Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators

W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (P_O) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the P_o of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.     

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

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 cystic 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. Together with the delta F508 (23% in this group), G542X, N1303K, and 1717-1G----A mutations, the identification of 92% of cystic fibrosis chromosomes of Ashkenazi origin becomes possible. Patients homozygous for the W1282X mutation (n = 16) and patients heterozygous for the delta F508 and W1282X mutations (n = 22) had similarly severe disease, reflected by pancreatic insufficiency, high incidence of meconium ileus (37% and 27%, respectively), early age at diagnosis, poor nutritional status, and variable pulmonary function. 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.     

Effect of lipopolysaccharide mutations and temperature on plasmid transformation efficiency in Pseudomonas aeruginosa

We have isolated, and characterized electrophoretically, two new lipopolysaccharide-defective (rough) mutants of Pseudomonas aeruginosa strain PAO. These strains, AK1401 and AK1414, together with two previously characterized isolates, AK1012 and AK1282, were used as recipients in transformation experiments with plasmid pR01614 DNA. The roughest mutant, AK1282, was not transformable, while the transformation efficiency of AK1012, and to a lesser extent the wild-type strain, was dependent upon the growth temperature. The two new isolates which are less rough than AK1012 were transformed at a frequency equivalent to that of the wild type-strain.     

切割HPV-6bE1和HPV-11E1通用核酶Rz 1282的体外活性鉴定

利用计算机分析 HPV- 6b E1和 HPV- 1 1 E1 m RNA的同源序列 ,设计出通用于两者的锤头状核酶—— Rz1 2 82 (HPV- 6b基因 1 2 82位 ) ,通过体外转录建立了体外大量制备 Rz1 2 82的方法 .体外的切割实验表明 ,Rz1 2 82可在体外准确和有效地切割 HPV- 6b/1 1 E1靶 RNA,形成 2 68nt/52nt和 2 31 nt/52 nt大小的切割产物 .对于 HPV- 6b,Km和 kcat值分别为 1 3.8nmol/L和 0 .0 7min-1;对于 HPV- 1 1 ,Km 和 kcat值分别为 2 3.0 nmol/L和 0 .2 4 min-1.结果表明 ,体外制备的 Rz1 2 82具有较好的特异催化切割活性 ,并通用于 HPV- 6b及 HPV- 1 1 .它有望发展成为在细胞内有效抑制HPV- 6b/1 1 DNA复制的核酸药物 .     

Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and predicts a carrier frequency of 1:29 in the Jewish Ashkenazi population.

To determine the distribution and frequency of cystic fibrosis (CF) mutations in the Israeli population, we have screened 96 patients for 11 relatively common mutations. Five mutations--delta F508, G542X, W1282X, N1303K, and 3849 + 10kb C-->T--were found to account for 97% of the CF alleles in the Ashkenazi Jews. In contrast, of the 11 mutations tested, only delta F508 was detected in Jewish patients of Sephardic or Oriental origin, accounting for 43% of the CF alleles. Four mutations--delta F508, G542X, W1282X, and N1303K--accounted for 55% of the CF alleles in Arab patients. In a pilot screening study, a random sample of 424 Ashkenazi individuals was analyzed for three mutations--delta F508, W1282X, and G542X. Thirteen individuals were detected as heterozygotes (six for delta F508 and seven for W1282X), predicting a heterozygote frequency of 1:29. This is similar to the frequency of carriers in the Caucasian population of northern European ancestry. On the basis of these data, the Ashkenazi population is considered to be a candidate for CF heterozygote screening.     

