parallel to the encasing graphene sheets. The same electrochemical synthesis strategy is also used for the preparation of a new acceptor-type GIC containing the cyclo-hexafluoropropane-1,3-bis(sulfonyl)amide anion, [CF2(CF2SO2)2N] -. The gallery heights of 0.85-0.86 nm are determined by powder X-ray diffraction for stage 2 and 3 products. These GICs are obtained by electrochemical oxidation of graphite in a nitromethane electrolyte. GICs containing the linear anion, [(CF3SO2)2N]- are also prepared in order to compare the gallery heights and the electron charge distributions that helps to understand the GIC stabilities within the graphene sheets. The compositions of GICs containing [CF2(CF 2SO2)2N]- are determined by thermogravimetric, fluorine and nitrogen elemental analyses. GICs of composition Cx[(C2F 5)3PF3] are prepared for the first time by the intercalation of tris(pentafluoroethyl)trifluorophosphate (FAP) anion, [(C 2F5)3PF3]- by electrochemical oxidation of graphite. Powder-XRD data indicate that products are of stages 2-4 with gallery heights of 0.82-0.86 nm. These GICs are characterized by the same methods using TGA and F ion-selective probe analyses.

Increased risk of recurrent thrombosis in patients with essential thrombocythemia carrying the homozygous JAK2 V617F mutation.

PubMed

De Stefano, Valerio; Za, Tommaso; Rossi, Elena; Vannucchi, Alessandro M; Ruggeri, Marco; Elli, Elena; Micò, Caterina; Tieghi, Alessia; Cacciola, Rossella R; Santoro, Cristina; Vianelli, Nicola; Guglielmelli, Paola; Pieri, Lisa; Scognamiglio, Francesca; Cacciola, Emma; Rodeghiero, Francesco; Pogliani, Enrico M; Finazzi, Guido; Gugliotta, Luigi; Leone, Giuseppe; Barbui, Tiziano

2010-02-01

Evidence suggests that the JAK2 V617F mutation is associated with an increased risk of first thrombosis in patients with essential thrombocythemia (ET). Whether this mutation is also a risk factor for recurrent thrombosis is currently unknown. To investigate the impact of the JAK2 V617F mutation on the risk of recurrent thrombosis in patients with ET, we carried out a multicentre retrospective cohort study. We recruited 143 patients with previous arterial (64.4%) or venous major thrombosis (34.8%) or both (0.8%); 98 of them (68.5%) carried the mutation. Thrombosis recurred in 43 of the patients (30%); overall, after adjustment for sex, age, presence of vascular risk factors, and treatment after the first thrombosis, the presence of the JAK2 mutation did not predict recurrence (multivariable hazard ratio, HR, 0.88, 95% CI 0.46-1.68). Indeed, the individuals homozygous for the JAK2 V617F (allele burden >50%) mutation had an increased risk of recurrence in comparison with wild-type patients (HR 6.15, 95% CI 1.51-24.92). In conclusion, a homozygous JAK2 V617F mutation is an independent risk factor for recurrent thrombosis in patients with ET.

Clinical features of Japanese polycythemia vera and essential thrombocythemia patients harboring CALR, JAK2V617F, JAK2Ex12del, and MPLW515L/K mutations.

PubMed

Okabe, Masahiro; Yamaguchi, Hiroki; Usuki, Kensuke; Kobayashi, Yutaka; Kawata, Eri; Kuroda, Junya; Kimura, Shinya; Tajika, Kenji; Gomi, Seiji; Arima, Nobuyoshi; Mori, Sinichiro; Ito, Shigeki; Koizumi, Masayuki; Ito, Yoshikazu; Wakita, Satoshi; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Dan, Kazuo; Inokuchi, Koiti

2016-01-01

The risk of complication of polycythemia vera (PV) and essential thrombocythemia (ET) by thrombosis in Japanese patients is clearly lower than in western populations, suggesting that genetic background such as race may influence the clinical features. This study aimed to clarify the relationship between genetic mutations and haplotypes and clinical features in Japanese patients with PV and ET. Clinical features were assessed prospectively among 74 PV and 303 ET patients. There were no clinical differences, including JAK2V617F allele burden, between PV patients harboring the various genetic mutations. However, CALR mutation-positive ET patients had a significantly lower WBC count, Hb value, Ht value, and neutrophil alkaline phosphatase score (NAP), and significantly more platelets, relative to JAK2V617F-positive ET patients and ET patients with no mutations. Compared to normal controls, the frequency of the JAK246/1 haplotype was significantly higher among patients with JAK2V617F, JAK2Ex12del, or MPL mutations, whereas no significant difference was found among CALR mutation-positive patients. CALR mutation-positive patients had a lower incidence of thrombosis relative to JAK2V617F-positive patients. Our findings suggest that JAK2V617F-positive ET patients and CALR mutation-positive patients have different mechanisms of occurrence and clinical features of ET, suggesting the potential need for therapy stratification in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

20 CFR 641.879 - What are the financial and performance reporting requirements for recipients?

Code of Federal Regulations, 2011 CFR

2011-04-01

... instructions for the preparation of this report. (OAA § 503(f)(3)). (1) Financial data must be reported on an accrual basis, and cumulatively by funding year of appropriation. Financial data may also be required on... for the preparation of this report. (OAA § 508). (d) In addition to the data required to be submitted...

Mutation and Evolutionary Rates in Adélie Penguins from the Antarctic

PubMed Central

Millar, Craig D.; Dodd, Andrew; Anderson, Jennifer; Gibb, Gillian C.; Ritchie, Peter A.; Baroni, Carlo; Woodhams, Michael D.; Hendy, Michael D.; Lambert, David M.

2008-01-01

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent. PMID:18833304

CLC-2 single nucleotide polymorphisms (SNPs) as potential modifiers of cystic fibrosis disease severity

PubMed Central

Blaisdell, Carol J; Howard, Timothy D; Stern, Augustus; Bamford, Penelope; Bleecker, Eugene R; Stine, O Colin

2004-01-01

Background Cystic fibrosis (CF) lung disease manifest by impaired chloride secretion leads to eventual respiratory failure. Candidate genes that may modify CF lung disease severity include alternative chloride channels. The objectives of this study are to identify single nucleotide polymorphisms (SNPs) in the airway epithelial chloride channel, CLC-2, and correlate these polymorphisms with CF lung disease. Methods The CLC-2 promoter, intron 1 and exon 20 were examined for SNPs in adult CF dF508/dF508 homozygotes with mild and severe lung disease (forced expiratory volume at one second (FEV1) > 70% and < 40%). Results PCR amplification of genomic CLC-2 and sequence analysis revealed 1 polymorphism in the hClC -2 promoter, 4 in intron 1, and none in exon 20. Fisher's analysis within this data set, did not demonstrate a significant relationship between the severity of lung disease and SNPs in the CLC-2 gene. Conclusions CLC-2 is not a key modifier gene of CF lung phenotype. Further studies evaluating other phenotypes associated with CF may be useful in the future to assess the ability of CLC-2 to modify CF disease severity. PMID:15507145