Transduction of envelope stress in Escherichia coli by the Cpx two-component system

Disruption of normal protein trafficking in the Escherichia coli cell envelope (inner membrane, periplasm, outer membrane) can activate two parallel, but distinct, signal transduction pathways. This activation stimulates the expression of a number of genes whose products function to fold or degrade the mislocalized proteins. One of these signal transduction pathways is a two-component regulatory system comprised of the histidine kinase CpxA and the response regulator, CpxR. In this study we characterized gain-of-function Cpx* mutants in order to learn more about Cpx signal transduction. Sequencing demonstrated that the cpx* mutations cluster in either the periplasmic, the transmembrane, or the H-box domain of CpxA. Intriguingly, most of the periplasmic cpx* gain-of-function mutations cluster in the central region of this domain, and one encodes a deletion of 32 amino acids. Strains harboring these mutations are rendered insensitive to a normally activating signal. In vivo and in vitro characterization of maltose-binding-protein fusions between the wild-type CpxA and a representative cpx* mutant, CpxA101, showed that the mutant CpxA is altered in phosphotransfer reactions with CpxR. Specifically, while both CpxA and CpxA101 function as autokinases and CpxR kinases, CpxA101 is devoid of a CpxR-P phosphatase activity normally present in the wild-type protein. Taken together, the data support a model for Cpx-mediated signal transduction in which the kinase/phosphatase ratio is elevated by stress. Further, the sequence and phenotypes of periplasmic cpx* mutations suggest that interactions with a periplasmic signaling molecule may normally dictate a decreased kinase/phosphatase ratio under nonstress conditions.     

High-level genetic diversity in the vapD chromosomal region of Helicobacter pylori

Helicobacter pylori isolates from different patients are characterized by diversity in the nucleotide sequences of individual genes, variation in genome size, and variation in gene order. Genetic diversity is particularly striking in vacuolating cytotoxin (vacA) alleles. In this study, five open reading frames (ORFs) were identified within a 4.2-kb region downstream from vacA in H. pylori 60190. One of these ORFs was closely related to the virulence-associated protein D (vapD) gene of Dichelobacter nodosus (64.9% nucleotide identity). A probe derived from vapD of H. pylori 60190 hybridized with only 19 (61.3%) of 31 H. pylori strains tested. Sequence analysis of the vapD region in vapD-negative H. pylori strains revealed that there were two different families of approximately 0.5-kb DNA segments, which were both unrelated to vapD. The presence of vapD was not associated with any specific family of vacA alleles. These findings are consistent with a recombinational population structure for H. pylori.     

Glycosylation is essential for functional expression of a human brain ecto-apyrase

The importance of N-linked glycosylation for the function and oligomerization of an E-type ATPase was examined by using tunicamycin and peptide N-glycosidase F, two agents used to prevent and remove glycosylations, respectively. The cDNA encoding a human ecto-apyrase (HB6), predicted to have seven N-linked glycosylation sites, was transiently expressed in mammalian COS cells and the resulting membrane preparations were treated with peptide N-glycosidase F (PNGase-F). PNGase-F caused a decrease in the apparent molecular weight of the protein (consistent with glycan removal) and a decrease in enzymatic activity over time. The ecto-apyrase was also expressed in the presence of tunicamycin, which completely prevented N-linked glycosylation, resulting in a nonglycosylated core protein devoid of ATP and ADP hydrolyzing activity. However, control and tunicamycin-treated cells expressed the enzyme to similar levels and localization. Interestingly, the quaternary structure of this E-type ATPase appears to be dependent upon the presence of glycan chains. The glycosylated ecto-apyrase exists as a homodimer in situ as assessed by both size-exclusion chromatography of detergent-solubilized ecto-apyrase and cross-linking of membrane-bound ecto-apyrase, in contrast to the enzymatically deglycosylated ecto-apyrase and the tunicamycin-treated ecto-apyrase. These results suggest that glycosylation is necessary for homooligomerization and nucleotide hydrolyzing activity, but not for expression and plasma membrane localization of the E-type ATPase. Similar results were obtained with another human ecto-apyrase, CD39, suggesting that the importance of glycosylation may be generalized to all membrane-bound E-type ATPases.     

Evaluation of energy and dietary intake estimates from a food frequency questionnaire using independent energy expenditure measurement and weighed food records

Background  

We have developed a food frequency questionnaire (FFQ) for the assessment of habitual diet, with special focus on the intake of fruit, vegetables and other antioxidant-rich foods and beverages. The aim of the present study was to evaluate the relative validity of the intakes of energy, food and nutrients from the FFQ.     

Candida albicans sensitization in patients with atopic bronchial asthma and atopic dermatitis

Candida albicans, a component of normal human microflora, can induce synthesis of specific IgE-antibodies in patients with atopic bronchial asthma and atopic dermatitis. The study included 25 patients with atopic dermatitis sensitized to C.albicans and 23 patients with atopic dermatitis non-sensitized to C.albicans. The sensitization was determined by the skin test and enzyme immunoassay. The patients had the history of atopic dermatitis exacerbation after taking food containing baking yeasts. Atopic dermatitis with sensitization to C.albicans is characterized by severe course correlating with the following indices: high total IgE (r = 0.6), level of IgE antibodies to C.albicans (r = 0.6), level of serum IgG (r = 0.46) and IgA (r = 0.33). Contrary to adults, children with sensitization to C.albicans had decreased relative number of CD4+, CD8+ and CD72+ of lymphocyte subpopulations. Thus, sensitization to C.albicans manifests in severe atopic dermatitis which in children is often associated with immune deficiency.     

Automated detection of bolus arrival and initiation of data acquisition in fast, three-dimensional, gadolinium-enhanced MR angiography

Automatic triggering of magnetic resonance (MR) angiography with detection of a contrast material bolus was evaluated. Signal intensity changes with time were tracked in a prescribed tracking or monitoring volume by a parallel signal processing unit that automatically started data acquisition once user-defined thresholds were exceeded. This technique, referred to as MR Smartprep, was reliable and avoided the inconsistencies of manual timing.