Effect of hyperglycemia on insulin receptor signaling in the cultured retinal Müller glial cells

Hyperglycemia and impaired insulin signaling are considered as major factors in the retinal pathology in diabetic retinopathy (DR). Numerous reports support that these two factors damage retinal glial as well as neuronal cells early in diabetes. However, it is not known whether diabetic induced hyperglycemia causes a depression to the insulin signaling. In this study we utilized a well characterized cultured Muller cells (TR-MUL) where we found a high expression of insulin receptor molecules. TR-MUL Cells were treated with high glucose, glutamate and hydrogen peroxide, and activated with insulin. Following treatments, cell lysates were analyzed by immunoblotting experiments for insulin receptor (IRβ) and insulin receptor substrate (IRS1). In addition, cell lysates were immunoprecipitated using antibodies against insulin receptor proteins to analyze tyrosine phosphorylation and serine phosphorylation of insulin receptor proteins. Results indicate that hyperglycemia did not affect the expression of insulin receptor proteins in cultured TR-MUL cells. Although, hyperglycemia seems to inhibit the interaction between IRS1 and IRβ. Hydrogen peroxide increased the tyrosine phosphorylation of insulin receptor proteins but excess glutamate could not affect the insulin receptor proteins indicating that glutamate may not cause oxidative stress in TR-MUL cells. Hyperglycemia lowered serine phosphorylation of IRS^ser632 and IRS^ser1101 however, IRS^ser307 was not affected. Thus, hyperglycemia may not affect insulin signaling through tyrosine phosphorylation of insulin receptor proteins but may inhibit the interactions between insulin receptor proteins. Hyperglycemia induced phosphorylation of various serine residues of IRS1 and their influence on insulin signaling needs further investigation in TR-MUL cells.     

Macronutrient balancing in free‐ranging populations of moose

At northern latitudes, large spatial and temporal variation in the nutritional composition of available foods poses challenges to wild herbivores trying to satisfy their nutrient requirements. Studies conducted in mostly captive settings have shown that animals from a variety of taxonomic groups deal with this challenge by adjusting the amounts and proportions of available food combinations to achieve a target nutrient balance. In this study, we used proportions‐based nutritional geometry to analyze the nutritional composition of rumen samples collected in winter from 481 moose (Alces alces) in southern Sweden and examine whether free‐ranging moose show comparable patterns of nutrient balancing. Our main hypothesis was that wild moose actively regulate their rumen nutrient composition to offset ecologically imposed variation in the nutritional composition of available foods. To test this, we assessed the macronutritional composition (protein, carbohydrates, and lipids) of rumen contents and commonly eaten foods, including supplementary feed, across populations with contrasting winter diets, spanning an area of approximately 10,000 km². Our results suggest that moose balanced the macronutrient composition of their rumen, with the rumen contents having consistently similar proportional relationship between protein and nonstructural carbohydrates, despite differences in available (and eaten) foods. Furthermore, we found that rumen macronutrient balance was tightly related to ingested levels of dietary fiber (cellulose and hemicellulose), such that the greater the fiber content, the less protein was present in the rumen compared with nonstructural carbohydrates. Our results also suggest that moose benefit from access to a greater variety of trees, shrubs, herbs, and grasses, which provides them with a larger nutritional space to maneuver within. Our findings provide novel theoretical insights into a model species for ungulate nutritional ecology, while also generating data of direct relevance to wildlife and forest management, such as silvicultural or supplementary feeding practices.     

Long- and short-term state-dependent foraging under predation risk: an indication of habitat quality

Animals living in environments of different quality will have different expectations of their future reproductive success and survival. This may affect the individual's risk-taking behaviour as manifest in the cost of predation. We investigated the foraging behaviour of starlings, Sturnus vulgaris, when perceived predation risk varied between patches. Short-term food availability varied between treatments and long-term differences in perceptions of environmental quality varied between groups of individuals. This corresponds to variation in the three components of the cost of predation (P): the predation risk (μ); the change in reproductive value with energy gain (∂F /∂e); and the reproductive value or fitness factor (F). The birds showed that they experienced a higher cost of predation while using the risky food patches (μ component) and in the high food treatment (∂F /∂e component). Furthermore, birds from a high-reward habitat revealed a higher P than birds from a poor habitat (F component). The results show that the costs of predation are possible to tease apart by using behavioural indicators. The method presented allows measurement of fitness prospects of individuals, which may have consequences for conservation, for example, to identify low-quality habitat. Copyright 2002 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour     

Is population structure in the European white stork determined by flyway permeability rather than translocation history?