Pseudonocardia nantongensis sp. nov., a novel endophytic actinomycete isolated from the coastal halophyte Tamarix chinensis Lour

A novel isolate, designated strain KLBMP 1282^T was isolated from the surface-sterilized leaves of a coastal halophyte Tamarix chinensis Lour., collected from Nantong, Jiangsu Province, east of China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that this strain belongs to the genus Pseudonocardia, being most closely related to Pseudonocardia kongjuensis LM 157^T (98.33 %), Pseudonocardia autotrophica IMSNU 20050^T (97.77 %), Pseudonocardia endophytica YIM 56035^T (97.63 %), Pseudonocardia ammonioxydans H9 ^T (97.62 %) and Pseudonocardia compacta IMSNU 20111^T (97.56 %); similarity to other type strains of the genus Pseudonocardia was <97.5 %. Chemotaxonomic data confirmed the affiliation of strain KLBMP 1282^T to the genus Pseudonocardia. Strain KLBMP 1282^T contained MK-8(H₄) as the predominant ubiquinone and iso-C_16:0 as the major fatty acid. The polar lipids detected in strain KLBMP 1282^T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, one unknown phospholipid and four unknown glycolipids. The DNA G + C content of strain KLBMP 1282^T was 73.1 mol %. The results of DNA–DNA hybridizations and the phylogenetic analysis, together with the phenotypic and biochemical tests, allowed the differentiation of strain KLBMP 1282^T from strains of other recognized Pseudonocardia species. Therefore, strain KLBMP 1282^T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia nantongensis sp. nov. is proposed. The type strain is KLBMP 1282^T (=KCTC 29053^T = NBRC 108677^T).     

Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor

Peptide nucleic acids (PNAs) containing an insert of three chiral monomers based on D-lysine ('chiral box') were synthesized and used as probes in Biospecific Interaction Analysis (BIA) for the recognition of DNA containing the W1282X point mutation of the cystic fibrosis gene. Hybridization experiments carried out in solution showed enhanced mismatch recognition when compared with the analogous achiral PNAs and oligonucleotides. The signal intensity was lower, but the selectivity of the Biacore response was found to be much higher than that observed with achiral PNAs. The newly designed chiral PNA probes were also found to hybridize with a 1:1 mixture of normal (N-W1282X) and mutated (M-W1282X) DNA oligomers immobilized on the biosensor, thus allowing discrimination not only between a normal and a mutated sequence (healthy/homozygous), but also between homo- and heterozygous individuals. These results suggest that 'chiral box' PNAs are potential powerful tools for the analysis of single point mutations of biological/biomedical relevance.     

Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the DeltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene

A satisfactory model describing the airway surface fluid (ASF) in the airways of persons with cystic fibrosis (CF) remains to be established due to theoretical challenges to both the "Hydration Hypothesis" and the "Salt Hypothesis." Irrespective of these models, inhaled hypertonic saline is often used to facilitate clearance of inspissated secretions. Hypertonicity induces interleukin-8 (IL-8) expression, a potent chemokine for neutrophils. The objectives of this study were: (i) to determine the relative contribution of three potential cis-regulatory elements in the regulation of NaCl-induced IL-8 production in BEAS-2B human bronchial epithelial cells, (ii) to compare NaCl-induced IL-8 expression in IB3-1 bronchial epithelial cells, which have the DeltaF508/W1282X mutation of the CF transmembrane conductance regulator (CFTR) gene, with that in C38 cells, which are IB3-1 cells stably transfected with a truncated but functional CFTR gene, and (iii) to compare equal osmolar concentrations of NaCl and D-sorbitol in the induction of IL-8 in all three cell types. In human bronchial epithelial cells, binding sites for NFkappaB, AP-1, and NF-IL6 in the 5'-flanking region of the IL-8 promoter are necessary for optimal NaCl induction of IL-8. Human bronchial epithelial cells with the DeltaF508/W1282X CFTR mutation produce an exaggerated amount of basal and NaCl-induced IL-8.     

Heart attacks, heart rate, and gel electrodes: the invention of ambulatory cardiology

Ambulatory cardiology began in 1959 in a department of pathology to answer a question raised at the autopsy table: are high heart rates in apparently healthy individuals a risk factor for developing coronary artery disease? This question led to the development of a miniature monitor and a new kind of electrode, which enabled clinicians to measure EKG signals during activity and over prolonged periods of time. These electrodes are now used universally for diagnosis and for monitoring the heart during a myriad of different activities and circumstances. The story of the development of the monitor and electrodes illustrates the ways in which ideas and discovery lead to applications and advances in medicine.     

