Characterization of oxidative stress in blood from diabetic vs. hypercholesterolaemic patients,using a novel synthesized marker

In the present study, we extend our novel concept of designing and using exogenous markers for the characterization of oxidative stress (OS) and OS-associated diseases. The aim was to use such a synthetic compound as a tool for studying OS in blood from diabetic and hypercholesterolaemic (Hc) patients. The marker used N-linoleoyl tyrosine (LT) was constructed from tyrosine and linoleic acid (LA); both components are known to be easily oxidized upon exposure to different types of reactive oxygen/nitrogen species (ROS/RNS), and to generate specific oxidized products, depending on the type of oxidants present in vivo. Using the LT probe, we showed that the ratios of oxidized LT to total LT (Ox-LT/LT) is significantly higher in blood samples obtained from diabetic patients, than in Hc patients or healthy control subjects. LC/MS analysis revealed that blood from diabetic patients oxidizes the marker with predominant formation of Ox-LT hydroperoxide (LT-OOH) and epoxide (epoxy-LT), where the LA moiety is oxidized to hydroperoxide and to epoxide, respectively. Analysis of oxysterol levels in these samples (GC/MS) revealed that the blood of both diabetic and Hc patients contained significantly more oxysterols than blood of control subjects. Consumption of pomegranate juice by diabetic patients for 3 months suppressed their blood capacity to oxidize the LT and similarly also reduced their blood oxysterol/total cholesterol ratio by 93%. The use of an exogenous marker to characterize OS in blood samples yields important information on the extent of OS, and can provide a fingerprint for the early identification of different pathological conditions associated with OS.     

Evolution of resident bird breeding phenology in a landscape with heterogeneous resource phenology and carryover effects

It is generally expected that, in environments with pronounced seasonal resource peaks, birds’ reproductive success will be maximised when nestlings’ peak food demand coincides with the timing of high food availability. However in certain birds that stay resident over winter, earlier breeding leads juveniles to join the winter flock earlier, which by the prior residence effect increases their success in breeding territory competition. This trade-off between reproduction and competition may explain why, in certain species, breeding phenology is earlier and asynchronous with the resource. This study extends a previous model of the evolution of breeding phenology in a single habitat type to a landscape with two habitat types: ‘early’ and ‘late’ resource phenology. The offspring’s natal habitat type has a carryover effect upon their competitive ability regardless of which habitat type they settle in to potentially breed. We find that, when the difference in resource phenology between habitats is small (weak carryover effect), breeding phenology in the late habitat evolves to occur earlier and more asynchronously than in the early habitat, to compensate for the competitive disadvantage to juveniles raised there. However if the difference is large (strong carryover effect), then the reproductive cost of earlier breeding outweighs the benefit of the compensation, so instead breeding phenology in the late habitat evolves to become more synchronous with the resource. Recruitment is generally asymmetric, from early to late habitat type. However if the early habitat is less frequent in the landscape or produces fewer offspring, then the asymmetry is reduced, and if there is some natal habitat-type fidelity, then recruitment can have an insular pattern, i.e. most recruits to each habitat type come from that same habitat type. We detail the different scenarios in which the different recruitment patterns are predicted, and we propose that they have implications for local adaptation.     

Incorporation of glycosylphosphatidylinositol-anchored granulocyte- macrophage colony-stimulating factor or CD40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles

The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4(+)- and CD8(+)-T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens.     

Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.     

Responses of Antarctic Iridaea cordata (Rhodophyta) tetraspores exposed to ultraviolet radiation

In Antarctica ozone depletion is highest during spring, coinciding with the reproduction of many seaweed species. Propagules are the life-stage of an alga most susceptible to environmental perturbations. Therefore, fertile thalli of Iridaea cordata (Turner) Bory (Rhodophyta) were collected in the eulittoral of King George Island (Antarctica) to examine spore susceptibility to ultraviolet radiation (UVR). In the laboratory, freshly released tetraspores were exposed to photosynthetically active radiation (PAR) (400–700 nm), PAR+UV-A (320–700 nm) or PAR+UV-A+UV-B (280–700 nm). Photosynthetic efficiency was measured during 1–8 h of exposure and after 48 h of recovery. Additionally, mycosporine-like amino acids (MAAs) and DNA damage were determined. Saturating irradiance of photosynthesis of freshly released tetraspores was 57 µmol photons m⁻² s⁻¹. Exposure to increasing fluence of PAR reduced photosynthetic efficiency. UVR further decreased the photosynthetic efficiencies of the tetraspores but spores were able to recover completely after UVR exposure and 2 days post-cultivation under low PAR. DNA damage was minimal and lesions were effectively repaired under photoreactivating light. Concentrations of the MAAs shinorine and palythine were higher in tetraspores treated with UVR than in spores only exposed to PAR. Generally, the tetraspores show a good UV tolerance. This flexible response of the tetraspores of this species to changing radiation conditions enables the alga to grow along a considerable depth gradient from the sublittoral to the eulittoral where they can be exposed to enhanced UVBR under conditions of stratospheric ozone depletion.     

