Lipid peroxidation and its inhibition in low density lipoproteins: quenching of cis-parinaric acid fluorescence.

The fluorescent polyunsaturated parinaric acid incorporated in LDL particles is highly sensitive to the concentration of peroxyl radicals in the aqueous medium, undergoing rapidly oxidative degradation, as detected by a quenching of fluorescence, without delay after radical generation in solution. Ascorbate, cysteine, and urate suppress the parinaric acid fluorescence decay promoted by peroxyl radicals generated at a constant rate (thermal decomposition of 2,2'-azo-bis(2-amidino-propane hydrochloride)) in a concentration-dependent manner. The chain-breaking efficiencies of these antioxidants are evaluated from the time interval (inhibition period) of parinaric acid protection from oxidative degradation. The results correlate with the inhibition periods of LDL oxidation as monitored by O2 consumption. Therefore, the sensitive and simple parinaric acid assay can be used as a semiquantitative screening test for the detection of potentially important water-soluble chain-breaking antioxidants. Conversely to O2 consumption, the absence of any initial lag phase of probe degradation attests to the sensitivity of the assay. An improved methodology based on second-derivative spectroscopy to follow the formation of conjugated diene isomers directly in the preparation without the need for lipid extraction also confirms the sensitivity of this assay. To assess the usefulness of parinaric acid assay, strong chain-breaking activities of caffeic and chlorogenic acids are reported.     

Partition of malathion in synthetic and native membranes

Partition coefficients of [14C]malathion in model and native membranes are affected by temperature, cholesterol content, and lipid chain length. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10-40 degrees C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol severely decreases partition and practically abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in malathion partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 225, 135 and 48 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. Partition values in native membranes decrease sequentially as follows: sarcoplasmic reticulum, mitochondria, brain microsomes, myelin and erythrocytes. This dependence parallels the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values.     

High throughput characterization of structural differences between closely related proteins in solution

Partitioning of a protein in an aqueous two-phase system (ATPS) is governed by interactions of the protein with aqueous media in the two phases. Here we describe how partitioning of proteins in a set of ATPS of different compositions can be used to quantify differences between 3D structures of closely related proteins. We also provide perspective on practical applications of the technology when comparative analysis of the higher-order structure of proteins is desired.     

Tracing geographical patterns of population differentiation in a widespread mangrove gastropod: genetic and geometric morphometrics surveys along the eastern African coast

In the present study, we assessed the inter‐ and intrapopulation genetic and morphological variation of Cerithidea decollata along the eastern coast of Africa. The population structure of C. decollata along the latitudinal gradient was examined by sequencing 420 bp of the mitochondrial cytochrome c oxidase I (COI) gene in 172 snails from 29 sites, in a combined analysis with geometric morphometrics in 1799 snails from 32 sites. Analysis of molecular variance and spatial analysis of molecular variance showed a moderate spatial population differentiation from Kenya to the Republic of South Africa, suggesting genetic divergence between the northern, central, and southern regions. This structure appears to be the result of life‐history traits combined with oceanographic features. Haplotype network and mismatch analysis suggest a recent population expansion during the Holsteinian interglacial period in the northern region and several colonization events in the central and southern regions. The morphometric approach suggests that morphological variation in shell shape is somewhat independent of the genetic divergence, revealing an overlap of shape across the latitudinal gradient but significant differences among‐population at a local level. This may indicate that similar ecological pressures are acting along the coast, leading to the occurrence of similar morphological characters. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 647–663.     

Changes in tree and liana communities along a successional gradient in a tropical dry forest in south-eastern Brazil

We investigated changes in species composition and structure of tree and liana communities along a successional gradient in a seasonally dry tropical forest. There was a progressive increase in tree richness and all tree structural traits from early to late stages, as well as marked changes in tree species composition and dominance. This pattern is probably related to pasture management practices such as ploughing, which remove tree roots and preclude regeneration by resprouting. On the other hand, liana density decreased from intermediate to late stages, showing a negative correlation with tree density. The higher liana abundance in intermediate stage is probably due to a balanced availability of support and light availability, since these variables may show opposite trends during forest growth. Predicted succession models may represent extremes in a continuum of possible successional pathways strongly influenced by land use history, climate, soil type, and by the outcomes of tree–liana interactions.     

A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism

In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.