Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid–Light-Harvesting Complex II Model Membranes

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.     

Clicking in Shallow Rivers: Short-Range Echolocation of Irrawaddy and Ganges River Dolphins in a Shallow,Acoustically Complex Habitat

Toothed whales (Cetacea, odontoceti) use biosonar to navigate their environment and to find and catch prey. All studied toothed whale species have evolved highly directional, high-amplitude ultrasonic clicks suited for long-range echolocation of prey in open water. Little is known about the biosonar signals of toothed whale species inhabiting freshwater habitats such as endangered river dolphins. To address the evolutionary pressures shaping the echolocation signal parameters of non-marine toothed whales, we investigated the biosonar source parameters of Ganges river dolphins (Platanista gangetica gangetica) and Irrawaddy dolphins (Orcaella brevirostris) within the river systems of the Sundarban mangrove forest. Both Ganges and Irrawaddy dolphins produced echolocation clicks with a high repetition rate and low source level compared to marine species. Irrawaddy dolphins, inhabiting coastal and riverine habitats, produced a mean source level of 195 dB (max 203 dB) re 1 µPa_pp whereas Ganges river dolphins, living exclusively upriver, produced a mean source level of 184 dB (max 191) re 1 µPa_pp. These source levels are 1–2 orders of magnitude lower than those of similar sized marine delphinids and may reflect an adaptation to a shallow, acoustically complex freshwater habitat with high reverberation and acoustic clutter. The centroid frequency of Ganges river dolphin clicks are an octave lower than predicted from scaling, but with an estimated beamwidth comparable to that of porpoises. The unique bony maxillary crests found in the Platanista forehead may help achieve a higher directionality than expected using clicks nearly an octave lower than similar sized odontocetes.     

Integrating multiple data sources to assess the distribution and abundance of bottlenose dolphins Tursiops truncatus in Scottish waters

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Expression of integrins α3β1 and α5β1 and GlcNAc β1,6 glycan branching influences metastatic melanoma cell migration on fibronectin

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of the best-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) and its products, β1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers. In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793 cell line) to the metastatic stage (WM1205Lu line), β1,6 glycosylation of melanoma cell surface proteins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatment with swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was stronger in metastatic cells, probably due to the higher content of GlcNAc β1,6-branched glycans on the main fibronectin receptors – integrins α5β1 and α3β1. Our results show that GlcNAc β1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.     

Trapping of European buprestid beetles in oak forests using visual and olfactory cues

Trapping approaches developed for the emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), were adapted for trapping several European oak buprestid species. These approaches included the use of natural leaf surfaces as well as green and purple plastic in sticky trap designs. Plastic surfaces were incorporated into novel ‘branch‐trap’ designs that each presented two 5 × 9‐cm² rectangular surfaces on a cardboard structure wrapped around the leaves of a branch. We used visual adult Agrilus decoys in an attempt to evoke male mating approaches toward the traps. Our first experiment compared the attractiveness of visual characteristics of the surfaces of branch‐traps. The second looked at the effect on trap captures of adding semiochemical lures, including manuka oil, (Z)‐3‐hexen‐1‐ol, and (Z)‐9‐tricosene. In total, 1 962 buprestid specimens including 14 species from the genus Agrilus were caught on 178 traps in a 22‐day time‐span. Overall, the green plastic‐covered branch‐traps significantly out‐performed the other trap designs. We further found that the presence of an EAB visual decoy placed on the trap surface often increased captures on these green traps, but this effect was stronger for certain Agrilus species than for others. The visual decoy was particularly important for the most serious pest detected, Agrilus biguttatus Fabricius, which was captured 13 times on traps with decoys, but only once without a decoy. There were some small but significant effects of odor treatment on the capture of buprestids of two common species, Agrilus angustulus Illiger and Agrilus sulcicollis Lacordaire. There were also 141 Elateridae specimens on these traps, which were not influenced by trap type or decoys. The results suggest that small branch‐traps of this nature can provide a useful new tool for monitoring of buprestids, which have the potential to be further optimized with respect to visual and olfactory cues.     

Development of a fluorescent monoclonal antibody‐based assay to measure the allosteric effects of synthetic peptides on self‐oligomerization of AGR2 protein

Many regulatory proteins are homo‐oligomeric and designing assays that measure self‐assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer‐associated oncoprotein AGR2. A two site‐sandwich microtiter assay (^2SMTA) was designed using a DyLight800‐labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self‐peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N‐terminal region of AGR2 increase in trans oligomer stability as defined using the ^2SMTA assay. A DSS‐crosslinking assay that traps the AGR2 dimer through K95‐K95 adducts confirmed that Δ45‐AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt‐AGR2, Δ45‐AGR2 (more stable dimer), and monomeric AGR2^E60A revealed that Δ45‐AGR2 was more active in binding to Reptin than either wt‐AGR2 or the AGR2^E60A mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential.     

The antioxidant acetylcysteine reduces oxidative stress by decreasing level of AOPPs

Oxidative stress and especially its connection with many diseases has been discussed much recently. Among markers of oxidative stress there appear new and quite specific ones called advanced oxidation protein products (AOPPs). We tried to influence the level of AOPPs by an antioxidant therapy with N-acetylcysteine. Fourteen individuals with many cardiovascular risk factors were examined. All these patients were administered acetylcysteine (NAC) 600 mg/day orally during 20 days. Before starting the therapy we determined AOPP, albumin cobalt binding (ACB), glucose, creatinine, urea, ALT, AST, cholesterol, LDL, HDL and triglycerides values in peripheral venous blood in all individuals. After finishing our intervention we determined AOPP, ACB and glucose level again. Our results show a statistically significant decrease in AOPP levels after 20-day N-acetylcysteine therapy (medians, initially 82.2, at study end 74.3 umol/l, p = 0.039). We demonstrate a significant decrease in AOPP levels after 20-day N-acetylcysteine therapy in dose 600 mg/day.     

Construction of a bicistronic proangiogenic expression vector and its application in experimental angiogenesis in vivo

Manipulation of angiogenesis in vivo is an example of successful gene therapy strategies. Overexpression of angiogenic genes like VEGF, FGF or PDGF causes new vessel formation and improves the clinical state of patients. Gene therapy is a very promising procedure but requires large amounts of pharmaceutical-grade plasmid DNA. In this regard we have constructed a bicistronic plasmid DNA vector encoding two proangiogenic factors, VEGF165 and FGF-2. The construct (pVIF) contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) which permits both genes to be translated from a single bicistronic mRNA. The IRES sequence allows for a high efficiency of gene expression in vivo. The pVIF vector was characterized in vitro and in vivo. In vivo angiogenesis studies showed that the bicistronic vector encoding two proangiogenic factors induces the formation of new vessels significantly more than pVEGF165 or pFGF-2 alone. In our opinion the combined proangiogenic approach with VEGF165 and FGF-2 is more powerful and efficient than single gene therapy. We also postulate that IRES sequence can serve as a useful device improving efficiency of gene therapy.