Mcl-1 and YY1 inhibition and induction of DR5 by the BH3-mimetic Obatoclax (GX15-070) contribute in the sensitization of B-NHL cells to TRAIL apoptosis

The pan Bcl-2 family antagonist Obatoclax (GX15-070), currently in clinical trials, was shown to sensitize TRAIL-resistant tumors to TRAIL-mediated apoptosis via the release of Bak and Bim from Mcl-1 or Bcl-2/Bcl-X_L complexes or by the activation of Bax, though other mechanisms were not examined. Herein, we hypothesize that Obatoclax-mediated sensitization to TRAIL apoptosis may also result from alterations of the apoptotic pathways. The TRAIL-resistant B-cell line Ramos was used as a model for investigation. Treatment of Ramos cells with obatoclax significantly inhibited the expression of several members of the Bcl-2 family, dissociated Bak from Mcl-1 and inhibited the NFκB activity. Cells treated with Mcl-1 siRNA were sensitized to TRAIL apoptosis. We examined whether the sensitization of Ramos to TRAIL by Obatoclax resulted from signaling of the DR4 and/or DR5. Transfection with DR5 siRNA, but not with DR4 siRNA, sensitized the cells to apoptosis following treatment with Obatoclax and TRAIL. The signaling via DR5 correlated with Obatoclax-induced inhibition of the DR5 repressor Yin Yang 1 (YY1). Transfection with YY1 siRNA sensitized the cells to TRAIL apoptosis following treatment with Obatoclax and TRAIL. Overall, the present findings reveal a new mechanism of Obatoclax-induced sensitization to TRAIL apoptosis and the involvement of the inhibition of NFκB activity and downstream Mcl-1 and YY1 expressions and activities.Key words: Obatoclax, TRAIL, YY1, DR5, lymphoma, immunosensitization     

The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

Pneumonia associated with Iegionnaires''s disease is initiated in humans after inhalation of contaminated aerosols. In the environment, Legionella pneumophila is thought to survive and multiply as an intracellular parasite within free-living amoeba. In the genome of L. pneumophila Lens, we identified a unique gene, tolC, encoding a protein that is highly homologous to the outer membrane protein TolC of Escherichia coli. Deletion of tolC by allelic exchange in L. pneumophila caused increased sensitivity to various drugs. The complementation of the tolC mutation in trans restored drug resistance, indicating that TolC is involved in multi-drug efflux machinery. In addition, deletion of tolC caused a significant attenuation of virulence towards both amoebae and macrophages. Thus, the TolC protein appears to play a crucial role in virulence which could be mediated by its involvement in efflux pump mechanisms. These findings will be helpful in unraveling the pathogenic mechanisms of L. pneumophila as well as in developing new therapeutic agents affecting the efflux of toxic compounds.     

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.     

Changes in Protein Synthesis and Morphology during Acid Adaptation of Propionibacterium freudenreichii

Survival of bacteria in changing environments depends on their ability to adapt to abiotic stresses. Microorganisms used in food technology face acid stress during fermentation processes. Similarly, probiotic bacteria have to survive acid stress imposed within the stomach in order to reach the intestine and play a beneficial role. Propionibacteria are used both as cheese starters and as probiotics in human alimentation. Adaptation to low pH thus constitutes a limit to their efficacy. Acid stress adaptation in the probiotic SI41 strain of Propionibacterium freudenreichii was therefore investigated. The acid tolerance response (ATR) was evidenced in a chemically defined medium. Transient exposure to pH 5 afforded protection toward acid challenge at pH 2. Protein neosynthesis was shown to be required for optimal ATR, since chloramphenicol reduced the acquired acid tolerance. Important changes in genetic expression were observed with two-dimensional electrophoresis during adaptation. Among the up-regulated polypeptides, a biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair were identified during the early stress response, while the universal chaperonins GroEL and GroES corresponded to a later response. The beneficial effect of ATR was evident at both the physiological and morphological levels. This study constitutes a first step toward understanding the very efficient ATR described in P. freudenreichii.     

Overexpression of the CT GalNAc transferase inhibits muscular dystrophy in a cleavage-resistant dystroglycan mutant mouse

Transgenic mice that express dystroglycan containing a serine to alanine point mutation at the normal site of cleavage (DG(S654A)) in their skeletal muscles fail to express endogenously cleaved dystroglycan and have muscular dystrophy [Neuromusc. Disord., in press]. Dystrophic DG(S654A) muscles have reduced binding of antibodies, including VIA4-1, that recognize carbohydrate antigens on alpha dystroglycan, a finding similar to muscles in some forms of congenital muscular dystrophy. Here we describe one DG(S654A) transgenic line where VIA4-1 antibody binding is absent in skeletal muscle. In theory, the absence of this carbohydrate antigen should inhibit later glycosylation events that would occur on the structure or structures this antibody binds to. One such modification is likely to be the CT carbohydrate antigen, which is present on alpha dystroglycan in muscles overexpressing the CT GalNAc transferase [Dev. Biol. 242 (2002) 58]. To test the relationship between the VIA4-1 and CT carbohydrate antigens, we made DG(S654A)/CT GalNAc transferase (DG(S654A)/CT) transgenic mice. Surprisingly, dystroglycan was cleaved, and alpha dystroglycan was glycosylated with the VIA4-1 antigen, in DG(S654A)/CT muscles. In addition, muscles in DG(S654A)/CT transgenic mice had little or no evidence of muscular dystrophy when compared to DG(S654A) littermates. These experiments demonstrate that the CT GalNAc transferase can affect the post-translational processing of dystroglycan and the extent of muscular dystrophy even in muscles where the VIA4-1 antigen is not present.     