European white stork are long considered to diverge to eastern and western migration pools as a result of independent overwintering flyways. In relatively recent times, the western and northern distribution has been subject to dramatic population declines and country‐specific extirpations. A number of independent reintroduction programs were started in the mid 1950s to bring storks back to historical ranges. Founder individuals were sourced opportunistically from the Eastern and Western European distributions and Algeria, leading to significant artificial mixing between eastern and western flyways. Here we use mitochondrial and microsatellite DNA to test the contention that prior to translocation, eastern and western flyways were genetically distinct. The data show a surprising lack of structure at any spatial or temporal scale suggesting that even though birds were moved between flyways, there is evidence of natural mixing prior to the onset of translocation activities. Overall a high retention of genetic diversity, high N_ef, and an apparent absence of recent genetic bottleneck associated with early 20th century declines suggest that the species is well equipped to respond to future environmental pressures.     

Characterization of the viral O-glycopeptidome: a novel tool of relevance for vaccine design and serodiagnosis

Viral envelope proteins mediate interactions with host cells, leading to internalization and intracellular propagation. Envelope proteins are glycosylated and are known to serve important functions in masking host immunity to viral glycoproteins. However, the viral infectious cycle in cells may also lead to aberrant glycosylation that may elicit immunity. Our knowledge of immunity to aberrant viral glycans and glycoproteins is limited, potentially due to technical limitations in identifying immunogenic glycans and glycopeptide epitopes. This work describes three different complementary methods for high-throughput screening and identification of potential immunodominant O-glycopeptide epitopes on viral envelope glycoproteins: (i) on-chip enzymatic glycosylation of scan peptides, (ii) chemical glycopeptide microarray synthesis, and (iii) a one-bead-one-compound random glycopeptide library. We used herpes simplex virus type 2 (HSV-2) as a model system and identified a simple O-glycopeptide pan-epitope, (501)PPA(GalNAc)TAPG(507), on the mature gG-2 glycoprotein that was broadly recognized by IgG antibodies in HSV-2-infected individuals but not in HSV-1-infected or noninfected individuals. Serum reactivity to the extended sialyl-T glycoform was tolerated, suggesting that self glycans can participate in immune responses. The methods presented provide new insight into viral immunity and new targets for immunodiagnostic and therapeutic measures.     

Uptake of 15N-labelled alanine,ammonium and nitrate in Pinus sylvestris L. ectomycorrhiza growing in forest soil treated with nitrogen,sulphur or lime

The uptake of ¹⁵N-labelled alanine, ammonium and nitrate was studied in ectomycorrhizal morphotypes of intact Pinus sylvestris seedlings. PCR-RFLP analysis of the ITS-region of fungal rDNA was used to identify the morphotypes. Seedlings were grown in forest soil collected at an experimental site in southern Sweden. The treatments compared were a control, N fertilisation (600 kg N ha^-1 as urea), sulfur application (1200 kg S ha^-1) and lime application (6000 kg CaCO₃ ha^-1). The forest, which had been dominated by Picea abies, was clear-cut two years before the forest soil was sampled. Soil was also collected from an adjacent standing forest. The aim of the present study was to detect changes in the ectomycorrhizal communities in forest soils and relate these changes to the functional parameter of uptake of nitrogen from organic (alanine and protein) and inorganic (ammonium and nitrate) sources.Liming resulted in the detection of a morphotype not found in other samples, and one morphotype was only found in samples from the standing forest (the fungi in these two morphotypes could not be identified). All mycorrhizal root tips showed a higher ¹⁵N concentration after exposure to different nitrogen forms than non-mycorrhizal long roots. Uptake of¹⁵ N from a labelled solution of alanine or ammonium was higher (about tenfold) than uptake from a ¹⁵N-labelled solution of nitrate. Uptake of ammonium and alanine varied between 0.2 and 0.5 mg N g^-1 h^-1 and between 0.1 and 0.33 mg N g^-1 h^-1, respectively, among the different morphotypes.In seedlings grown in the control soil and in soil from standing forest, alanine and ammonium were taken up to a similar degree from a supply solution by all morphotypes, whereas ammonium uptake was higher than alanine uptake in seedlings grown in lime-treated soil (about twofold) and, to a lesser extent, in the nitrogen- and sulfur-treated soils. The higher ammonium uptake by morphotypes from the limed soil was confirmed in pure culture studies. In cases where ammonium was used as the N source, an isolate of the S. variegatus morphotype collected in the limed soil produced more biomass compared with isolates of S. variegatus collected in nitrogen- or sulphur-treated soil. One isolate of a silvery white morphotype produced about equal amounts of biomass on alanine and ammonium, whereas all S. variegatus isolated performed better with ammonium as their N source. Based on the results it is hypothesised that liming can induce a shift in the ectomycorrhizal community, favouring individuals that mainly utilise inorganic nitrogen over those that primarily utilise organic nitrogen.     

