Contrasting success in the restoration of plant and phytophagous beetle assemblages of species-rich mesotrophic grasslands

Over the last 60 years changes to the management of species-rich mesotrophic grasslands have resulted in the large-scale loss and degradation of this habitat across Europe. Restoration of such grasslands on agriculturally improved pastures provides a potentially valuable approach to the conservation of these threatened areas. Over a four-year period a replicated block design was used to test the effects of seed addition (green hay spreading and brush harvest collection) and soil disturbance on the restoration of phytophagous beetle and plant communities. Patterns of increasing restoration success, particularly where hay spreading and soil disturbance were used in combination, were identified for the phytophagous beetles. In the case of the plants, however, initial differences in restoration success in response to these same treatments were not followed by subsequent temporal changes in plant community similarity to target mesotrophic grassland. It is possible that the long-term consequences of the management treatments would not be the establishment of beetle and plant communities characteristic of the targets for restoration. Restoration management to enhance plant establishment using hay spreading and soil disturbance techniques would, however, still increase community similarity in both taxa to that of species-rich mesotrophic grasslands, and so raise their conservation value.

The stimulation of mitogenic signaling pathways by N-POMC peptides

The N-terminal fragment of pro-opiomelancortin (POMC) has been shown previously to act as an adrenal mitogen. However, little is known about the molecular mechanisms by which mitogenesis is stimulated, although it has been shown that N-POMC1-28 Stimulates the ERK pathway in human H295R cells. We have investigated signaling stimulated by N-POMC1-28 and N-POMC1-49 in the mouse Y1 cell line and found that both peptides stimulate ERK phosphorylation with maximal stimulation being achieved within 5 min. Similar results were observed for both MEK and c-Raf phosphorylation, although N-POMC1-49 stimulated the phosphorylation of Akt more robustly than N-POMC1-28. We also investigated the expression of tyrosine kinase receptors in adrenal cells. PCR utilizing degenerate primers was performed on cDNA from both Y1 cells and rat adrenal tissue. Sequencing of 114 clones from each cDNA population revealed the expression of a number of receptors, several of which have not been described previously in the adrenal. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

A novel method for production of lipid hydroperoxide- or oxysterol-rich low-density lipoprotein

LDL oxidation may be important in atherosclerosis. Extensive oxidation of LDL by copper induces increased uptake by macrophages, but results in decomposition of hydroperoxides, making it more difficult to investigate the effects of hydroperoxides in oxidised LDL on cell function. We describe here a simple method of oxidising LDL by dialysis against copper ions at 4 degrees C, which inhibits the decomposition of hydroperoxides, and allows the production of LDL rich in hydroperoxides (626 +/- 98 nmol/mg LDL protein) but low in oxysterols (3 +/- 1 nmol 7-ketocholesterol/mg LDL protein), whilst allowing sufficient modification (2.6 +/- 0.5 relative electrophoretic mobility) for rapid uptake by macrophages (5.49 +/- 0.75 mu g I-125-labelled hydroperoxide-rich LDL vs. 0.46 +/- 0.04 mu g protein/mg cell protein in 18 h for native LDL). By dialysing under the same conditions, but at 37 degrees C, the hydroperoxides are decomposed extensively and the LDL becomes rich in oxysterols. This novel method of oxidising LDL with high yield to either a hydroperoxide- or oxysterol-rich form by simply altering the temperature of dialysis may provide a useful tool for determining the effects of these different oxidation products on cell function. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo

The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)(4)AhxB (Ahx is 6-aminohexanoic acid and B is beta-alanine) CPP-PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)(4)AhxB-PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)(4)AhxB-PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)(4)AhxB-PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R(9)F(2) CPP significantly decreased the efficacy of the resulting PPMO (CPP-PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Role of gamma-MSH peptides in the regulation of adrenal steroidogenesis

Alpha-, beta- and gamma-melanocyte stimulating hormones (MSHs) are peptides derived from the ACTH precursor, pro-opiomelanocortin. All three peptides have been highly conserved throughout evolution but their exact biological function in mammals is still largely obscure. In recent years, there has been a surge of interest in alpha-MSH and its role in the regulation of feeding. Gamma-MSH by contrast has been shown to be involved in the regulation of adrenal steroidogenesis and also has effects on the cardiovascular and renal systems. This review will provide an overview of the role that gamma-MSH peptides play in the regulation of adrenal steroidogenesis. (c) 2005 Elsevier Inc. All rights reserved.

Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3D(pol)) in vitro

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 313 (VTg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV ere has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 313 peptides has now been determined, and the role of the FMDV ere (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

The role of the melanocortin 3 receptor in mediating the effects of gamma-MSH peptides on the adrenal

The pro-opiomelanocortin (POMC)-derived peptides, pro-gamma-MSH (16K fragment), and Lys-gamma(3)-MSH, have been shown to potentiate the steroidogenic action of corticotrophin (ACTH) on the adrenal cortex. Using a continuously perfused adrenal cell column system, we have tested the hypothesis that gamma-MSH peptides exert their effect through the Melanocortin 3 Receptor (MC3-R), since this is the only known receptor to have high affinity for gamma-MSH peptides and has been suggested to be expressed in the rat adrenal. To investigate this hypothesis we tested whether the MC3-R agonist MTII and antagonist SHU9119 could mimic or block the actions of pro-gamma-MSH. We found that MTII could not mimic, and SHU9119 could not block pro-gamma-MSH mediated potentiation of ACTH-induced steroidogenesis. These results suggest that the MC3-R is not involved in mediating the potentiation effect, adding further evidence to the argument that another melanocortin receptor exists.

