Bim inhibits autophagy by recruiting beclin 1 to microtubules

Bim is a proapoptotic BH3-only Bcl-2 family member.?In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in?vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites.     

Molecular Epidemiology of Chronic Pseudomonas aeruginosa Airway Infections in Cystic Fibrosis

Background/Methods

The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years.

Results

Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient.

Conclusion

The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.     

Integrative control of coronary resistance vessel tone by endothelin and angiotensin II is altered in swine with a recent myocardial infarction

Several studies have indicated an interaction between the renin-angiotensin (ANG II) system and endothelin (ET) in the regulation of vascular tone. Previously, we have shown that both ET and ANG II exert a vasoconstrictor influence on the coronary resistance vessels of awake normal swine. Here, we investigated whether the interaction between ANG II and ET exists in the control of coronary resistance vessel tone at rest and during exercise using single and combined blockade of angiotensin type 1 (AT(1)) and ET(A)/ET(B) receptors. Since both circulating ANG II and ET levels are increased after myocardial infarction (MI), we investigated if the interaction between these systems is altered after MI. In awake healthy swine, coronary vasodilation in response to ET(A)/ET(B) receptor blockade in the presence of AT(1) blockade was similar to vasodilation produced by ET(A)/ET(B) blockade under control conditions. In awake swine with a 2- to 3-wk-old MI, coronary vasodilator responses to individual AT(1) and ET(A)/ET(B) receptor blockade were virtually abolished, despite similar coronary arteriolar AT(1) and ET(A) receptor expression compared with normal swine. Unexpectedly, in the presence of AT(1) blockade (which had no effect on circulating ET levels), ET(A)/ET(B) receptor blockade elicited a coronary vasodilator response. These findings suggest that in normal healthy swine the two vasoconstrictor systems contribute to coronary resistance vessel control in a linear additive manner, i.e., with negligible cross-talk. In contrast, in the remodeled myocardium, cross-talk between ANG II and ET emerges, resulting in nonlinear redundant control of coronary resistance vessel tone.     

Enucleation: a possible mechanism of cancer cell death

There are few major morphologies of cell death that have been described so far: apoptosis (type I), cell death associated with autophagy (type II), necrosis (type III) and anchorage‐dependent mechanisms—anoikis. Here, we show for the first time a possibly novel mechanism inducing tumour cell death under in vitro conditions—enucleation. We pursued the influence of colloidal suspensions of Fe₃O₄ nanoparticles on tumour cell lines (SK‐BR‐3 and MCF‐7 breast cancer cell lines) grown according to standard cell culture protocols. Magnetite nanoparticles were prepared by combustion synthesis and double layer coated with oleic acid. Scanning and transmission electron microscopy revealed that tumour cells developed a network of intracytoplasmic stress fibres, which induce extrusion of nuclei, and enucleated cells die. Normal adult mesenchymal stem cells, used as control, did not exhibit the same behaviour. Intact nuclei were found in culture supernatant of tumour cells, and were visualized by immunofluorescence. Enucleation as a potential mechanism of tumour cell death might open new horizons in cancer biology research and development of therapeutic agents capable of exploiting this behaviour.     

Lactuca capensis reverses memory deficits in Aβ1‐42‐induced an animal model of Alzheimer's disease

We investigated the neuropharmacological effects of the methanolic extract from Lactuca capensis Thunb. leaves (100 and 200 mg/kg) for 21 days on memory impairment in an Alzheimer's disease (AD) rat model produced by direct intraventricular delivery of amyloid‐β1‐42 (Aβ1‐42). Behavioural assays such as Y‐maze and radial arm maze test were used for assessing memory performance. Aβ1‐42 decreased cognitive performance in the behavioural tests which were ameliorated by pre‐treatment with the methanolic extract. Acetylcholinesterase activity and oxidant–antioxidant balance in the rat hippocampus were abnormally altered by Aβ1‐42 treatment while these deficits were recovered by pre‐treatment with the methanolic extract. In addition, rats were given Aβ1‐42 exhibited in the hippocampus decreased brain‐derived neurotrophic factor (BDNF) mRNA copy number and increased IL‐1β mRNA copy number which was reversed by the methanolic extract administration. These findings suggest that the methanolic extract could be a potent neuropharmacological agent against dementia via modulating cholinergic activity, increasing of BDNF levels and promoting antioxidant action in the rat hippocampus.     

