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.     

Yeast Proteolytic Activity During High and Low Gravity Wort Fermentations and its Effect on Head Retention

The aim was to discover the effect of high gravity brewing on yeast protease activity during fermentation, on the loss of hydrophobic polypeptides from wort during fermentation, and on the foam stability of stored beer. The hydrophobic polypeptide content of low (10° Plato) gravity worts showed a steady decline throughout fermentation, but for the 20° Plato wort there was a rapid decline over the first 8 days of fermentation, followed by little change over the remaining period. The decrease in hydrophobic polypeptides was greater in the high gravity fermentation. Proteinase A increased during fermentations with the highest levels being present at the end of fermentations. High gravity fermentations exhibited levels of yeast protease that from the 3rd to 11th day of fermentation were at least twice the values of the low gravity fermentations. The high gravity brewed beer contained significantly higher levels of proteinase A activity than the low gravity brewed beer. The inclusion of FERMCAP™, an antifoam, in high gravity wort did not affect either the hydrophobic polypeptide levels or foam stability of the resultant beer. This suggests that proteinase A, rather than fermenter foaming, must be the major contributor to the lack of foam stability of high gravity brewed beer. Head retention measurements conducted on the high and low gravity brewed bottled beers, over a five month period, demonstrated a steady decline in foam stability for both beers. The declines in head retention did not occur in high and low gravity beers that had been pasteurised.     

Shear-induced 1-D alignment of alumina nanoparticles in coatings

Atomic Force Microscopy and Scanning Electron Microscopy were used to study shear-induced alignment of alumina and silica nanoparticles in two-component polyurethane clear coatings. 1-D strings of nanoparticles, formed in an extended pearl-necklace fashion were observed near the surfaces of cured films at nanoparticle volume fractions less than 0.05. This alignment is affected by the shear conditions of the application method. When applied by spraying, linear particle strings as long as 5 cm were observed in the direction of shear. Nanoparticle strings were also found, to a lesser extent, when coatings were applied by a drawdown method. The phenomenon was not observed in coatings applied with minimal shear. These particle string formations, in addition to affecting the performance of coatings, may have broader implications in the field of nanomaterials. Our literature searches so far have not uncovered reports of stable, 1-D nanoparticle arrangements with same degree of linearity produced under simple shear with compositions having very low particle loadings. Presented at the 2006 FutureCoat! conference, sponsored by the Federation of Societies for Coatings Technology, in New Orleans, LA, on November 1–3, 2006.     

The Propensity of Wooden Crevices to Smoldering Ignition by Firebrands

Fire Technology - Fires occurring at the wildland-urban interface (WUI) have rapidly increased in frequency and severity over the past few decades. As a result of these extreme fires, multiple...     

HYDROPHOBIC POLYPEPTIDE EXTRACTION DURING HIGH GRAVITY MASHING— EXPERIMENTAL APPROACHES FOR ITS IMPROVEMENT

The aim was to establish if a substantial increase in hydrophobic polypeptides could be achieved during high gravity mashing. When worts with gravities ranging from 5–20°P were analysed for hydrophobic polypeptide content it was found that there was no appreciable increase in hydrophobic polypeptide levels. Remashing of the spent grains from low and high gravity mashes demonstrated that this resulted from inefficient extraction of hydrophobic polypeptide levels during the mashing process. For example, wort produced from remashed high gravity spent grains contained 150 mg/L hydrophobic polypeptides compared to only 10 mg/L in the low gravity remashed spent grains. Experiments were conducted, employing standard mashing techniques, in an attempt to increase the extraction of hydrophobic polypeptides during high gravity mashing. Thus the use of gypsum, proteolytic stands, varying liquor to grist ratios and wheat malt addition were all investigated for their effect on hydrophobic polypeptide extraction during high and low gravity mashing. Wort analysis demonstrated that none of the techniques employed had a significant effect on hydrophobic polypeptide extraction. When wort from remashed spent grains was used as mashing in liquor for a fresh mash and the resultant worts analysed for hydrophobic polypeptides it was observed that no increase in hydrophobic polypeptide extraction was achieved. For example, wort from the remashed high gravity spent grains, containing 140 mg/L hydrophobic polypeptides, when used as mashing-in liquor, produced no increase in hydrophobic polypeptide levels in the resultant high gravity wort (230 mg/L) when compared to a high gravity wort produced using distilled water as mashing-in liquor (255 mg/L). It is therefore concluded that a saturation point has been reached and no more hydrophobic polypeptides can be extracted during mashing regardless of the procedures employed.     

Imiquimod, a patient-applied immune-response modifier for treatment of external genital warts

Genital human papillomavirus infection is one of the most common sexually transmitted diseases. Imiquimod is a new agent, an immune-response modifier, that has been demonstrated to have potent in vivo antiviral and antitumor effects in animal models. The present prospective, multicenter, double-blind, randomized, vehicle-controlled trial evaluated the efficacy and safety of daily patient-applied imiquimod for up to 16 weeks for the treatment of external genital warts. Wart recurrence was investigated during a 12-week treatment-free follow-up period. In the intent-to-treat analysis, baseline warts cleared from 49 of 94 (52%) patients treated with 5% imiquimod cream, 13 of 90 (14%) patients treated with 1% imiquimod cream, and 3 of 95 (4%) vehicle-treated patients; the differences between the groups treated with vehicle and imiquimod were significant (P < 0.0001). For subjects who completed the follow-up period, recurrence rates after a complete response were 19% (9 of 48 patients) in the 5% imiquimod cream group, 17% (2 of 12) in the 1% imiquimod cream group, and 0% (0 of 3) in the vehicle-treated group. There were no systemic reactions, although local skin reactions (generally of mild or moderate severity) were common, particularly in the 5% imiquimod cream group. Local reactions caused two patients to discontinue treatment. The most frequently reported local skin reactions were erythema, excoriation or flaking, and erosion. Patient-applied 5% imiquimod cream is effective for the treatment of external genital warts and has a favorable safety profile.     

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL-11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.