Savory peptides present in Moromi obtained from soy sauce fermentation of yellow soybean

The presence of savory peptides in moromi has been investigated. Moromi was prepared by fermenting yellow soybean using Aspergillus oryzae as the starter at the first step (mold fermentation) and 20% brine solution at the next step (brine fermentation). The moromi was then ultrafiltered stepwise using membranes with MW cut-offs of 10,000, 3,000, and 500 Da, respectively. The fraction with MW < 500 Da was chromatographed using Sephadex G-25 SF to yield four fractions, 1-4. Analysis of soluble peptides, NaCl content, alpha-amino nitrogen, amino acid composition, peptide profile using CE coupled with DAD, taste profile and free glutamic acid content, were performed for each fraction. Fraction 2 contained a relatively high total glutamic acid content, but a relatively low free glutamic acid content and had the highest umami taste. This fraction also had more peptides containing non-aromatic amino acids than the other fractions. The peptides present in fraction 2 may play a role, at least in part, in its intense umami taste.

Supramolecular parallel beta-sheet and amyloid-like fibril forming peptides using delta-aminovaleric acid residue

Four terminally blocked tripeptides containing delta-aminovaleric acid residue self-assemble to form supramolecular beta-sheet structures as are revealed from their FT-IR data. Single crystal X-ray diffraction studies of two representative peptides also show that they form parallel beta-sheet structures. Self-aggregation of these beta-sheet forming peptides leads to the formation of fibrillar structures, as is evident from scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images. These peptide fibrils bind to a physiological dye, Congo red and exhibit a typical green-gold birefringence under polarized light, showing close resemblance to neurodegenerative disease causing amyloid fibrils. (c) 2005 Elsevier Ltd. All rights reserved.

Spontaneous condensation in DNA-polystyrene-b-poly(l-lysine) polyelectrolyte block copolymer mixtures

We investigated the condensation of calf thymus DNA by amphiphilic polystyrene(m)-b-poly(l-lysine)(n) block copolymers (PSm-b- PLys(n), m, n = degree of polymerization), using small-angle X-ray scattering, polarized optical microscopy and laser scanning confocal microscopy. Microscopy studies showed that the DNA condenses in the form of fibrillar precipitates, with an irregular structure, due to electrostatic interactions between PLys and DNA. This is not modified by the presence of hydrophobic PS block. Scattering experiments show that the structure of the polyplexes corresponds to a local order of DNA rods which becomes more compact upon increasing n. It can be concluded that for DNA/ PSm-b- PLys(n) polyplexes, the balance between the PLys block length and the excess charge in the system plays an essential role in the formation of a liquid crystalline phase.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes, Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin I cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins. (C) 2009 Elsevier Inc. All rights reserved.

Differential uptake of subfractions of triglyceride-rich lipoproteins by THP-1 macrophages

It is well known that raised plasma triglycerides (TG) are positively linked to the development of coronary heart disease. However, triglycerides circulate in a range of distinct lipoprotein subtractions and the relative atherogenicity of these subtractions is not clear. In this study, three fractions of triglyceride rich lipoprotein (TRL) were isolated from normolipidaemic males according to their differing Svedberg flotation (S-f) rates: chylomicron (CM, S-f > 400), very low-density lipoprotein (VLDL)-1 (S-f 60-400) and VLDL-2 (S-f 20-60). These fractions were incubated with THP-1 monocyte-derived macrophages for determination of cholesterol and TG accumulation, in the presence and absence of the lipoprotein lipase (LPL) inhibitor orlistat. Expression of LDL receptor related protein (LRP) and apolipoprotein B48 receptor (apoB48R) was also examined in both differentiating monocytes, and monocyte-derived macrophages, incubated with TRL. VLDL-I caused a significantly greater accumulation of TG within macrophages compared to VLDL-2. Binding studies also tended to show a greater preference for VLDL-1. No change in expression of LRP or apoB48R was observed in fully differentiated macrophages incubated with VLDL-1, VLDL-2 or CM, although a greater expression of LRP mRNA was observed in differentiating monocytes exposed to VLDL-1, compared to those incubated with CM or VLDL-2. TG loading in response to all three TRL fractions was blocked by orlistat, suggesting that it is likely that the major pathway for uptake of TG was hydrolysis by LPL. Calculations suggested that direct uptake of particles accounts for between 12 and 25% of total TAG uptake. In conclusion, THP monocyte-derived macrophages demonstrate a preference for VLDL-1, both through the LPL pathway and by direct uptake of whole particles. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) A1 from human macrophage foam cells

High circulating levels of triglyceride-rich lipoproteins (TGRL) represent an independent risk factor for coronary artery disease. Here, we show that TGRL inhibit the efflux of cholesterol from 'foam cell' macrophages to lipid-poor apolipoprotein (apo) A1, and may thereby inhibit arterial reverse cholesterol transport and promote the formation of atherosclerotic lesions. Human (THP-1) monocyte-derived macrophages were pre-incubated (48h) with acetylated low-density lipoprotein (AcLDL) to provide a foam cell model of cholesterol efflux to apoA1. Pre-incubation of macrophage 'foam cells' with TGRL (0-200 mug/ml, 0-24 h) inhibited the efflux of exogenously radiolabelled ([H-3]), endogenously synthesised ([C-14]) and cellular cholesterol mass to lipid-poor apoA1, but not control medium, during a (subsequent) efflux period. This inhibition is dependent upon the length of prior exposure to, and concentration of, TGRL employed, but is independent of changes in intracellular triglyceride accumulation or turnover of the cholesteryl ester pool. Despite the negative impact of TGRL on cholesterol efflux, major proteins involved in this process-namely apoE, ABCA1, SR-B1 and caveolin-1-were unaffected by TGRL pre-incubation, suggesting that exposure to these lipoproteins inhibits an alternate, and possibly novel, anti-atherogenic pathway. (C) 2003 Elsevier Ireland Ltd. All rights reserved.