Ion-Ion interactions at the selectivity filter. Evidence from K(+)-dependent modulation of tetraethylammonium efficacy in Kv2.1 potassium channels

In the Kv2.1 potassium channel, binding of K(+) to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na(+) current, K(+) interacts with the TEA modulation site at concentrations 相似文献   

Analysis of the RNase T1 mediated cleavage of an immobilized gapped heteroduplex via fluorescence correlation spectroscopy

We report a new method for studying the activity of hydrolytic enzymes. Fluorescence correlation spectroscopy was used to observe online the hydrolyzation of a rhodamine B-labeled substrate by ribonuclease T1. A gapped heteroduplex substrate - a hybrid of a ribooligonucleotide and two smaller complementary deoxyribooligonucleotides - was immobilized via biotin to a streptavidin-coated surface of a coverslip. The reported method opens the possibility to study the cleavage of small substrates differing only slightly in molecular weight from the enzyme reaction product. The use of fluorescence correlation spectroscopy allows the detection of very low enzyme concentrations (down to 10(-21) mol 0.05 fM of RNase T1, corresponding to about 600 RNase T1 molecules in 0.02 ml).     

Phage display of RNase A and an improved method for purification of phages displaying RNases

Functional ribonuclease A was presented on the surface of the filamentous phage M13 by fusion to the minor coat protein. RNase activity of the fusion protein was shown by a zymogram assay. In addition, we established a modified method for preparing RNase-displaying phages without contaminating host RNases.     

Common genetic variants and modification of penetrance of BRCA2-associated breast cancer

The considerable uncertainty regarding cancer risks associated with inherited mutations of BRCA2 is due to unknown factors. To investigate whether common genetic variants modify penetrance for BRCA2 mutation carriers, we undertook a two-staged genome-wide association study in BRCA2 mutation carriers. In stage 1 using the Affymetrix 6.0 platform, 592,163 filtered SNPs genotyped were available on 899 young (<40 years) affected and 804 unaffected carriers of European ancestry. Associations were evaluated using a survival-based score test adjusted for familial correlations and stratified by country of the study and BRCA2*6174delT mutation status. The genomic inflation factor (λ) was 1.011. The stage 1 association analysis revealed multiple variants associated with breast cancer risk: 3 SNPs had p-values<10(-5) and 39 SNPs had p-values<10(-4). These variants included several previously associated with sporadic breast cancer risk and two novel loci on chromosome 20 (rs311499) and chromosome 10 (rs16917302). The chromosome 10 locus was in ZNF365, which contains another variant that has recently been associated with breast cancer in an independent study of unselected cases. In stage 2, the top 85 loci from stage 1 were genotyped in 1,264 cases and 1,222 controls. Hazard ratios (HR) and 95% confidence intervals (CI) for stage 1 and 2 were combined and estimated using a retrospective likelihood approach, stratified by country of residence and the most common mutation, BRCA2*6174delT. The combined per allele HR of the minor allele for the novel loci rs16917302 was 0.75 (95% CI 0.66-0.86, ) and for rs311499 was 0.72 (95% CI 0.61-0.85, ). FGFR2 rs2981575 had the strongest association with breast cancer risk (per allele HR = 1.28, 95% CI 1.18-1.39, ). These results indicate that SNPs that modify BRCA2 penetrance identified by an agnostic approach thus far are limited to variants that also modify risk of sporadic BRCA2 wild-type breast cancer.     

Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.     

A posterior tale

A vignette is presented that naively suggests that the posterior distribution of a parameter may not always contain everything that is needed for inference about that parameter. The resolution of this apparent paradox is discussed as well as its relation to real-life problems involving data monitoring of clinical trials.     

A systematic approach for testing expression of human full-length proteins in cell-free expression systems

Background  

The growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems.