Isolation and 2‐D‐DIGE proteomic analysis of intracellular and extracellular forms of Listeria monocytogenes

The pathogenicity of Listeria monocytogenes is related to its ability of invading and multiplying in eukaryotic cells. Its main virulence factors are now well characterized, but limited proteomic data is available concerning its adaptation to the intracellular environment. In this study, L. monocytogenes EGD (serotype 1/2a) grown in human THP‐1 monocytes (24 h) were successfully separated from host organelles and cytosolic proteins by differential and isopycnic centrifugation. For control, we used cell homogenates spiked with bacteria grown in broth. Proteomes from both forms of bacteria were compared using a 2‐D‐DIGE approach followed by MALDI‐TOF analysis to identify proteins. From 1684 distinct spots, 448 were identified corresponding to 245 distinct proteins with no apparent contamination of host proteins. Amongst them, 61 show underexpression (stress defense; transport systems, carbon metabolism, pyrimidines synthesis, D ‐Ala‐D ‐Ala ligase) and 22 an overexpression (enzymes involved in the synthesis of cell envelope lipids, glyceraldehyde‐3‐phosphate, pyruvate and fatty acids). Our proteomic analysis of intracellular L. monocytogenes (i) suggests that bacteria thrive in a more favorable environment than extracellularly, (ii) supports the concept of metabolic adaptation of bacteria to intracellular environment and (iii) may be at the basis of improved anti‐Listeria therapy.     

The role of gap junction communication and oxidative stress in the propagation of toxic effects among high-dose α-particle-irradiated human cells

We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase.     

Human ability to detect kinship in strangers' faces: effects of the degree of relatedness

The resemblance between human faces has been shown to be a possible cue in recognizing the relatedness between parents and children, and more recently, between siblings. However, the general inclusive fitness theory proposes that kin-selective behaviours are also relevant to more distant relatives, which requires the detection of larger kinship bonds. We conducted an experiment to explore the use of facial clues by ‘strangers’, i.e. evaluators from a different family, to associate humans of varying degrees of relatedness. We hypothesized that the visual capacity to detect relatedness should be weaker with lower degrees of relatedness. We showed that human adults are capable of (although not very efficient at) assessing the relatedness of unrelated individuals from photographs and that visible facial cues vary according to the degree of relatedness. This sensitivity exists even for kin pair members that are more than a generation apart and have never lived together. Collectively, our findings are in agreement with emerging knowledge on the role played by facial resemblance as a kinship cue. But we have progressed further to show how the capacity to distinguish between related and non-related pairs applies to situations relevant to indirect fitness.     

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein,survival of motor neuron

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5′ cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.     

Leaf senescence is accompanied by an early disruption of the microtubule network in Arabidopsis

The dynamic assembly and disassembly of microtubules (MTs) is essential for cell function. Although leaf senescence is a well-documented process, the role of the MT cytoskeleton during senescence in plants remains unknown. Here, we show that both natural leaf senescence and senescence of individually darkened Arabidopsis (Arabidopsis thaliana) leaves are accompanied by early degradation of the MT network in epidermis and mesophyll cells, whereas guard cells, which do not senesce, retain their MT network. Similarly, entirely darkened plants, which do not senesce, retain their MT network. While genes encoding the tubulin subunits and the bundling/stabilizing MT-associated proteins (MAPs) MAP65 and MAP70-1 were repressed in both natural senescence and dark-induced senescence, we found strong induction of the gene encoding the MT-destabilizing protein MAP18. However, induction of MAP18 gene expression was also observed in leaves from entirely darkened plants, showing that its expression is not sufficient to induce MT disassembly and is more likely to be part of a Ca(2+)-dependent signaling mechanism. Similarly, genes encoding the MT-severing protein katanin p60 and two of the four putative regulatory katanin p80s were repressed in the dark, but their expression did not correlate with degradation of the MT network during leaf senescence. Taken together, these results highlight the earliness of the degradation of the cortical MT array during leaf senescence and lead us to propose a model in which suppression of tubulin and MAP genes together with induction of MAP18 play key roles in MT disassembly during senescence.     

Both Hepatic and Body Iron Stores Are Increased in Dysmetabolic Iron Overload Syndrome. A Case-Control Study

Background & Aims

Hepatic iron is increased in dysmetabolic iron overload syndrome (DIOS). Whether this reflects elevated body iron stores is still debated. The study was aimed at assessing body iron stores in DIOS patients by calculating the amount of mobilized iron (AMI).

Methods

We conducted a prospective case-control study comparing AMI in 12 DIOS patients and 12 overweight normoferritinemic subjects matched on BMI and age. All participants were phlebotomized until serum ferritin dropped ≤ 50μg/L.

Results

The two groups were comparable with respect to metabolic abnormalities and differed according to serum ferritin levels only. AMI was significantly (p<0.0001) higher in DIOS (2.5g±0.7) than in controls (0.8g±0.3). No side effects were related to phlebotomies.     

RNA localization in the cytoplasm

RNA localization in subcytoplasmic areas is a process known for more than twenty years, and more than a hundred RNAs have now been shown to be spatially regulated. In most cases, RNA localization is involved in cell polarity, either by reading spatial clues and translating them into a spatial regulation of gene expression, or more directly by controlling cytoskeletal polarity. In this review, the various functions of RNA localization will be presented, and by analyzing two examples, Ash1 mRNA in yeast and retroviral genomic RNAs in mammals, the reader will be taken step by step into the detailed mechanisms of this fascinating process.