Effects of long-term open-air exposure to fluoride,nitrogen compounds and SO2 on visible symptoms,pollutant accumulation and ultrastructure of Scots pine and Norway spruce seedlings

 Effects of SO₂, aqueous fluoride (NaF) and a solution of nitrogen compounds (NH₄NO₃) on the visible symptoms, pollutant accumulation and ultrastructure of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.] seedlings were studied in an open-air experiment lasting for 3 consecutive years. Visible injury symptoms were most pronounced in combination exposures and whenever F was applied. Visible symptoms correlated well with needle pollutant concentrations. Exposure to NaF increased needle F contents particularly when F was applied with SO₂ or NH₄NO₃. This suggests that a reduction in N or SO₂ emissions, in F polluted areas, could improve the condition of conifers via decreased accumulation of phytotoxic F in the needles. Norway spruce needles accumulated 2 – 10 times as much S and F as those of Scots pine. Microscopic observations showed various changes in the needle mesophyll cell ultrastructure. In both species, exposure to SO₂ increased significantly the amount of cytoplasmic vacuoles, suggesting detoxification of excess sulphate or low pH. F treatments resulted in a significant enlargement of plastoglobuli in Scots pine and a darkening of plastoglobuli in Norway spruce. All exposures enhanced the accumulation of lipid bodies. An increased portion of translucent plastoglobuli was most pronounced in N treatments. Many of the ultrastructural changes and visible symptoms appeared only as number of years exposed increased, indicating that long-term experiments are needed. Both visible symptoms and ultrastructural changes pointed to the more pronounced sensitivity of Norway spruce compared to Scots pine. Ultrastructural results mostly supported earlier qualitative observations of F, N and SO₂ effects on needle mesophyll cell ultrastructure. However, no reduction of thylakoids in SO₂ containing exposure or curling of thylakoids in F exposure could be detected in the present study. Received: 5 December 1994 / Accepted: 28 April 1995     

Quantitative Organization of GABAergic Synapses in the Molecular Layer of the Mouse Cerebellar Cortex

In the cerebellar cortex, interneurons of the molecular layer (stellate and basket cells) provide GABAergic input to Purkinje cells, as well as to each other and possibly to other interneurons. GABAergic inhibition in the molecular layer has mainly been investigated at the interneuron to Purkinje cell synapse. In this study, we used complementary subtractive strategies to quantitatively assess the ratio of GABAergic synapses on Purkinje cell dendrites versus those on interneurons. We generated a mouse model in which the GABA_A receptor α1 subunit (GABA_ARα1) was selectively removed from Purkinje cells using the Cre/loxP system. Deletion of the α1 subunit resulted in a complete loss of GABA_AR aggregates from Purkinje cells, allowing us to determine the density of GABA_AR clusters in interneurons. In a complementary approach, we determined the density of GABA synapses impinging on Purkinje cells using α-dystroglycan as a specific marker of inhibitory postsynaptic sites. Combining these inverse approaches, we found that synapses received by interneurons represent approximately 40% of all GABAergic synapses in the molecular layer. Notably, this proportion was stable during postnatal development, indicating synchronized synaptogenesis. Based on the pure quantity of GABAergic synapses onto interneurons, we propose that mutual inhibition must play an important, yet largely neglected, computational role in the cerebellar cortex.     

Living at the edge of the front; reduced predation risk to invasive round goby in a Great Lakes tributary

The objective of this research was to determine if the highly invasive round goby (Neogobius melanostomus) experiences lower predation risk during early stages of invasion. We compared round goby predation rates between a recently invaded area (occupied for ≈1 year) and a longer established area (≈7 years) of the Trent River, Ontario, Canada. Tethering trials were conducted in three habitat types, and comparable habitats in the two areas were similar in water temperature, velocity and depth. Predation rates of tethered round gobies were on average 27% lower in the recently invaded area. Reduced predation in the recently invaded area may be due to the short duration of round goby occupancy and/or differences in predator communities between the two study areas. Data before the round goby invasion suggest that predator communities were similar between the two range areas, but differences in predator abundance cannot be ruled out as a potential mechanism. Other possible mechanisms include a numerical or learned response by predators over time to a novel prey item. Reduced predation rate during the initial stages of invasion may contribute to the fitness of individuals that migrate into areas not previously occupied, and thus facilitate successful range expansion.     

A fuzzy guided genetic algorithm for operon prediction

Motivation: The operon structure of the prokaryotic genome isa critical input for the reconstruction of regulatory networksat the whole genome level. As experimental methods for the detectionof operons are difficult and time-consuming, efforts are beingput into developing computational methods that can use availablebiological information to predict operons. Method: A genetic algorithm is developed to evolve a startingpopulation of putative operon maps of the genome into progressivelybetter predictions. Fuzzy scoring functions based on multiplecriteria are used for assessing the ‘fitness’ ofthe newly evolved operon maps and guiding their evolution. Results: The algorithm organizes the whole genome into operons.The fuzzy guided genetic algorithm-based approach makes it possibleto use diverse biological information like genome sequence data,functional annotations and conservation across multiple genomes,to guide the organization process. This approach does not requireany prior training with experimental operons. The predictionsfrom this algorithm for Escherchia coli K12 and Bacillus subtilisare evaluated against experimentally discovered operons forthese organisms. The accuracy of the method is evaluated usingan ROC (receiver operating characteristic) analysis. The areaunder the ROC curve is around 0.9, which indicates excellentaccuracy. Contact: roschen_csir{at}rediffmail.com