A simple method to assess the oxidative susceptibility of low density lipoproteins

BACKGROUND: Oxidative modification of low density lipoproteins (LDL) is recognized as one of the major processes involved in atherogenesis. The in vitro standardized measurement of LDL oxidative susceptibility could thus be of clinical significance. The aim of the present study was to establish a method which would allow the evaluation of oxidative susceptibility of LDL in the general clinical laboratory. RESULTS: LDL was isolated from human plasma by selective precipitation with amphipathic polymers. The ability of LDL to form peroxides was assessed by measuring thiobarbituric acid reactive substances (TBARS) after incubation with Cu(2+) and H(2)O(2). Reaction kinetics showed a three-phase pattern (latency, propagation and decomposition phases) which allowed us to select 150 min as the time point to stop the incubation by cooling and EDTA addition. The mixture Cu(2+)/H(2)O(2) yielded more lipoperoxides than each one on its own at the same time end-point. Induced peroxidation was measured in normal subjects and in type 2 diabetic patients. In the control group, results were 21.7 ± 1.5 nmol MDA/mg LDL protein, while in the diabetic group results were significantly increased (39.0 ± 3.0 nmol MDA/mg LDL protein; p < 0.001). CONCLUSION: a simple and useful method is presented for the routine determination of LDL susceptibility to peroxidation in a clinical laboratory

Knockout studies reveal an important role of <i>plasmodium</i> lipoic acid protein ligase a1 for asexual blood stage parasite survival

Lipoic acid (LA) is a dithiol-containing cofactor that is essential for the function of a-keto acid dehydrogenase complexes. LA acts as a reversible acyl group acceptor and 'swinging arm' during acyl-coenzyme A formation. The cofactor is post-translationally attached to the acyl-transferase subunits of the multienzyme complexes through the action of octanoyl (lipoyl): &lt;i&gt;N&lt;/i&gt;-octanoyl (lipoyl) transferase (LipB) or lipoic acid protein ligases (LplA). Remarkably, apicomplexan parasites possess LA biosynthesis as well as scavenging pathways and the two pathways are distributed between mitochondrion and a vestigial organelle, the apicoplast. The apicoplast-specific LipB is dispensable for parasite growth due to functional redundancy of the parasite's lipoic acid/octanoic acid ligases/transferases. In this study, we show that &lt;i&gt;LplA1&lt;/i&gt; plays a pivotal role during the development of the erythrocytic stages of the malaria parasite. Gene disruptions in the human malaria parasite &lt;i&gt;P.falciparum&lt;/i&gt; consistently were unsuccessful while in the rodent malaria model parasite &lt;i&gt;P. berghei&lt;/i&gt; the &lt;i&gt;LplA1&lt;/i&gt; gene locus was targeted by knock-in and knockout constructs. However, the &lt;i&gt;LplA1&lt;/i&gt; &lt;sup&gt;(-)&lt;/sup&gt; mutant could not be cloned suggesting a critical role of LplA1 for asexual parasite growth &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. These experimental genetics data suggest that lipoylation during expansion in red blood cells largely occurs through salvage from the host erythrocytes and subsequent ligation of LA to the target proteins of the malaria parasite

The influence of soil communities on the temperature sensitivity of soil respiration

Soil respiration represents a major carbon flux between terrestrial ecosystems and the atmosphere, and is expected to accelerate under climate warming. Despite its importance in climate change forecasts, however, our understanding of the effects of temperature on soil respiration (RS) is incomplete. Using a metabolic ecology approach we link soil biota metabolism, community composition and heterotrophic activity, to predict RS rates across five biomes. We find that accounting for the ecological mechanisms underpinning decomposition processes predicts climatological RS variations observed in an independent dataset (n = 312). The importance of community composition is evident because without it RS is substantially underestimated. With increasing temperature, we predict a latitudinal increase in RS temperature sensitivity, with Q10 values ranging between 2.33 ±0.01 in tropical forests to 2.72 ±0.03 in tundra. This global trend has been widely observed, but has not previously been linked to soil communities

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests

In Vivo Regulation of Glycogen Synthase Kinase-3β (GSK3β) by Serotonergic Activity in Mouse Brain

The goal of this study was to determine if serotonergic activity, which is impaired in depression, regulates the phosphorylation of glycogen synthase kinase-3β (GSK3β) in mouse brain in vivo. GSK3β is inhibited by phosphorylation on serine-9 and is a target of the mood stabilizer lithium. Following administration to mice of d-fenfluramine to stimulate serotonin (5HT) release and reduce its reuptake, and clorgyline to inhibit 5HT catabolism, levels of phospho-Ser9-GSK3β were 300–400% of control levels in the prefrontal cortex, hippocampus, and striatum. Treatment with monoamine reuptake inhibitors fluoxetine and imipramine also increased the level of phospho-Ser9-GSK3β. Using receptor selective agonists and antagonists, 5HT1A receptors were found to mediate increases, and 5HT2 receptors decreases, in phospho-Ser9-GSK3β levels. This indicates that serotonergic regulation of the phosphorylation of GSK3β is achieved by a balance between the opposing actions of these 5HT receptor subtypes. These findings demonstrate for the first time that serotonergic activity regulates the phosphorylation of GSK3β and show that this regulation occurs in mammalian brain in vivo. These results raise the possibility that impaired inhibitory control of GSK3β may occur in conditions where serotonergic activity is dysregulated, such as in mood disorders

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib.

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers

Contribution of Direct Heating, Thermal Conduction and Perfusion During Radiofrequency and Microwave Ablation

Both radiofrequency (RF) and microwave (MW) ablation devices are clinically used for tumor ablation. Several studies report less dependence on vascular mediated cooling of MW compared to RF ablation. We created computer models of a cooled RF needle electrode, and a dipole MW antenna to determine differences in tissue heat transfer

Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast

Acknowledgments We thank Rebecca Shapiro for creating CaLC1819, CaLC1855 and CaLC1875, Gillian Milne for help with EM, Aaron Mitchell for generously providing the transposon insertion mutant library, Jesus Pla for generously providing the hog1 hst7 mutant, and Cathy Collins for technical assistance.Peer reviewedPublisher PD