Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity

We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery

Premature Senescence and Increased TGFβ Signaling in the Absence of Tgif1

Transforming growth factor β (TGFβ) signaling regulates cell cycle progression in several cell types, primarily by inducing a G1 cell cycle arrest. Tgif1 is a transcriptional corepressor that limits TGFβ responsive gene expression. Here we demonstrate that primary mouse embryo fibroblasts (MEFs) lacking Tgif1 proliferate slowly, accumulate increased levels of DNA damage, and senesce prematurely. We also provide evidence that the effects of loss of Tgif1 on proliferation and senescence are not limited to primary cells. The increased DNA damage in Tgif1 null MEFs can be partially reversed by culturing cells at physiological oxygen levels, and growth in normoxic conditions also partially rescues the proliferation defect, suggesting that in the absence of Tgif1 primary MEFs are less able to cope with elevated levels of oxidative stress. Additionally, we show that Tgif1 null MEFs are more sensitive to TGFβ-mediated growth inhibition, and that treatment with a TGFβ receptor kinase inhibitor increases proliferation of Tgif1 null MEFs. Conversely, persistent treatment of wild type cells with low levels of TGFβ slows proliferation and induces senescence, suggesting that TGFβ signaling also contributes to cellular senescence. We suggest that in the absence of Tgif1, a persistent increase in TGFβ responsive transcription and a reduced ability to deal with hyperoxic stress result in premature senescence in primary MEFs

Vitamin C and E Supplementation Effects in Professional Soccer Players Under Regular Training

Exercise training is known to induce an increase in free radical production potentially leading to enhanced muscle injury. Vitamins C and E are well known antioxidants that may prevent muscle cell damage. The purpose of this study was to determine the effects of these supplemental antioxidant vitamins on markers of oxidative stress, muscle damage and performance of elite soccer players. Ten male young soccer players were divided into two groups. Supplementation group (n = 5) received vitamins C and E supplementation daily during the pre-competitive season (S group), while the placebo group (PL group, n = 5) received a pill containing maltodextrin. Both groups performed the same training load during the three-month pre-season training period. Erythrocyte antioxidant enzymes glutathione reductase, catalase and plasma carbonyl derivatives did not show any significant variation among the experimental groups. Similarly, fitness level markers did not differ among the experimental groups. However, S group demonstrated lower lipid peroxidation and muscle damage levels (p < 0.05) compared to PL group at the final phase of pre-competitive season. In conclusion, our data demonstrated that vitamin C and E supplementation in soccer players may reduce lipid peroxidation and muscle damage during high intensity efforts, but did not enhance performance

Audio-Visual Speech Timing Sensitivity Is Enhanced in Cluttered Conditions

Events encoded in separate sensory modalities, such as audition and vision, can seem to be synchronous across a relatively broad range of physical timing differences. This may suggest that the precision of audio-visual timing judgments is inherently poor. Here we show that this is not necessarily true. We contrast timing sensitivity for isolated streams of audio and visual speech, and for streams of audio and visual speech accompanied by additional, temporally offset, visual speech streams. We find that the precision with which synchronous streams of audio and visual speech are identified is enhanced by the presence of additional streams of asynchronous visual speech. Our data suggest that timing perception is shaped by selective grouping processes, which can result in enhanced precision in temporally cluttered environments. The imprecision suggested by previous studies might therefore be a consequence of examining isolated pairs of audio and visual events. We argue that when an isolated pair of cross-modal events is presented, they tend to group perceptually and to seem synchronous as a consequence. We have revealed greater precision by providing multiple visual signals, possibly allowing a single auditory speech stream to group selectively with the most synchronous visual candidate. The grouping processes we have identified might be important in daily life, such as when we attempt to follow a conversation in a crowded room

Role of Surface Area, Primary Particle Size, and Crystal Phase on Titanium Dioxide Nanoparticle Dispersion Properties

