Environmental signals implicated in Dr fimbriae release by pathogenic Escherichia coli

Afa/Dr diffusely adhering Escherichia coli have been shown to cause urinary tract infections and enteric infections. Virulence of Dr-positive IH11128 bacteria is associated with the presence of Dr fimbriae. In this report, we show for the first time that the Dr fimbriae are released in the extracellular medium in response to multiple environmental signals. Production and secretion of Dr fimbriae are clearly thermoregulated. A comparison of the amounts of secreted fimbriae showed that the secretion is drastically increased during anaerobic growth in minimal medium. The effect of anaerobiosis on secretion seemed to depend on both the growth phase and the culture medium. The secretion was maximal during the logarithmic-phase growth and corresponded to 27 and 57% of total Dr fimbriae produced by bacteria grown in mineral medium+glucose and LB broth, respectively. Thus, the anaerobic environment of the colon would favour the secretion of Dr fimbriae during bacterial multiplication. The controlled release of the Dr fimbriae, which is carried out in the absence of cellular lysis, appears independent of the action of proteases or a process of maturation. The mechanism employed in the liberation of Dr fimbriae thus seems different from that described for the adhesins FHA and Hap of Bordetella pertussis and Haemophilus influenzae.     

Oxidation of methoxyphenethylamines by cytochrome P450 2D6. Analysis of rate-limiting steps

Cytochrome P450 (P450) 2D6 is involved in the oxidation of a large fraction ( approximately 30%) of drugs used by humans and also catalyzes the O-demethylation of the model substrates 3- and 4-methoxyphenethylamine followed by subsequent ring hydroxylation to dopamine. Burst kinetics were not observed; rate-limiting step(s) must occur prior to product formation. Rates of reduction of ferric P450 2D6 were stimulated by 3- or 4-methoxyphenethylamine or the inhibitor quinidine; reduction is not the most rate-limiting step. The non-competitive intramolecular deuterium isotope effect, an estimate of the intrinsic isotope effect, for 4-methoxyphenethylamine O-demethylation was 9.6. Intermolecular non-competitive deuterium isotope effects of 3.1-3.8 were measured for k(cat) and k(cat)/K(m) for both O-demethylation reactions, implicating at least partially rate-limiting C-H bond breaking. Simulation of steady-state kinetic data yielded a catalytic mechanism dominated by the rates of (i) Fe(2+)O(2)(-) protonation (plus O-O bond scission) and (ii) C-H bond breaking, consistent with the appearance of the spectral intermediates in the steady state, attributed to iron-oxygen complexes. However, all the rates of individual steps (or rates of combined steps) are considerably higher than k(cat), and the contributions of several steps must be considered in understanding rates of the P450 2D6 reactions.     

Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (plLL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli. the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in ihe chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (WIBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic as proteins fused to the maltose-binding protein (WIBP) and purified in large scale. The MBP-HspA and WlBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H, py/or/-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.     

Induction of colitis in cftr-/- mice results in bile duct injury

It is unknown why some patients with inflammatory bowel disease develop primary sclerosing cholangitis. We have recently shown that patients with primary sclerosing cholangitis have an increased prevalence of mutations in the gene responsible for cystic fibrosis (CFTR) compared with individuals with inflammatory bowel disease alone. Our aim was to examine whether induction of colitis by oral dextran leads to bile duct injury in mice heterozygous or homozygous for mutations in CFTR. The effect of oral administration of docosahexaenoic acid to correct a fatty acid imbalance associated with cystic fibrosis was also examined to determine whether this would prevent bile duct inflammation. Wild-type mice and mice heterozygous and homozygous for CFTR mutations were given dextran orally for 14 days to induce colitis. Bile duct injury was quantitated by blinded histological scoring and measurement of serum alkaline phosphatase activity. The effect of pretreatment with docosahexaenoic acid for 7 days was examined. Treatment of mice with 100 mg dextran/day for 9 days followed by 85 mg/day for 5 days resulted in a significant increase in bile duct injury as determined by histological scoring in homozygous cystic fibrosis mice compared with wild-type mice (P = 0.005). The bile duct injury seen in cystic fibrosis mice was reflected in a threefold increase in serum alkaline phosphatase (P = 0.0006). Pretreatment with oral docosahexaenoic acid decreased both histological evidence of bile duct injury and serum alkaline phosphatase levels. In the setting of colitis, loss of CFTR function leads to bile duct injury.     

