Detection of more than 50 different CFTR mutations in a large group of German cystic fibrosis patients

We have conducted a comprehensive study of the molecular basis of cystic fibrosis (CF) in 350 German CF patients. A screening approach based on single-strand conformation analysis and direct sequencing of genomic polymerase chain reaction products has allowed us to detect the molecular defects on 95.4% of the CF chromosomes within the coding region and splice sites of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The spectrum of sequence changes comprises 54 different mutations, including 17 missense mutations, 14 nonsense mutations, 11 frameshift mutations, 10 splice site variants and two amino acid deletions. Eleven of these mutations have not previously been described. Our results reflect the marked mutational heterogeneity of CF in a large sample of patients from a non-isolated population.     

Subtype-Selective Immunoprecipitation of the β2-Adrenergic Receptor

Most antibodies known to interact with beta-adrenergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1 raised against a synthetic peptide corresponding to the C-terminus of the beta 2-adrenergic receptor of hamster, is selective for the beta 2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the beta 2-adrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with beta 2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both beta 1- and beta 2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate beta 2-, but not beta 1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The beta-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycosylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.     

Absence of seasonal variation in great tit offspring sex ratios

When the timing of breeding affects the reproductive value of sons and daughters differently, parents are expected to increase their fitness by changing the offspring sex ratio during the course of the breeding season. Previous studies have shown that in great tits Parus major hatching date has a stronger effect on the fitness of juvenile males than on that of juvenile females. We tested whether this difference was reflected in a seasonal decline in the proportion of sons per breeding attempt. Although offspring sex ratio was more variable than would be expected from a binomial distribution, there was no significant relationship between the proportion of sons and the laying date of the clutch. Moreover, individual females did not adjust the sex ratio of their offspring following an experimental delay of breeding. This study therefore fails to demonstrate adaptive seasonal variation in great tit offspring sex ratios.     

Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells

Leptospira interrogans is a spirochete that is responsible for leptospirosis, a zoonotic disease. This bacterium possesses an unusual LPS that has been shown to use TLR2 instead of TLR4 for signaling in human cells. The structure of its lipid A was recently deciphered. Although its overall hexa-acylated disaccharide backbone is a classical feature of all lipid A forms, the lipid A of L. interrogans is peculiar. In this article, the functional characterization of this lipid A was studied in comparison to whole parental leptospiral LPS in terms of cell activation and use of TLR in murine and human cells. Lipid A from L. interrogans did not coagulate the Limulus hemolymph. Although leptospiral lipid A activated strongly murine RAW cells, it did not activate human monocytic cells. Results obtained from stimulation of peritoneal-elicited macrophages from genetically deficient mice for TLR2 or TLR4 clearly showed that lipid A stimulated the cells through TLR4 recognition, whereas highly purified leptospiral LPS utilized TLR2 as well as TLR4. In vitro experiments with transfected human HEK293 cells confirmed that activation by lipid A occurred only through murine TLR4-MD2 but not through human TLR4-MD2, nor murine or human TLR2. Similar studies with parental leptospiral LPS showed that TLR2/TLR1 were the predominant receptors in human cells, whereas TLR2 but also TLR4 contributed to activation in murine cells. Altogether these results highlight important differences between human and mouse specificity in terms of TLR4-MD2 recognition that may have important consequences for leptospiral LPS sensing and subsequent susceptibility to leptospirosis.     

Previtamin D3 with a trans-fused decalin CD-ring has pronounced genomic activity

Deletion of C19 in the structure of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] does not substantially alter the biological potency but prevents the conversion between the vitamin and the previtamin form. Hence, this modification allows the study of locked previtamin and vitamin forms. The locked 19-nor-1,25(OH)2-previtamin D3 analog (19-nor-previtamin D) had a low biological activity and was a rather weak activator of the genomic signal transduction pathway. 19-Nor-trans-decalin-1,25(OH)2-vitamin D3 (19-nor-TD-vitamin D), characterized by the presence of a trans-fused decalin CD-ring system, was 10-fold more potent than the parent compound and was a potent activator of the genomic signal transduction pathway. Surprisingly, the previtamin, 19-nor-trans-decalin-1,25(OH)2-previtamin D3 (19-nor-TD-previtamin D), was as potent as 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation and represents the first previtamin structure with pronounced vitamin D-like activity. Furthermore, this compound interacted as efficiently as 1,25(OH)2D3 with the vitamin D receptor (VDR), retinoid X receptor (RXR), coactivators, and DNA, which illustrated its potent ability to activate the genomic signal transduction pathway. Analysis of the transactivation potency of 12 VDR point mutants after stimulation with 19-nor-TD-previtamin D revealed that this analog used the same contact points within the receptor as did 1,25(OH)2D3. This could be confirmed by modeling analysis of this compound in the ligand binding pocket of VDR. In conclusion, a previtamin D3 analog is presented with genomic activities equivalent to 1,25(OH)2D3.     

Nest desertion: a trade-off between current and future reproduction

Life history theory predicts that parents should desert a reproductive attempt if the costs of rearing this brood exceed the expected benefits. Thus, if the value of the current breeding attempt is reduced, for example through an unexpected reduction in size, parents are expected to reconsider whether it is worth continuing investing in their brood. With regard to nest desertion behaviour two predictions can be made: individuals are (1) more likely to desert if the reduction in clutch size is large and (2) less likely to desert if the reduction is at a late stage of breeding. We investigated the threshold at which nest desertion takes place by experimentally reducing great tit, Parus major, clutches to different sizes and at different stages of the incubation period. The results were in accordance with the predictions: clutch desertion rates were negatively related to the number of eggs that remained in the nest, and nest desertion was less likely nearer the end of the incubation period. In addition, we estimated the fitness consequences of nest desertion behaviour. For this purpose we made one group of birds desert in favour of a replacement clutch and another group rear a reduced brood. The latter were more likely to produce a second clutch after the first-brood fledglings had left the nest. As a consequence, the number of fledglings produced over the entire breeding season did not differ between the two experimental groups. We also counted the number of recruits and breeding adults in the following breeding season and found that the experimental groups did not differ in local recruitment and adult survival. Therefore, the results did not indicate that parents improved their fitness by deserting a reduced clutch.Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved .     

Gene and cell therapy for heart disease

Heart disease is the most common cause of morbidity and mortality in Western society and the incidence is projected to increase significantly over the next few decades as our population ages. Heart failure occurs when the heart is unable to pump blood at a rate to commensurate with tissue metabolic requirements and represents the end stage of a variety of pathological conditions. Causes of heart failure include ischemia, hypertension, coronary artery disease, and idiopathic dilated cardiomyopathy. Hypertension and ischemia both cause infarction with loss of function and a consequent contractile deficit that promotes ventricular remodeling. Remodeling results in dramatic alterations in the size, shape, and composition of the walls and chambers of the heart and can have both positive and negative effects on function. In 30-40% of patients with heart failure, left ventricular systolic function is relatively unaffected while diastolic dysfunction predominates. Recent progress in our understanding of the molecular and cellular bases of heart disease has provided new therapeutic targets and led to novel approaches including the delivery of proteins, genes, and cells to replace defective or deficient components and restore function to the diseased heart. This review focuses on three such strategies that are currently under development: (a) gene transfer to modulate contractility, (b) therapeutic angiogenesis for the treatment of ischemia, and (c) embryonic and adult stem cell transfer to replace damaged myocardium.