Proteomic analysis of activity-dependent synaptic plasticity in hippocampal neurons

Following long-term treatment with bicuculline and tetrodotoxin (TTX) aimed at modifying synaptic activity in cultured neurons, we used a proteomic approach to identify the associated changes in protein expression. The neurons were left untreated, or treated with bicuculline or TTX, and fractionated by means of differential detergent extraction, after which the proteins in each fraction were separated by means of two-dimensional (2D) gel electrophoresis, and 57 proteins of interest were identified by mass spectrometry. The proteins that showed altered expression and/or post-translational modifications include proteins or enzymes involved in regulating cell and protein metabolism, the cytoskeleton, or mitochondrial activity. These results suggest that extensive alterations in neuronal protein expression take place as a result of increased or decreased synaptic activity.     

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.     

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.     

Entomopathogenic fungi from Argentina for the control of Schistocerca cancellata (Orthoptera: Acrididae) nymphs: fungal pathogenicity and enzyme activity

The South American locust Schistocerca cancellata (Serville) was the most serious agricultural pest in Argentina during the first half of the last century and remains as a threat when preventive control measures are relaxed in the outbreak area. In this study, we analysed in the laboratory, the effectiveness of 26 fungal strains (isolated from both insects and soil collected in Argentina) for S. cancellata control and determined the relationship between the chitinase, protease and lipase levels in these fungi and their insecticidal activities. We observed that Beauveria bassiana (isolate LPSC 1067) caused the highest mortality (90±1.03%), the highest values of chitinolytic, proteolytic and lipolytic activity were 6.13±0.05, 2.56±0.11 and 2.33±0.47, respectively, and the lowest median lethal time was 5.96 days. This is the first time that a wide variability in chitinase, protease and lipase activity as well as in virulence has been reported in a representative sample of different entomopathogenic fungal strains from Argentina.     

Heparin-like heparan sulfate from rabbit cartilage

Glycosaminoglycans were extracted from both young rabbit growth plate (GRP) and articular (ART) cartilage tissues and enzymatically treated to selectively eliminate chondroitin sulfates and hyaluronic acid. The procedure avoided any fractionation step that could enrich the extract with over- or under-sulfated species. Isolated heparan sulfate (HS) was characterized by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy to quantify their specific structural features and/or by mass spectrometry to establish the disaccharide composition. Both GRP and ART HSs, despite differing in their yield (GRP at least 100 times greater than ART), exhibited a surprisingly high degree of sulfation. Quantitative two-dimensional heteronuclear single-quantum coherence-NMR analysis of GRP HS revealed unusually high N-sulfated glucosamine and 2-O-sulfated iduronic acid contents, similar to heparin. The unique pentasaccharide sequence of the binding site for antithrombin was also detected in a significant amount. High-performance liquid chromatography mass spectrometry analysis of the enzymatic digests with a cocktail of heparin lyases of both cartilaginous HSs confirmed the NMR results. As well as the discovery of an unusual HS structure in the two different types of rabbit cartilage, the feasibility of the analytical method adopted here has been demonstrated within this study. Such a method can be used to isolate and analyze HS from both normal and pathologic tissues. Characterization of healthy and pathological HS structures will contribute to improve the understanding of diseases related to malfunctions of HS biosynthesis and/or metabolism.     

Transient expression of <Emphasis Type="Italic">Human papillomavirus</Emphasis> type 16 L2 epitope fused to N- and C-terminus of coat protein of <Emphasis Type="Italic">Potato virus X</Emphasis> in plants

Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108–120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2_108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2_108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2_108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2_108-120 epitope were found after both methods of vaccine delivery.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Enhancing the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic Arabidopsis thaliana via iterative metabolic engineering and genetic crossing

The synthesis and accumulation of long chain polyunsaturated fatty acids such as eicosapentaenoic acid has previously been demonstrated in the seeds of transgenic plants. However, the obtained levels are relatively low, indicating the need for further studies and the better definition of the interplay between endogenous lipid synthesis and the non-native transgene-encoded activities. In this study we have systematically compared three different transgenic configurations of the biosynthetic pathway for eicosapentaenoic acid, using lipidomic profiling to identify metabolic bottlenecks. We have also used genetic crossing to stack up to ten transgenes in Arabidopsis. These studies indicate several potential approaches to optimize the accumulation of target fatty acids in transgenic plants. Our data show the unexpected channeling of heterologous C20 polyunsaturated fatty acids into minor phospholipid species, and also the apparent negative metabolic regulation of phospholipid-dependent ??6-desaturases. Collectively, this study confirms the benefits of iterative approaches to metabolic engineering of plant lipid synthesis.     

Diketopiperazines produced by endophytic fungi found in association with two Asteraceae species

Diketopiperazine (DKP) derivatives, named colletopiperazine, fusaperazine C and E as well as four known DKPs were isolated from cultures of Colletotrichum gloeosporioides, Penicillium crustosum, both endophytic fungi isolated from Viguiera robusta, and a Fusarium spp., an endophyte of Viguiera arenaria, respectively. Their structures were established on the basis of their spectroscopic data. Conformational analysis of two known DKPs showed that folded conformations were as energetically stable as the extended one.