Putative identification of an amphipathic alpha-helical sequence in hemolysin of Escherichia coli (HlyA) involved in transmembrane pore formation

Abstract Escherichia coli hemolysin is a pore-forming protein belonging to the RTX toxin family. Cysteine scanning mutagenesis was performed to characterize the putative pore-forming domain of the molecule. A single cysteine residue was introduced at 48 positions within the sequence spanning residues 170-400 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that several amino acids in this domain are inserted into the lipid bilayer during pore formation. An amphipathic alpha-helix spanning residues 272-298 was identified that may line the aqueous pore. The importance of this sequence was highlighted by the introduction of two prolines at positions 284 and 287. Disruption of the helix structure did not affect binding properties, but totally abolished the hemolytic activity of the molecule.     

Effect of creatine phosphate supplementation on anaerobic working capacity and body weight after two and six days of loading in men and women

The purpose of this study was to determine the effects of 2 and 6 days of creatine phosphate loading on anaerobic working capacity (AWC) and body weight (BW) in men and women. Sixty-one men (n = 31) and women (n = 30) randomly received 1 of 3 treatments (4 x 5 g.d(-1) x 6 days) using a double blind design: (a) 18 g dextrose as placebo (PL); (b) 5.0 g Cr + 20 g dextrose (Cr); or (c) 5.0 g Cr + 18 g dextrose + 4 g of sodium and potassium phosphates (CrP). AWC was determined at baseline and following 2 and 6 days of supplementation using the Critical Power Test. BW increased significantly over time, and the mean value for the men was significantly greater compared to that for women, but there were no interactions (p > 0.05). There were gender-specific responses for AWC expressed in both absolute values (kJ) and relative to BW (kJ. kg(-1)), with the women demonstrating no significant interactions. For the men, CrP loading significantly increased AWC following 2 days (23.8%) and 6 days (49.8%) of supplementation vs. PL (kJ and kJ.kg(-1)). Cr supplementation increased AWC 13-15% in both genders compared to PL (1.1%- 3.0% decline); although this result was not statistically significant, it may have some practical significance.     

A novel claudin 16 mutation associated with childhood hypercalciuria abolishes binding to ZO-1 and results in lysosomal mistargeting

Mutations in the gene coding for the renal tight junction protein claudin 16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, an autosomal recessive disorder of renal Ca(2+) and Mg(2+) handling that progressively leads to chronic renal failure, with nephrolithiasis having been reported in heterozygous carriers. Screening a cohort of 11 families with idiopathic hypercalciuria identified a novel homozygous mutation in the claudin 16 gene in two families. In contrast to classical symptoms of familial hypomagnesemia with hypercalciuria and nephrocalcinosis, the patients displayed serious but self-limiting childhood hypercalciuria with preserved glomerular filtration rate. The mutation results in inactivation of a PDZ-domain binding motif, thereby disabling the association of the tight junction scaffolding protein ZO-1 with claudin 16. In contrast to wild-type claudin 16, the mutant no longer localizes to tight junctions in kidney epithelial cells but instead accumulates in lysosomes. Thus, mutations at different intragenic sites in the claudin 16 gene may lead to particular clinical phenotypes with a distinct prognosis. Mutations in claudin 16 that affect interaction with ZO-1 lead to lysosomal mistargeting, providing-for the first time, to our knowledge-insight into the molecular mechanism of a disease-associated mutation in the claudin 16 gene.     

The passage of an ion through a synthetic ion channel

The simulated system consisted of a fatty acid bilayer membrane dividing two electrolyte layers each containing ions, and a channel composed of linked 15-crown-5 ether rings. The Na⁺ and F^?  ions in the aqueous electrolyte layers were too large to enter the channel, but the Li⁺ ions entered and were transported. Conditions that optimised the passive, electric-field-induced transport of Li⁺ ions through the channel were investigated. It was calculated and rationalised that the higher the numerical value of the electrostatic charge on the oxygen atoms of the crown ether rings, the more easily does the channel convey the Li⁺ ions.     

A novel microarray approach reveals new tissue-specific signatures of known and predicted mammalian microRNAs

Microarrays to examine the global expression levels of microRNAs (miRNAs) in a systematic in-parallel manner have become important tools to help unravel the functions of miRNAs and to understand their roles in RNA-based regulation and their implications in human diseases. We have established a novel miRNA-specific microarray platform that enables the simultaneous expression analysis of both known and predicted miRNAs obtained from human or mouse origin. Chemically modified 2′-O-(2-methoxyethyl)-(MOE) oligoribonucleotide probes were arrayed onto Evanescent Resonance (ER) microchips by robotic spotting. Supplementing the complementary probes against miRNAs with carefully designed mismatch controls allowed for accurate sequence-specific determination of miRNA expression profiles obtained from a panel of mouse tissues. This revealed new expression signatures of known miRNAs as well as of novel miRNAs previously predicted using bioinformatic methods. Systematic confirmation of the array data with northern blotting and, in particular, real-time PCR suggests that the described microarray platform is a powerful tool to analyze miRNA expression patterns with rapid throughput and high fidelity.