Single-strand conformation polymorphism (SSCP) analysis of exon 11 of the CFTR gene reliably detects more than one third of non-ΔF508 mutations in German cystic fibrosis patients

Summary In Central Europe, the F508 deletion accounts for approximately 75% of mutations in the cystic fibrosis transmembrane conductance regulator gene causing cystic fibrosis. The remainder comprise a large number of individually infrequent mutations whose detection requires a disproportionately large effort. However, a sizeable proportion of non-F508 mutations have been found to cluster within exon 11. We have taken advantage of this clustering to detect a total of five previously described point mutations present on 26/72 (36%) non-F508 chromosomes by polymerase chain reaction/direct sequencing of exon 11. These exon 11 mutations were then subjected to single-strand conformation polymorphism (SSCP) analysis, which was shown (i) to discriminate reliably between mutant and wildtype alleles and (ii) to generate reproducible mutation-specific band patterns. This analysis thus represents the first attempt to assess SSCP analysis retrospectively, and serves to illustrate the potential of this screening technique in diagnostic medicine.     

Molecular genetic analysis of 67 patients with duchenne/becker muscular dystrophy

Summary A total of 56 Duchenne muscular dystrophy (DMD) patients and 11 Becker muscular dystrophy (BMD) patients was analyzed by extended multiplex amplification of the DMD/BMD gene; deletions were found in 60% of these patients. The data obtained were used to test the frameshift hypothesis and to compare the distribution of familial versus isolated cases. A significant correlation was found between deletions and isolated cases. Additional experiments were performed in order to determine the deletion breakpoints more precisely. These data are a prerequisite for carrier analysis in the respective families by detection or exclusion of aberrant cDNA fragments derived from ectopic lymphocyte RNA. This diagnostic technique is illustrated by 5 examples.     

The engrailed locus of Drosophila: structural analysis of an embryonic transcript

cDNA clones originating from the engrailed gene of Drosophila have been isolated from recombinant phage libraries that were made using poly(A)+ RNA extracted from early embryos. The DNA sequence of one of these clones includes a homeo box, a 180 bp sequence present in several other Drosophila genes important in formation of body pattern during development. The homeo boxes found in the other Drosophila genes, as well as in cognate sequences from a wide range of segmented animals, including higher vertebrates, are highly conserved. By contrast, the homeo box within the engrailed gene diverges substantially and, unlike the other homeo boxes, is interrupted by an intervening sequence. The engrailed homeo box is located near the 3' end of a 1700 bp open reading frame. If translated, this sequence would produce a protein of unusual composition. We also show that a neighboring gene has a large region with strong homology to engrailed, and that it also contains a homeo box.     

Preparation of Insect Chromosomes for Immunolabeling

We describe a method for isolating chromosomes from testes of the desert locust, Schistocerca gregaria, and their subsequent incubation with antibodies directed against chromosomal proteins. The procedure involves hypotonic pretreatment of the germ cells, centrifugation onto coverslips in a cytocentrifuge and immunolabeling, while still unfixed, using a chromatin-stabilizing buffer. In the present case, an antibody specific for the acetylated isoforms of his tone H4 was tested. After the antibody treatment, the preparations are fixed using formaldehyde, stained with a DNA-specific fluorescent dye and mounted. Analysis of the preparations revealed good preservation of chromosome structure in prophase spermatogonia and late prophase I spermatocytes. Fully condensed chromosomes were not observed and are probably lost during preparation. The bright fluorescence of the autosomes indicates that the reaction between the antibody against acetylated histone H4 and its chromosomal antigen is not impeded. In contrast, the X univalent remained unstained with the exception of a small terminal band. Thus, cytospin preparations of locust germ cells allow high resolution immunolabeling with antibodies against chromosome-associated proteins.     

How multisite phosphorylation impacts the conformations of intrinsically disordered proteins

Phosphorylation of intrinsically disordered proteins (IDPs) can produce changes in structural and dynamical properties and thereby mediate critical biological functions. How phosphorylation effects intrinsically disordered proteins has been studied for an increasing number of IDPs, but a systematic understanding is still lacking. Here, we compare the collapse propensity of four disordered proteins, Ash1, the C-terminal domain of RNA polymerase (CTD2’), the cytosolic domain of E-Cadherin, and a fragment of the p130Cas, in unphosphorylated and phosphorylated forms using extensive all-atom molecular dynamics (MD) simulations. We find all proteins to show V-shape changes in their collapse propensity upon multi-site phosphorylation according to their initial net charge: phosphorylation expands neutral or overall negatively charged IDPs and shrinks positively charged IDPs. However, force fields including those tailored towards and commonly used for IDPs overestimate these changes. We find quantitative agreement of MD results with SAXS and NMR data for Ash1 and CTD2’ only when attenuating protein electrostatic interactions by using a higher salt concentration (e.g. 350 mM), highlighting the overstabilization of salt bridges in current force fields. We show that phosphorylation of IDPs also has a strong impact on the solvation of the protein, a factor that in addition to the actual collapse or expansion of the IDP should be considered when analyzing SAXS data. Compared to the overall mild change in global IDP dimension, the exposure of active sites can change significantly upon phosphorylation, underlining the large susceptibility of IDP ensembles to regulation through post-translational modifications.     

Clinical-scale generation of multi-specific anti-fungal T cells targeting Candida,Aspergillus and mucormycetes

Background aimsInvasive fungal infections, in particular, infections caused by Candida, Aspergillus and mucormycetes, are a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation. Adoptive transfer of donor-derived anti-fungal T cells shows promise to restore immunity and to offer a cure. Because T cells recognize only specific epitopes, the low rate of patients in which the causal fungal pathogen can be identified and the considerable number of patients with co-infection with several genera or species of fungi significantly limit the application of adoptive immunotherapy.MethodsUsing the interferon-γ secretion assay, we isolated multi-specific human anti-fungal T cells after simultaneous stimulation with cellular extracts of Aspergillus fumigatus, Candida albicans and Rhizopus oryzae. Cells were phenotypically and functionally characterized by flow cytometry.ResultsOf a total of 1.1 × 10⁹ peripheral blood mononuclear cells, a median number of 5.2 × 10⁷ CD3⁺CD4⁺ T cells was generated within 12 days. This cell population consisted of activated memory T_H1 cells and reproducibly responded to a multitude of Aspergillus spp., Candida spp. and mucormycetes with interferon-γ production. On re-stimulation, the generated T cells proliferated and enhanced anti-fungal activity of phagocytes and showed reduced alloreactivity compared with the original cell fraction.ConclusionsOur rapid and simple method of simultaneously generating functionally active multi-specific T cells that recognize a wide variety of medically relevant fungi may form the basis for future clinical trials investigating adoptive immunotherapy in allogeneic hematopoietic stem cell transplantation recipients with invasive fungal infection.     

Odor Qualities and Thresholds of Physiological Metabolites of 1,8‐Cineole as an Example for Structure?Activity Relationships Considering Chirality Aspects

The present study aimed at analyzing the odor properties of a group of physiological human metabolites of the odorant 1,8‐cineole: 2,3‐dehydro‐, α2,3‐epoxy‐, α/β2‐hydroxy‐, α3‐hydroxy‐, 4‐hydroxy‐, 7‐hydroxy‐, 9‐hydroxy‐, 2‐oxo‐, and 3‐oxo‐1,8‐cineole. These metabolites constitute a group of structurally closely related molecules, which differ mainly in nature and position of O‐containing functional groups. They thus offer the possibility to correlate odor properties with molecular structure, i.e., to establish structure? odor relationships of compounds that are biologically generated from a potent odorant as parent substance. Generally, the metabolites preserved the eucalyptus‐like odor quality of 1,8‐cineole but showed additional odor notes such as sweet, citrus‐like, plastic‐like, earthy, musty, and faecal, which made them distinguishable. The individual enantiomers of chiral molecules also exhibited different odors. With the exception of 2,3‐dehydro‐1,8‐cineole, all metabolites showed a highly decreased odor threshold in comparison to 1,8‐cineole. The determination of odor qualities and odor thresholds was accomplished by gas chromatography/olfactometry (GC/O) on achiral and chiral GC capillaries. The results were correlated with common theories on structure? odor relationships.     

Low genetic diversity of a high mountain burnet moth species in the Pyrenees

The burnet moth Zygaena anthyllidis, endemic to the high elevations of the Pyrenees, is vulnerable to land-use. In order to identify conservation priorities based on an assessment of genetic diversity within populations and gene flow among populations, we examined Z. anthyllidis’ genetic variability and differentiation based on allozyme electrophoresis from seven populations scattered across its entire range. In comparison to other mountain Lepidoptera, the populations studied exhibit a low level of genetic diversity. Remarkable between-population differentiation (F _ST = 0.053), the presence of private alleles, and the lack of significant isolation-by-distance pattern characterises the genetic make-up of the species. We interpreted the pattern of genetic differentiation as a consequence of low dispersal power in combination with insufficient landscape connectivity. Ongoing land-use change might reinforce genetic differentiation due to habitat fragmentation and additionally affect negatively allozyme variability at shifting range margins, i.e. the capacity to adapt to changing environments. We therefore suggest creating a network of suitable habitats at the landscape scale to facilitate genetic exchange and to conserve the species’ overall genetic variability.