Structural analysis of the 26S proteasome by cryoelectron tomography

The 26S proteasome is the key enzyme of intracellular protein degradation in eukaryotic cells. It is a multisubunit complex of 2.5 MDa confining the proteolytic action to an inner compartment with tightly controlled access. Structural studies of this intriguing molecular machine have been hampered by its intrinsic instability and its dynamics. Here we have used an unconventional approach to obtain a three-dimensional structure of the holocomplex uncompromised by preparation-induced alterations and unbiased by any starting model. We have performed a tomographic reconstruction, followed by averaging over approx. 150 individual reconstructions, of Drosophila 26S proteasomes suspended in a thin layer of amorphous ice.     

Laboratory appraisal of optimal gaseous conditions for growth of zoonotic Helicobacter felis ATCC 49179

An attempt was made to assess the hitherto undescribed optimal gaseous conditions for growth of zoonotic Helicobacter felis, focusing on the ratio of spiral-forms amongst the whole cells examined. The largest mean colony diameter was obtained under the gaseous condition of O₂ 12% and CO₂ 10%. In analyzing the five day old colonies, the highest percentage of spiral forms (85.5%) was observed under the condition of O₂ 18% and CO₂ 5%. In contrast, the lowest percentage of spiral forms (2.3%) was demonstrated under the condition of O₂ 1% and CO₂ 10%. The condition of O₂ 12% and CO₂ 10% was concluded to be optimal for obtaining cells with the largest colony sizes, although colonies proliferated under such conditions definitely contain many more coccoid cells than spiral forms. In culturing H. felis strains, optimal gaseous conditions should be employed according to the purposes or preferences of study designs.     

Panton–Valentine Leukocidin Enhances the Severity of Community-Associated Methicillin-Resistant Staphylococcus aureus Rabbit Osteomyelitis

Background

Extensive spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the United States, and the concomitant increase in severe invasive staphylococcal infections, including osteomyelitis, in healthy children, has led to renewed interest in Panton-Valentine leukocidin (PVL). However, the pathogenetic role of PVL in staphylococcal infections remains controversial, possibly because it depends on the site of infection.

Methodology/Principal Findings

We compared the course of experimental rabbit osteomyelitis due to the PVL-positive CA-MRSA strain USA 300 (LAC) and its PVL-negative isogenic derivative (LACΔpvl), using a low and a high inoculum (8×10⁵ and 4×10⁸ CFU). With the low inoculum, bone infection was less frequent on day 7 (D7) and day 28 (D28) with LACΔpvl than with LAC (respectively 12/19 and 18/19 animals, p = 0.042). With the high inoculum of both strains, all the animals were infected on D7 and the infection persisted on D28 in almost every case. However, tibial bacterial counts and the serum CRP concentration fell significantly between D7 and D28 with LACΔpvl but not with LAC. Respectively 67% and 60% of LAC-infected rabbits had bone deformation and muscle/joint involvement on D7, compared to 0% and 7% of LACΔpvl-infected rabbits (p = 0.001 and p = 0.005 respectively). Between D0 and D28, the anti-PVL antibody titer increased significantly only with the high inoculum of LAC.

Conclusions/Significance

PVL appears to play a role in the persistence and rapid local extension of rabbit osteomyelitis, in keeping with the greater severity of human bone infections due to PVL-positive S. aureus. The possible therapeutic implications of these findings are discussed.     

Development of antibiotic resistance and up-regulation of the antimutator gene pfpI in mutator Pseudomonas aeruginosa due to inactivation of two DNA oxidative repair genes (mutY, mutM)

Prevention and correction of oxidative DNA lesions in Pseudomonas aeruginosa is ensured by the DNA oxidative repair system (GO). Single inactivation of mutT, mutY and mutM involved in GO led to elevated mutation rates (MRs) that correlated to increased development of resistance to antibiotics. In this study, we constructed a double mutant in mutY and mutM (PAOMY-Mgm) and characterized the phenotype and the gene expression profile using microarray and RT-PCR. PAOMY-Mgm presented 28-fold increases in MR compared with wild-type reference strain PAO1. In comparison, the PAOMYgm (mutY) single mutant showed only a fivefold increase, whereas the single mutant PAOMMgm (mutM) showed a nonsignificant increase in MR compared with PAO1 and the single mutants. Mutations in the regulator nfxB leading to hyperexpression of MexCD-OprJ efflux pump were found as the mechanism of resistance to ciprofloxacin in the double mutant. A better fitness of the mutator compared with PAO1 was found in growth competition experiments in the presence of ciprofloxacin at concentrations just below minimal inhibitory concentration. Up-regulation of the antimutator gene pfpI, that has been shown to provide protection to oxidative stress, was found in PAOMY-Mgm compared with PAO1. In conclusion, we showed that MutY and MutM are cooperating in the GO of P. aeruginosa, and that oxidative DNA lesions might represent an oxidative stress for the bacteria.