3-(4-Aminobutyn-1-yl)pyridines: binding at alpha 4 beta 2 nicotinic cholinergic receptors

The binding of a series pyridylbutynylamines 6 was examined at alpha4beta2 nACh receptors. Structural modifications, comparing 6 with pyridyl ethers 2, did not consistently result in parallel effects on receptor affinity, suggesting possible differences in their modes of binding. Furthermore, the binding of amine 6a seemed to be accounted for by the newer vector pharmacophore models.     

(+/-)8-Amino-5,6,7,8-tetrahydroisoquinolines as novel antinociceptive agents

Several amine-substituted 8-amino-5,6,7,8-tetrahydroisoquinolines were examined as conformationally-constrained analogs of the nicotinic cholinergic (nACh) 3-(aminomethyl)pyridines. Although these ligands failed to bind at nACh receptors, the N-ethyl-N-methyl analog 3d was found to be at least equipotent with nicotine in rodent tests of antinociception. The mechanism of action of 3d is currently unknown.     

Composition,Chemical Variability and Biological Activity of Cymbopogon schoenanthus Essential Oil from Central Algeria

Cymbopogon schoenanthus (L.) Spreng . (Poaceae) is an aromatic plant whose aerial parts and rhizome produced an essential oil with pleasant odor. A chemical variability has been observed depending of the countries where the plant grows wild, including Algeria. The chemical compositions of 24 oil samples isolated from plants harvested in Central Algeria have been investigated, to evidence homogeneity or chemical variability within a given area of harvest. Twenty of these were dominated by cis‐ and trans‐p‐menth‐2‐en‐1‐ols (22.6 %±3.6 and 14.3 %±1.7, resp.) beside four atypical compositions. Otherwise, aerial parts and rhizomes produced similar essential oils. Lastly, a fair antimicrobial activity was measured against Staphylococcus aureus strain, while the antioxidant potential was low.     

Extracellular xylanase production by Fusarium species in solid state fermentation

Fusarium sp. has been shown to be a promising organism for enhanced production of xylanases. In the present study, xylanase production by 21 Fusarium sp. isolates (8 Fusarium culmorum, 4 Fusarium solani, 6 Fusarium verticillioides and 3 Fusarium equiseti) was evaluated under solid state fermentation (SSF). The fungal isolate Fusarium solani SYRN7 was the best xylanase producer among the tested isolates. The effects of some agriculture wastes (like wheat straw, wheat bran, beet pulp and cotton seed cake) and incubation period on xylanase production by F. solani were optimized. High xylanase production (1465.8 U/g) was observed in wheat bran after 96 h of incubation. Optimum pH and temperature for xylanase activity were found to be 5 and 50 degrees C, respectively.     

McMaster-Toronto Arthritis Patient Preference Disability Questionnaire sensitivity to change in low back pain: influence of shifts in priorities

Objective

To assess the sensitivity to change of the McMaster Toronto Arthritis Patient Preference Disability Questionnaire (MACTAR) in chronic low back pain (CLBP) and shifts in patients'' priorities of disabling activities over time.

Methods

A prospective longitudinal survey of 100 patients (38 males) with CLBP in a tertiary care teaching hospital. Evaluation at baseline and 6 months by the MACTAR, Quebec Back Pain Disability Questionnaire (QUEBEC), Hospital Anxiety and Depression scale (HAD), Fear-Avoidance Beliefs Questionnaire (FABQ), Coping Strategies Questionnaire (CSQ), and pain and handicap visual analogue scales (VASs). Patients'' perceived improvement or worsening of condition was assessed at 6 months. Effect size (ES) and Standardized response mean (SRM) and effect size (ES) were used to evaluate sensitivity to change of the MACTAR.

Results

The MACTAR SRM and ES values (SRM = 0.25; ES = 0.37) were among the highest for the instruments evaluated. For patients considering their condition as improved, the SRM was 0.66 and the ES 1. The 3 disability domains, classified by the International Classification of Functioning, Disability and Health (ICF), most often cited as priorities at baseline remained the most cited at follow-up: mobility (40.9% of patients); community, social and civic life (22.7%); and domestic life (22.4%). At 6 months, 48 patients shifted their priorities, for a decrease in MACTAR SRM and ES values for patients considering their condition improved and an increase in these values for those considering their condition deteriorated.

Conclusions

Although the MACTAR has similar sensitivity to change as other outcome measures widely used in CLBP, shifts in patient priorities over time are common and influence scores and sensitivity to change.