Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease

Tyrosinase is a key enzyme in the synthesis of melanin in skin and hair and has also been proposed to contribute to the formation of neuromelanin (NM). The presence of NM, which is biochemically similar to melanin in peripheral tissues, identifies groups of neurons susceptible in Parkinson's disease (PD). Whether tyrosinase is beneficial or detrimental to neurons is unclear; whilst the enzyme activity of tyrosinase generates dopamine-quinones and other oxidizing compounds, NM may form a sink for such radical species. In the present study, we demonstrated that tyrosinase is expressed at low levels in the human brain. We found that mRNA, protein and enzyme activity are all present but at barely detectable levels. In cell culture systems, expression of tyrosinase increases neuronal susceptibility to oxidizing conditions, including dopamine itself. We related these in vitro observations to the human disease by assessing whether there was any genetic association between the gene encoding tyrosinase and idiopathic PD. We found neither genotypic or haplotypic association with three polymorphic markers of the gene. This argues against a strong genetic association between tyrosinase and PD, although the observed contribution to cellular toxicity suggests that a biochemical association is likely.     

Kin interactions and changing social structure during a population outbreak of feral house mice

Populations of feral house mice (Mus domesticus L.) in Australia undergo multiannual fluctuations in density, and these outbreaks may be partly driven by some change in behavioural self-regulation. In other vertebrate populations with multiannual fluctuations, changes in kin structure have been proposed as a causal mechanism for changes in spacing behaviour, which consequently result in density fluctuations. We tested the predictions of two alternative conceptual models based on kin selection in a population of house mice during such an outbreak. Both published models (Charnov & Finerty 1980; Lambin & Krebs 1991) propose that the level of relatedness between interacting individuals affects their behavioural response and that this changes with population density, though the nature of this relationship differs between the two models. Neither of the models was consistent with all observed changes in relatedness between interacting female mice; however, our results suggested that changes in kin structure still have potential for explaining why mouse outbreaks begin. Therefore, we have developed a variant of one of these conceptual models suggesting that the maintenance of female kin groups through the preceding winter significantly improves recruitment during the subsequent breeding season, and is therefore necessary for mouse outbreaks. We provide six testable predictions to falsify this hypothesis.     

Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.     

Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix

Archaea contain one or more proteins with homology to eukaryotic ORC/Cdc6 proteins. Sequence analysis suggests the existence of at least two subfamilies of these proteins, for which we propose the nomenclature ORC1 and ORC2. We have determined crystal structures of the ORC2 protein from the archaeon Aeropyrum pernix in complexes with ADP or a non-hydrolysable ATP analogue, ADPNP. Between two crystal forms, there are three crystallographically independent views of the ADP complex and two of the ADPNP complex. The protein molecules in the three complexes with ADP adopt very different conformations, while the two complexes with ADPNP are the same. These structures indicate that there is considerable conformational flexibility in ORC2 but that ATP binding stabilises a single conformation. We show that the ORC2 protein can bind DNA, and that this activity is associated with the C-terminal domain of the protein. We present a model for the interaction of the winged helix (WH) domain of ORC2 with DNA that differs from that proposed previously for Pyrobaculum aerophilum ORC/Cdc6.     

Application of genome-wide single nucleotide polymorphism typing: simple association and beyond

The International HapMap Project and the arrival of technologies that type more than 100,000 SNPs in a single experiment have made genome-wide single nucleotide polymorphism (GW-SNP) assay a realistic endeavor. This has sparked considerable debate regarding the promise of GW-SNP typing to identify genetic association in disease. As has already been shown, this approach has the potential to localize common genetic variation underlying disease risk. The data provided from this technology also lends itself to several other lines of investigation; autozygosity mapping in consanguineous families and outbred populations, direct detection of structural variation, admixture analysis, and other population genetic approaches. In this review we will discuss the potential uses and practical application of GW-SNP typing including those above and beyond simple association testing.     

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

A large T cell invagination with CD2 enrichment resets receptor engagement in the immunological synapse

T cell activation is driven by the TCR and complemented by costimulation. We have studied the dynamics of ligand-engagement of the costimulatory receptor CD2 in T cell/APC couples. Thousands of ligand-engaged CD2 molecules were included in a large T cell invagination at the center of the cellular interface within 1 min of cell couple formation. The structure and regulation of this invagination shared numerous features with phagocytosis and macropinocytosis. Three observations further characterize the invagination and the inclusion of CD2: 1) numerous ligand-engaged receptors were enriched in and internalized through the T cell invagination, none as prominently as CD2; 2) dissolution of the T cell invagination and CD2 engagement were required for effective proximal T cell signaling; and 3) the T cell invagination was uniquely sensitive to the affinity of the TCR for peptide-MHC. Based on this characterization, we speculate that the T cell invagination, aided by CD2 enrichment, internalizes parts of the TCR signaling machinery to reset T cell signaling upon agonist-mediated, stable APC contact.     

Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid

AIMS: The aim of the present study was to isolate and characterize a bacterium, strain EN3, capable of using diesel oil as a major carbon and energy source, and to analyse the enhancement of diesel oil degradation by this organism using synthetic mycolic acid (2-hexyl-3-hydroxyldecanoic acid). METHOD AND RESULTS: An actinomycete with the ability to degrade diesel oil was isolated from oil contaminated soil and characterized. The strain had phenotypic properties consistent with its classification in the genus Rhodococcus showing a 16S rRNA gene similarity of 99.7% with Rhodococcus baikonurensis DSM 44587(T). The ability of the characterized strain to degrade diesel oil at various concentrations (1000, 5000, 10 000 and 20 000 mg l(-1)) was determined. The effect of synthetic mycolic acid on the biodegradation of diesel oil was investigated at the 20 000 mg l(-1) concentration; the surfactant was added to the flask cultures at three different concentrations (10, 50 and 100 mg l(-1)) and degradation followed over 7 days. Enhanced degradation was found at all three concentrations of the surfactant. In addition, the enhancement of diesel oil degradation by other surfactants was observed. CONCLUSIONS: The synthetic mycolic acid has potential for the remediation of petroleum-contaminated sites from both an economic and applied perspective as it can stimulate biodegradation at low concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the synthesized mycolic acid can be used for potential applications in the bioremediation industries, for example, in oil spill clean-up, diesel fuel remediation and biostimulation.