HvPIP1;6, a barley (Hordeum vulgare L.) plasma membrane water channel particularly expressed in growing compared with non-growing leaf tissues

The aim of the present study was to identify water channel(s) which are expressed specifically in the growth zone of grass leaves and may facilitate growth-associated water uptake into cells. Previously, a gene had been described (HvEmip) which encodes a membrane intrinsic protein (MIP) and which is particularly expressed in the base 1 cm of barley primary leaves. The functionality of the encoding protein was not known. In the present study on leaf 3 of barley (Hordeum vulgare L.), a clone was isolated, termed HvPIP1;6, which has 99% amino acid sequence identity to HvEmip and belongs to the family of plasma membrane intrinsic proteins (PIPs). Expression of HvPIP1;6 was highest in the elongation zone, where it accounted for >85% of expression of known barley PIP1s. Within the elongation zone, faster grower regions showed higher expression than slower growing regions. Expression of HvPIP1;6 was confined to the epidermis, with some expression in neighboring mesophyll cells. Expression of HvPIP1;6 in Xenopus laevis oocytes increased osmotic water permeability 4- to 6-fold. Water channel activity was inhibited by pre-incubation of oocytes with 50 microM HgCl(2) and increased following incubation with the phosphatase inhibitor okadaic acid or the plant hormone ABA. Plasma membrane preparations were analyzed by Western blots using an antibody that recognized PIP1s. Levels of PIP1s were highest in the elongation and adjacent non-elongation zone. The developmental expression profile of HvPIP2;1, the only known barley water channel belonging to the PIP2 subgroup, was opposite to that of HvPIP1;6.     

Myostatin (MSTN) gene duplications in Atlantic salmon (Salmo salar): evidence for different selective pressure on teleost MSTN-1 and -2

Whereas the negative muscle regulator myostatin (MSTN) in mammals is almost exclusively expressed in the muscle by a single encoding gene, teleost fish possess at least two MSTN genes which are differentially expressed in both muscular and non-muscular tissues. Duplicated MSTN-1 genes have previously been identified in the tetraploid salmonid genome. From Atlantic salmon we succeeded in isolating the paralogous genes of MSTN-2, which shared about 70% identity with MSTN-1a and -1b. The salmon MSTN-2a cDNA encoded a predicted protein of 363 residues and included the conserved C-terminal bioactive domain. MSTN-2a seemed to be primarily expressed in the brain, and a functional role of teleost MSTN-2 in the neurogenesis similar to the inhibitory action of the closely related GDF-11 in the mammalian brain was proposed. In contrast, a frame-shift mutation in exon 1 of salmon MSTN-2b would lead to the synthesis of a putatively non-functional truncated protein. The absence of processed MSTN-2b mRNA in the examined tissues indicated that this gene has become a non-functional pseudogene. The differential, but partially overlapping, expression patterns of salmon MSTN-2a, -1a and -1b in muscular and non-muscular tissues are probably due to the different arrangement of the potential cis-acting regulatory elements identified in their putative promoter regions. Single and paired E-boxes in the MSTN-1b promoter were shown to bind both homo-and hetero-dimers of the myogenic regulatory factor MyoD and E47 in vitro of importance for initiating the myogenic program. Analyses of nucleotide substitution patterns indicated that the teleost MSTNs essentially have evolved under purifying selection, but a subset of amino acid sites under positive selective pressure were identified within the MSTN1 branch. The results may reflect the evolutionary forces related to adoption of the different functional roles proposed for the teleost MSTN isoforms. The phylogenetic analysis of multiple vertebrate MSTNs suggested at least two separate gene duplication events in the fish lineage. Linkage analysis of polymorphic microsatellites within intron 2 of salmon MSTN-1a and -1b mapped the two genes to different linkage groups in agreement with the tetraploid origin of the duplicated salmonid MSTN-1 and MSTN-2 genes.     

A novel potential therapeutic avenue for autism: design, synthesis and pharmacophore generation of SSRIs with dual action

Autism symptoms are currently modulated by Selective Serotonin Reuptake Inhibitors (SSRIs). SSRIs slow onset of action limits their efficiency. The established synergistic activity of SSRIs and 5HT(1B/1D) autoreceptors antagonists motivated us to incorporate SSRIs and 5HT(1B/1D) antagonists in one 'hybrid' molecule. A library of virtual 'hybrid' molecules was designed using the tethering technique. A pharmacophore model was generated derived from 16 structurally diverse SSRIs (K(i)=0.013-5000 nM) and used as 3D query. Compounds with fit values (≥2) were chosen for synthesis and subsequent in vitro biological evaluation. Our pharmacophore model is a promising milestone to a class of SSRIs with dual action.     

Design, synthesis and structure-activity relationships of (indo-3-yl) heterocyclic derivatives as agonists of the CB1 receptor. Discovery of a clinical candidate

We report an expansion of the structure-activity relationship (SAR) of a novel series of indole-3-heterocyclic CB1 receptor agonists. Starting from the potent but poorly soluble lead, 1, a rational approach was taken in order to balance solubility, hERG activity and potency while retaining the desired long duration of action within the mouse tail flick test. This led to the discovery of compound 38 which successfully progressed into clinical development.