Role for p21-activated kinase PAK4 in development of the mammalian heart

The serine-threonine kinase PAK4 plays a pivotal role in cell proliferation, survival, and control of the cytoskeleton. Mice that lack Pak4 die in midgestation prior to embryonic day E11 from unidentified causes. Analysis of PAK4 protein levels demonstrated that it was highly expressed in the whole embryo and in the developing heart but became low in the hearts of adult mice. In this study we analyzed development of the heart in conventional and conditional Pak4 knockout mice and embryos. We found that in conventional Pak4 knockout mice cardiogenesis is strongly affected from early developmental stages and by E9.5, hearts of Pak4?/? embryos developed multiple profound deficits. Conditional deletion of Pak4 in the progenitors of the secondary heart field led to abnormal development of the outflow tract, in which the pulmonary artery had a smaller diameter, and the aortal wall was thinner than in wildtype mice. The conditional knockout mice also displayed the characteristic enlargement of the right ventricles and right atria. Pak4?/? embryos and cardiomyocytes in which PAK4 was depleted exhibited low levels of LIMK1, a protein that plays key roles in cytoskeletal organization. Knock down of PAK4 in cultured cardiomyocytes led to severely compromised sarcomeric structure and deficits in contraction. These results indicate that PAK4 functions, including control of actin dynamics, are necessary for normal development of the heart.     

Stoffwechselprodukte von Mikroorganismen

Zusammenfassung Bei Neurospora crassa, arg-5, ota, aga wird die Desferricoprogenbildung durch Zusatz von Fe³⁺, Ga³⁺, Al³⁺ und V³⁺ im Medium unterdrückt. Bei Anwesenheit von Cr³⁺ und Co³⁺ verhält sich die Mutante wie unter Eisenmangelbedingungen. Eine angelaufene Coprogenbiosynthese wird innerhalb von 24 h mit Fe-Coprogen (10 M), Ga-Coprogen (10 M) und Al-Coprogen (>100 M) reprimiert. Die Coprogenaufnahme entspricht einer Sättigungskinetik, die Aufnahme von Desferricoprogen und Fe³⁺ folgt dagegen einer Diffusionsgeraden. Die verschiedenen Metall-Coprogene verhalten sich bei der Aufnahme kompetitiv und werden nach folgender Affinitätsreihe angereichert: Ga>Fe>Al>V>Cr>Co. Die gleiche Reihenfolge wird eingehalten bei der Regulation der Desferricoprogenbiosynthese. Ein Modell für die Aufnahme, das auf der Stabilität der Metall-Coprogene basiert, wird vorgeschlagen.

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Metabolic products of microorganisms120. Uptake of iron by Neurospora crassa II. Regulation of the biosynthesis of sideramines and inhibition of iron transport by metal analogues of coprogen

Summary The production of desferricoprogen in Neurospora crassa, arg-5, ota, aga is suppressed by addition of Fe³⁺, Ga³⁺, Al³⁺, and V³⁺ to the medium. In the presence of Cr³⁺ and Co³⁺, the mutant behaves as under iron-deficient conditions. Once started, the biosynthesis of coprogen is suppressed within 24 h by Fe-Coprogen (10 M), Ga-Coprogen (10 M), and Al-Coprogen (>100 M). The uptake of Coprogen corresponds to a saturation kinetic, whereas the uptake of desferricoprogen and Fe³⁺ is in accordance with a diffusion line. The different metal analogues of coprogen exhibit competitive behavior during the uptake, and are concentrated by the cells in the following order of affinity: Ga>Fe>Al>V>Cr>Co, which seems to be the same sequence in the regulation of the desferricoprogen biosynthesis. A model for uptake, based on the stability of the metal coprogens, is proposed.