Characterizing nanoparticle dispersions and understanding the effect of parameters that alter dispersion properties are important for both environmental applications and toxicity investigations. The role of particle surface area, primary particle size, and crystal phase on TiO2 nanoparticle dispersion properties is reported. Hydrodynamic size, zeta potential, and isoelectric point (IEP) of ten laboratory synthesized TiO2 samples, and one commercial Degussa TiO2 sample (P25) dispersed in different solutions were characterized. Solution ionic strength and pH affect titania dispersion properties. The effect of monovalent (NaCl) and divalent (MgCl2) inert electrolytes on dispersion properties was quantified through their contribution to ionic strength. Increasing titania particle surface area resulted in a decrease in solution pH. At fixed pH, increasing the particle surface area enhanced the collision frequency between particles and led to a higher degree of agglomeration. In addition to the synthesis method, TiO2 isoelectric point was found to be dependent on particle size. As anatase TiO2 primary particle size increased from 6 nm to 104 nm, its IEP decreased from 6.0 to 3.8 that also results in changes in dispersion zeta potential and hydrodynamic size. In contrast to particle size, TiO2 nanoparticle IEP was found to be insensitive to particle crystal structure

Viral Mediated Redirection of NEMO/IKKγ to Autophagosomes Curtails the Inflammatory Cascade

The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response

An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied <it>in vitro </it>but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on <it>in vitro </it>systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the <it>in vitro </it>model system and model toxicant, respectively.</p> <p>Results</p> <p>The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.</p> <p>Conclusions</p> <p>Untargeted profiling of the polar and apolar metabolites of <it>in vitro </it>cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.</p

Ganglioside composition and histology of a spontaneous metastatic brain tumour in the VM mouse

Glycosphingolipid abnormalities have long been implicated in tumour malignancy and metastasis. Gangliosides are a family of sialic acid-containing glycosphingolipids that modulate cell–cell and cell–matrix interactions. Histology and ganglioside composition were examined in a natural brain tumour of the VM mouse strain. The tumour is distinguished from other metastatic tumour models because it arose spontaneously and metastasizes to several organs including brain and spinal cord after subcutaneous inoculation of tumour tissue in the flank. By electron microscopy, the tumour consisted of cells (15 to 20 μm in diameter) that had slightly indented nuclei and scant cytoplasm. The presence of smooth membranes with an absence of junctional complexes was a characteristic ultrastructural feature. No positive immunostaining was found for glial or neuronal markers. The total ganglioside sialic acid content of the subcutaneously grown tumour was low (12.6 ± 0.9 μg per 100 mg dry wt, n= 6 separate tumours) and about 70% of this was in the form of N-glycolylneuraminic acid. In contrast, the ganglioside content of the cultured VM tumour cells was high (248.4 ± 4.4 μg, n= 3) and consisted almost exclusively of N-acetylneuraminic acid. The ganglioside pattern of the tumour grown subcutaneously was complex, while GM3, GM2, GM1, and GD1a were the major gangliosides in the cultured tumour cells. This tumour will be a useful natural model for evaluating the role of gangliosides and other glycolipids in tumour cell invasion and metastasis. © 2001 Cancer Research Campaign http://www.bjcancer.co

Treatment with a corticotrophin releasing factor 2 receptor agonist modulates skeletal muscle mass and force production in aged and chronically ill animals

<p>Abstract</p> <p>Background</p> <p>Muscle weakness is associated with a variety of chronic disorders such as emphysema (EMP) and congestive heart failure (CHF) as well as aging. Therapies to treat muscle weakness associated with chronic disease or aging are lacking. Corticotrophin releasing factor 2 receptor (CRF2R) agonists have been shown to maintain skeletal muscle mass and force production in a variety of acute conditions that lead to skeletal muscle wasting.</p> <p>Hypothesis</p> <p>We hypothesize that treating animals with a CRF2R agonist will maintain skeletal muscle mass and force production in animals with chronic disease and in aged animals.</p> <p>Methods</p> <p>We utilized animal models of aging, CHF and EMP to evaluate the potential of CRF2R agonist treatment to maintain skeletal muscle mass and force production in aged animals and animals with CHF and EMP.</p> <p>Results</p> <p>In aged rats, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater extensor digitorum longus (EDL) force production, EDL mass, soleus mass and soleus force production compared to age matched untreated animals. In the hamster EMP model, we demonstrate that treatment with a CRF2R agonist for up to 5 months results in greater EDL force production in EMP hamsters when compared to vehicle treated EMP hamsters and greater EDL mass and force in normal hamsters when compared to vehicle treated normal hamsters. In the rat CHF model, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater EDL and soleus muscle mass and force production in CHF rats and normal rats when compared to the corresponding vehicle treated animals.</p> <p>Conclusions</p> <p>These data demonstrate that the underlying physiological conditions associated with chronic diseases such as CHF and emphysema in addition to aging do not reduce the potential of CRF2R agonists to maintain skeletal muscle mass and force production.</p