Binding Properties of the Galactose-detecting Lectin Pseudomonas aeruginosa Agglutinin (PA-IL) to Skeletal Muscle Fibres. Quantitative Precipitation and Precipitation Inhibition Assays

Pseudomonas aeruginosa agglutinin (PA-IL) staining and the influence of various carbohydrates on lectin binding to muscle sections were investigated quantitatively using a scanning and integrating microspectrophotometre. A strong dose-dependent inhibition of PA-IL staining in the sections was recorded with galabiose (Gal1-4Gal) while lactose (Gal1-4Glc) had no inhibitory effect. The affinity of PA-IL to Gal1 carbohydrates was studied by ELISA using immobilized glycoconjugates in which Gal1 glycans were attached to bovine serum albumin or ceramide. PA-IL exhibited strong binding to both simple glycoconjugates having a single Gal moiety and to di- and trisaccharides with terminal Gal1 at the non-reducing end. In all cross-sectioned muscle fibres incubated with PA-IL, the staining was present as a honeycomb-shaped network through the entire cytoplasm. Further, a dense punctuate staining could be shown in most fibres. A similar staining pattern was noticed after incubation with a monoclonal antibody against ryanodine receptors and with biotinylated ryanodine suggesting that the network could represent the sarcoplasmic reticulum. Further, Western blots of a sarcoplasmic reticulum preparation showed multiple bands after incubation with PA-IL. It may therefore be proposed that glycoconjugates carrying terminal Gal1 show affinity for PA-IL and are located to the sarcoplasmic reticulum.     

Overview on coenzyme Q10 as adjunctive therapy in chronic heart failure. Rationale, design and end-points of "Q-symbio"--a multinational trial

Energy starvation of the myocardium is probably a dominant feature of heart failure and attention has been directed towards agents which may stabilize myocardial metabolism and maintain adequate energy stores. A reduced myocardial tissue content of the essential redox-component and natural antioxidant Coenzyme Q10 (CoQ10) has been detected in patients with heart failure and the observed level of CoQ10 deficiency was correlated to the severity of heart failure. CoQ10 fulfills various criteria of an obvious adjunct in patients with symptomatic heart failure: it is devoid of significant side effects and it improves symptoms and quality of life. Till this date, several double-blind placebo-controlled trials with CoQ10 supplementation in more than 1000 patients have been positive and statistically significant with respect to various clinical parameters, e.g. improvement in NYHA Class, exercise capacity and reduced hospitalisation frequency. Also treatment with CoQ10 led to a significant improvement of relevant hemodynamic parameters. In only 3 out of 13 double-blind studies comprising 10% of the total number of patients treated the results were neutral. Thus, based on the available controlled data CoQ10 is a promising, effective and safe approach in chronic heart failure. This is why a double-blind multicenter trial with focus on morbidity and mortality has been planned to start in 2003: Q-SYMBIO. Patients in NYHA classes III to IV (N=550) receiving standard therapy are being randomized to treatment with CoQ10 100 mg t.i.d. or placebo in parallel groups. End-points in a short-term evaluation phase of 3 months include symptoms, functional capacity and biomarker status (BNP). The aim of a subsequent 2-year follow-up study is to test the hypothesis that CoQ10 may reduce cardiovascular morbidity (unplanned cardiovascular hospitalisation due to worsening heart failure) and mortality as a composite endpoint. This trial should help to establish the future role of CoQ10 as part of a maintenance therapy in patients with chronic heart failure.     

Serum coenzyme Q10 concentrations in healthy men supplemented with 30 mg or 100 mg coenzyme Q10 for two months in a randomised controlled study

Serum coenzyme Q10 (Q10) concentrations were evaluated in healthy male volunteers supplemented with 30 mg or 100 mg Q10 or placebo as a single daily dose for two months in a randomised, double-blind, placebo-controlled study. Median baseline serum Q10 concentration in 99 men was 1.26 mg/l (10%, 90% fractiles: 0.82, 1.83). Baseline serum Q10 concentration did not depend on age, while borderline significant positive associations were found for body weight and smoking 1-10 cigarettes/d. Supplementation with 30 mg or 100 mg Q10 resulted in median increases in serum Q10 concentration of 0.55 mg/l and 1.36 mg/l, respectively, compared with a median decrease of 0.23 mg/l with placebo. The changes in the Q10 groups were significantly different from that in the placebo group, and the increase in the 100 mg Q10 group was significantly greater than that in the 30 mg Q10 group. The change in serum Q10 concentration in the Q10 groups did not depend on baseline serum Q10 concentration, age, or body weight.     

Cuticular proteins from the horseshoe crab,Limulus polyphemus

Proteins were purified from the carapace cuticle of a juvenile horseshoe crab, Limulus polyphemus, and several of them were characterized by amino acid sequence determination. The proteins are small (7-16 kDa) and their isoelectric points range from 6.5 to 9.2. They have high contents of tyrosine, ranging from 13.5 to 35.4%. Some of the proteins show sequence similarity to cuticular proteins from other arthropod groups, with the most pronounced similarity to proteins from the cuticle of the spider Araneus diadematus. Two proteins show sequence similarity to a hexamerin storage protein from Blaberus discoidalis.     

Estimating time to pregnancy from current durations in a cross-sectional sample

A new design for estimating the distribution of time to pregnancy is proposed and investigated. The design is based on recording current durations in a cross-sectional sample of women, leading to statistical problems similar to estimating renewal time distributions from backward recurrence times. Non-parametric estimation is studied in some detail and a parametric approach is indicated. The results are illustrated on Monte Carlo simulations and on data from a recent European collaborative study. The role and applicability of this approach is discussed.     

Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.     

RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators

APOBEC1 is the catalytic component of an RNA editing complex but shows homology to activation-induced cytidine deaminase (AID), a protein whose function is to potentiate diversification of immunoglobulin gene DNA. Here, we show that APOBEC1 and its homologs APOBEC3C and APOBEC3G exhibit potent DNA mutator activity in an E. coli assay. Indeed, like AID, these proteins appear to trigger DNA mutation through dC deamination. However, each protein exhibits a distinct local target sequence specificity. The results reveal the existence of a family of potential active dC/dG mutators, with possible implications for cancer.     

Hrs-2 regulates receptor-mediated endocytosis via interactions with Eps15

Hrs-2, via interactions with SNAP-25, plays a regulatory role on the exocytic machinery. We now show that Hrs-2 physically interacts with Eps15, a protein required for receptor-mediated endocytosis. The Hrs-2/Eps15 interaction is calcium dependent, inhibited by SNAP-25 and alpha-adaptin, and results in the inhibition of receptor-mediated endocytosis. Immunoelectron microscopy reveals Hrs-2 localization on the limiting membrane of multivesicular bodies, organelles in the endosomal pathway. These data show that Hrs-2 regulates endocytosis, delineate a biochemical pathway (Hrs-2-Eps15-AP2) in which Hrs-2 functions, and suggest that Hrs-2 acts to provide communication between endo- and exocytic processes.     

Characterization of in vitro chlamydial cultures in low-oxygen atmospheres

To mimic in vivo conditions during chlamydial infections, Chlamydia trachomatis serovar D and Chlamydia pneumoniae CWL029 were cultured in low-oxygen atmospheres containing 4% O(2), with parallel controls cultured in atmospheric air. Both were enriched with 5% CO(2). The results showed a dramatic increase in the growth of C. pneumoniae but not of C. trachomatis.