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119. Mitt.: Schindler, P.W., Zähner, H.: Europ. J. Biochem. (im Druck, 1973).     

Role of molecular biology in understanding the formation of congenital cardiomyopathies in humans

New insights into the genetics of congenital heart diseases in human beings have been obtained these past years. The identification of genes for heart defects have led to a new clinical approach of these malformations in children and their families. These progresses have been made by means of positional cloning as well as through the analysis of mouse models. These findings uncovered a new complexity in the development of cardiac defects and led to revise the different classifications for congenital heart defects. Pediatric cardiologists have also improved their efficiency in defining cardiac phenotypes in affected individuals and in pedigrees with recurrent malformations. At the present time, clinical consequences of these findings remain sparse, though in particular cases genetic counselling has been dramatically improved.     

The mouse as a model for heart morphogenesis in mammals: the origin of myocytes and studies with cardiac explants

The heart is one of the first organs to form during embryogenesis since its circulatory function is critical from early stages for embryo survival. In man, morphological events are affected by molecular perturbations, which can lead to a congenital heart defect. It is important therefore to understand not only the molecular signals, but also the morphological events, which govern myocardial cell identity. The study of transgenic mouse lines, Mlc1v-nlacZ-24 and Mlc3f-nlacZ-2, has led to the identification of a new precardiac territory, the anterior heart field, which has also been described recently in birds, and which contributes to the myocardium of the arterial pole of the heart. The use of explant cultures also indicates that pharyngeal mesoderm participates in the formation of the outflow tract and right ventricle and shows that the primitive heart tube has a predominantly left ventricular identity. We have also shown that Fgf-10 is expressed in the anterior heart field, where a role for FGF signaling in arterial pole morphogenesis is suggested by inhibitor experiments. Finally explant cultures have been employed to examine the acquisition of left-right atrial identity. The Mlc3f-nlacZ-2 line, which marks the right atrium, allowed us to determine the time window during which left-right signaling confers left-right atrial identity.     

Clenbuterol induced changes in cholesterol and triglyceride levels of gastrocnemius, pectoralis and heart of rat under work induced stress

Work induced stress led to decreased cholesterol and fluctuating triglyceride levels in gastrocnemius and pectoralis muscles in rats. But the drug (clenbuterol, 2 mg kg(-1) day(-1)) treatment increased cholesterol and triglyceride levels in both the muscles. However, heart showed decreased cholesterol and increased triglyceride level in the animals under work stress, but at the same time drug treatment led to a significant increase in levels of the two lipid fractions, inferring towards deleterious effect of the drug on heart.     

Effect of emotional and pain stress on the contractile function of the hypertrophied heart muscle

Rats with compensatory hypertrophy of the heart and control animals were subjected to emotional painful stress (EPS). It was established that EPS led to the lowering of the main indicators of the contractile function of an isolated papillary muscle and reduced the resistance of the function under study to excess/Na+ and H+ forcing out Ca2+ from the binding sites on the sarcolemma. Compensatory hypertrophy of the heart itself was accompanied by a reduction of the myocardial contractility but the increase of the concentration of Na+ and H+ in the perfusate led to a far greater depression of the contractile parameters than in the myocardium of the control animals. Contractile function of the hypertrophied myocardium after stress turned out to be reduced to the level close to that seen in heart insufficiency.     

Aortic banding in rat as a model to investigate malnutrition associated with heart failure

Heart failure is a severe pathology, which has displayed a dramatic increase in the occurrence of patients with chronic heart disease in developed countries, as a result of increases in the population's average age and in survival time. This pathology is associated with severe malnutrition, which worsens the prognosis. Although the cachexia associated with chronic heart failure is a well-known complication, there is no reference animal model of malnutrition related to heart failure. This study was designed to evaluate the nutritional status of rats in a model of loss of cardiac function obtained by ascending aortic banding. Cardiac overload led to the development of cardiac hypertrophy, which decompensates to heart failure, with increased brain natriuretic peptide levels. The rats displayed hepatic dysfunction and an associated renal hypotrophy and renal failure, evidenced by the alteration in renal function markers such as citrullinemia, creatininemia, and uremia. Malnutrition has been evidenced by the alteration of protein and amino acid metabolism. A muscular atrophy with decreased protein content and increased amino acid concentrations in both plasma and muscle was observed. These rats with heart failure displayed a multiorgan failure and malnutrition, which reflected the clinical situation of human chronic heart failure.     