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.     

Biogeography of functional trait diversity in the Taiwanese reef fish fauna

The richness of Taiwanese reef fish species is inversely correlated to latitude as a direct consequence of the abiotic environment and its effects on benthic habitats. However, to date, no studies have investigated the variations in the diversity of traits (FD) linked with the role of these fishes in the ecosystem. FD is usually considered more sensitive than species richness in detecting early changes in response to disturbances, and therefore could serve as an indicator of ecological resilience to environmental changes. Here, we aim to characterize FD in the Taiwanese reef fish fauna and to document its regional variations. Six traits were used to categorize the 1,484 reef fish species occurring in four environmentally contrasted regions around Taiwan. The number of unique trait combinations (FEs), their richness (FRic), their redundancy (FR), their over‐redundancy (FOR), and their vulnerability (FV) were compared among these regions. Overall, 416 FEs were identified. Their number decreased from south to north in step with regional species richness but FRic remained similar among regions. FR and FOR were higher to the south. At the local scale, variations in FEs and FRic are in concordance with the worldwide pattern of FD. High‐latitude, impoverished fish assemblages, offer a range of trait combinations similar to diversified tropical assemblages. Increasing diversity in the latter mainly contributes to raising FR and supports already over‐redundant entities. High vulnerability makes many combinations highly sensitive to species loss, and was higher at intermediate latitudes when using a fine resolution in trait categories. It suggests that the loss of FEs may first be characterized by an increase in their vulnerability, a pattern that could have been overlooked in previous global scale analyses. Overall, this study provides new insights into reef fish trait biogeography with potential ramifications for ecosystem functioning.     

CRTAM: A molecule involved in epithelial cell adhesion

Class I‐restricted T cell associated molecule (CRTAM) is a member of the immunoglobulin superfamily that complies with the structural characteristics of the JAM family of proteins and is phylogenetically more closely related to nectin‐like proteins. Here we demonstrate for the first time, that CRTAM is expressed in epithelial cells along the lateral membrane and is important for early cell–cell contacts and cell–substrate interactions. CRTAM is sensitive to intermediate filament disruption and treatment of monolayers with soluble CRTAM enhances cell–cell dissociation and lowers transepithelial electrical resistance. Incubation of newly plated cells with anti‐CRTAM antibody decreases the formation of cell aggregates and promotes cell detachment. Co‐cultures of epithelial cells and fibroblasts that lack CRTAM expression and in vitro binding assays, demonstrate the participation of CRTAM in homotypic and heterotypic trans‐interactions. Hence we conclude that CRTAM is a molecule involved in epithelial cell adhesion. J. Cell. Biochem. 111: 111–122, 2010. © 2010 Wiley‐Liss, Inc.     

Fast Growth May Impair Regeneration Capacity in the Branching Coral Acropora muricata

Regeneration of artificially induced lesions was monitored in nubbins of the branching coral Acropora muricata at two reef-flat sites representing contrasting environments at Réunion Island (21°07′S, 55°32′E). Growth of these injured nubbins was examined in parallel, and compared to controls. Biochemical compositions of the holobiont and the zooxanthellae density were determined at the onset of the experiment, and the photosynthetic efficiency (F_v/F_m) of zooxanthellae was monitored during the experiment. Acropora muricata rapidly regenerated small lesions, but regeneration rates significantly differed between sites. At the sheltered site characterized by high temperatures, temperature variations, and irradiance levels, regeneration took 192 days on average. At the exposed site, characterized by steadier temperatures and lower irradiation, nubbins demonstrated fast lesion repair (81 days), slower growth, lower zooxanthellae density, chlorophyll a concentration and lipid content than at the former site. A trade-off between growth and regeneration rates was evident here. High growth rates seem to impair regeneration capacity. We show that environmental conditions conducive to high zooxanthellae densities in corals are related to fast skeletal growth but also to reduced lesion regeneration rates. We hypothesize that a lowered regenerative capacity may be related to limited availability of energetic and cellular resources, consequences of coral holobionts operating at high levels of photosynthesis and associated growth.     

Humoral Immunity Links Candida albicans Infection and Celiac Disease

Objective

The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI.

Methods

Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1.

Results

CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides.

Conclusions

Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.