Role of TGF-β1 and MAP Kinases in the Antiproliferative Effect of Aspirin in Human Vascular Smooth Muscle Cells

Background

We aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-β1).

Methodology/Principal Findings

VSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-β1 was able to reverse this effect. ASA (2 mM) induced TGF-β1 secretion; however it was unable to induce Smad activation. ASA increased p38^MAPK phosphorylation in a TGF-β1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-β1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged.

Conclusions/Significance

In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-β1 and p38^MAPK. Pre-surgical serum levels of TGF- β1 from bypass patients who took aspirin at antiplatelet doses did not change.     

CD8+ T Cells Cause Disability and Axon Loss in a Mouse Model of Multiple Sclerosis

Background

The objective of this study was to test the hypothesis that CD8⁺ T cells directly mediate motor disability and axon injury in the demyelinated central nervous system. We have previously observed that genetic deletion of the CD8⁺ T cell effector molecule perforin leads to preservation of motor function and preservation of spinal axons in chronically demyelinated mice.

Methodology/Principal Findings

To determine if CD8⁺ T cells are necessary and sufficient to directly injure demyelinated axons, we adoptively transferred purified perforin-competent CD8⁺ spinal cord-infiltrating T cells into profoundly demyelinated but functionally preserved perforin-deficient host mice. Transfer of CD8⁺ spinal cord-infiltrating T cells rapidly and irreversibly impaired motor function, disrupted spinal cord motor conduction, and reduced the number of medium- and large-caliber spinal axons. Likewise, immunodepletion of CD8⁺ T cells from chronically demyelinated wildtype mice preserved motor function and limited axon loss without altering other disease parameters.

Conclusions/Significance

In multiple sclerosis patients, CD8⁺ T cells outnumber CD4⁺ T cells in active lesions and the number of CD8⁺ T cells correlates with the extent of ongoing axon injury and functional disability. Our findings suggest that CD8⁺ T cells may directly injure demyelinated axons and are therefore a viable therapeutic target to protect axons and motor function in patients with multiple sclerosis.     

Proteotyping of human haptoglobin by MALDI-TOF profiling: Phenotype distribution in a population of toxic oil syndrome patients

Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981 as a consequence of the ingestion of an aniline-adulterated oil illegally marketed as edible. TOS affected more than 20 000 people and produced over 400 deaths in the first 18 months after the outbreak. There is evidence that genetic factors could play a role in the susceptibility of individuals towards the disease. Recently, we suggested that haptoglobin (Hp) polymorphism could also play a role in TOS. To provide a rapid method for high-throughput Hp phenotyping, we developed a two-step MALDI-TOF procedure that allows specific identification of the three common Hp alpha chains. Resolution of the homologous alpha-1s and alpha-1f chains, which have a mass difference of only 0.043 Da, is obtained after guanidination of the protein with O-methylisourea. We applied this procedure to the study of the distribution of the Hp alleles HP(1s), HP(1f), HP(2) in a control versus a TOS-affected population, both originally exposed to the toxic oil. The MALDI-TOF proteotyping method was validated by a parallel analysis of the serum samples by 2-DE. Data obtained from 54 TOS cases and 48 control individuals indicate significant differences in the distribution of Hp phenotypes in the two populations.     

A phyloproteomic characterization of in vitro autophosphorylation in calcium-dependent protein kinases

Calcium-dependent protein kinases (CDPKs) are a novel class of signaling molecules that have been broadly implicated in relaying specific calcium-mediated responses to biotic and abiotic stress as well as developmental cues in both plants and protists. Calcium-dependent autophosphorylation has been observed in almost all CDPKs examined, but a physiological role for autophosphorylation has not been demonstrated. To date, only a handful of autophosphorylation sites have been mapped to specific residues within CDPK amino acid sequences. In an attempt to gain further insight into this phenomenon, we have mapped autophosphorylation sites and compared these phosphorylation patterns among multiple CDPK isoforms. From eight CDPKs and two CDPK-related kinases from Arabidopsis thaliana and Plasmodium falciparum, 31 new autophosphorylation sites were characterized, which in addition to the previously described sites, allowed the identification of five conserved loci. Of the 35 total sites analyzed approximately one-half were observed in the N-terminal variable domain. Homology models were generated for the protein kinase and calmodulin-like domains, each containing two of the five conserved sites, to allow intelligent speculation regarding subsequent lines of investigation.     

Mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62

Signaling cascades that control adipogenesis are essential in the regulation of body weight and obesity. The adaptor p62 controls pathways that modulate cell differentiation. We report here that p62(-/-) mice develop mature-onset obesity, leptin resistance, as well as impaired glucose and insulin intolerance. The metabolic rate was significantly reduced in p62(-/-) nonobese mice, which displayed increased mRNA levels of PPAR-gamma and reduced levels of UCP-1 in adipose tissue. Basal activity of ERK was enhanced in fat from nonobese mutant mice. Embryo fibroblasts from p62(-/-) mice differentiated better than the wild-type controls into adipocytes, which was abrogated by pharmacological inhibition of the ERK pathway. p62 is induced during adipocyte differentiation and inhibits ERK activation by direct interaction. We propose that p62 normally antagonizes basal ERK activity and adipocyte differentiation and that its loss leads to the hyperactivation of ERK that favors adipogenesis and obesity.     

Integration of Retrotransposons-Based Markers in a Linkage Map of Barley

A deeper understanding of random markers is important if they are to be employed for a range of objectives. The sequence specific amplified polymorphism (S-SAP) technique is a powerful genetic analysis tool which exploits the high copy number of retrotransposon long terminal repeats (LTRs) in the plant genome. The distribution and inheritance of S-SAP bands in the barley genome was studied using the Steptoe × Morex (S × M) double haploid (DH) population. Six S-SAP primer combinations generated 98 polymorphic bands, and map positions were assigned to all but one band. Eight putative co-dominant loci were detected, representing 16 of the mapped markers. Thus at least 81 of the mapped S-SAP loci were dominant. The markers were distributed along all of the seven chromosomes and a tendency to cluster was observed. The distribution of S-SAP markers over the barley genome concurred with the knowledge of the high copy number of retrotransposons in plants. This experiment has demonstrated the potential for the S-SAP technique to be applied in a range of analyses such as genetic fingerprinting, marker assisted breeding, biodiversity assessment and phylogenetic analyses.     

The impacts of climate change in coastal marine systems

Anthropogenically induced global climate change has profound implications for marine ecosystems and the economic and social systems that depend upon them. The relationship between temperature and individual performance is reasonably well understood, and much climate-related research has focused on potential shifts in distribution and abundance driven directly by temperature. However, recent work has revealed that both abiotic changes and biological responses in the ocean will be substantially more complex. For example, changes in ocean chemistry may be more important than changes in temperature for the performance and survival of many organisms. Ocean circulation, which drives larval transport, will also change, with important consequences for population dynamics. Furthermore, climatic impacts on one or a few 'leverage species' may result in sweeping community-level changes. Finally, synergistic effects between climate and other anthropogenic variables, particularly fishing pressure, will likely exacerbate climate-induced changes. Efforts to manage and conserve living marine systems in the face of climate change will require improvements to the existing predictive framework. Key directions for future research include identifying key demographic transitions that influence population dynamics, predicting changes in the community-level impacts of ecologically dominant species, incorporating populations' ability to evolve (adapt), and understanding the scales over which climate will change and living systems will respond.     

MIDAS: software for analysis and visualisation of interallelic disequilibrium between multiallelic markers

Background  

Various software tools are available for the display of pairwise linkage disequilibrium across multiple single nucleotide polymorphisms. The HapMap project also presents these graphics within their website. However, these approaches are limited in their use of data from multiallelic markers and provide limited information in a graphical form.     

Uncoupling the central spindle-associated function of the chromosomal passenger complex from its role at centromeres

Survivin is a component of the chromosomal passenger complex (CPC) that plays a role in maintenance of an active spindle checkpoint and in cytokinesis. To study whether these different functions can be attributed to distinct domains within the Survivin protein, we complemented Survivin-depleted cells with a variety of point- and deletion-mutants of Survivin. We show that an intact baculovirus IAP repeat (BIR) domain is required for proper spindle checkpoint functioning, but dispensable for cytokinesis. In line with this, mutants lacking an intact BIR domain localized normally to the central spindle, but their localization to inner centromeres was severely perturbed. Consequently, these mutants failed to recruit Aurora B, Borealin/Dasra B, and BubR1 to centromeres and kinetochores, but they had retained the ability to recruit Aurora B and Borealin/Dasra B to the midzone and midbody. Thus, the C terminus of Survivin is sufficient for central spindle localization and execution of cytokinesis, but the additional presence of a functional BIR domain is essential for centromere targeting and spindle checkpoint function. Importantly, our data show that the function of the CPC at the centromere can be separated from its function at the central spindle and that execution of cytokinesis does not require prior concentration of the CPC at centromeres.