Role of nitric oxide in heart failure

The benefit effects of nitric oxide (NO) donors in acute heart failure have led to the development of vasodilators as treatment of chronic heart failure. However, the mechanisms involved in the effects of NO are complex and still discussed. In chronic heart failure, the eNOS downregulation in vascular endothelium explains the alteration of endothelial function. In addition, in the myocardium, cytokines induce the expression of inducible nitric oxide synthase (iNOS) which increase NO production by myocytes and surrounding cells. This excess of NO production, associated with anion superoxide synthesis, limits the inotropic properties of catecholamines and exert proapoptotic effects. The role of NO donors in heart failure treatment is still controversial but by reducing preload they improve patient's symptoms. Beside blockade of the renin-angiotensin system, the angiotensin converting enzyme inhibitors act via the inhibition of bradykinin degradation which increase NO levels. Finally, vascular endothelial NO expression is improved by exercise training and participates in the improvement of exercise capacity in patients with chronic heart failure involved in cardiac readaptation program.     

Immunologic identification of a glycogen synthase 93,000-dalton subunit from rat heart and liver

A polyclonal sheep antibody to rat heart glycogen synthase has been used for immunoblot analysis and immunoprecipitation of both rat heart and liver synthase. The purified antibody completely inhibits glycogen synthase activity in rat heart preparations and specifically blots to a 93-kDa band in the 10,000 X g supernatants of both heart and liver homogenates. Immunoprecipitation of in vitro translation products from rat heart or liver poly(A+) RNA yields a unique band with a molecular mass of 93 kDa. Thus the subunit molecular mass of active glycogen synthase in rat heart is 93 kDa. In rat liver at least one form of glycogen synthase also appears to have a molecular mass of 93 kDa. Protocols used to purify rat liver synthase yield a subunit of 80-87 kDa, which retains activity, but which is no longer recognized by the antibody. This suggests that 1) a specific antigenic sequence has been proteolytically removed from the NH2 or COOH terminus of the protein, or 2) that limited proteolysis has led to a conformational change in the enzyme such that the antibody binding site is no longer recognized. Either or both of these possibilities represent a significant alteration in the enzyme due to proteolysis. In vitro studies using synthase preparations having molecular masses less than 93 kDa must be interpreted with caution due to possible structural changes which occur during purification which may alter the regulation or covalent modification of synthase.     

Peptidergic control of the heart of the stick insect, Baculum extradentatum

The dorsal vessel of the Vietnamese stick insect, Baculum extradentatum, consists of a tubular heart and an aorta that extends anteriorly into the head. Alary muscles, associated with the heart, are anchored to the body wall with attachments to the dorsal diaphragm. Alary muscle contraction draws haemolymph into the heart through incurrent ostia. Excurrent ostia lie on the dorsal vessel in the last thoracic and in each of the first two abdominal segments. Muscle fibers are associated with these excurrent ostia. Crustacean cardioactive peptide (CCAP)- and proctolin-like immunoreactivity is present in axons of the segmental nerves that project to the dorsal vessel, and in processes extending over the heart and alary muscles. Proctolin-like immunoreactive processes are also localized to the valves of the incurrent ostia and to the excurrent ostia. Neither the link nerve neurons, nor the lateral cardiac neurons, stain positively for these peptides. Physiological assays reveal dose-dependent increases in heart beat frequency in response to CCAP and proctolin. Isolating the dorsal vessel from the ventral nerve cord led to a change in the pattern of heart contractions, from a tonic, stable heart beat, to one which was phasic. The tonic nature was restored by the application of CCAP.