Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I–F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I–F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans- homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2- and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM-mediated signaling.     

Belowground heathland responses after 2 years of combined warming, elevated CO2 and summer drought

Terrestrial ecosystems are exposed to atmospheric and climatic changes including increases in atmospheric CO₂ concentration, temperature and alterations of precipitation patterns, which are predicted to continue with consequences for ecosystem services and functioning in the future. In a field scale experiment on temperate heathland, manipulation of precipitation and temperature was performed with retractable curtains, and atmospheric CO₂ concentration was increased by FACE. The combination of elevated CO₂ and warming was expected to affect belowground processes additively, through increased belowground sequestration of labile carbohydrates due to elevated CO₂ in combination with temperature increased process rates. Together, these changes might increase microbial activity and availability of plant nutrients. Two years after the start of the experiment, belowground processes responded significantly to the treatments. In the combined temperature and CO₂ treatment the dissolved organic nitrogen concentration decreased and the ammonium concentration increased, but this release of nutrients was not mirrored by plant parameters. Microbial biomass carbon and microbial enrichment with ¹³C and ¹⁵N (1 year after ¹³C ₂ ¹⁵ N-glycine was injected into the soil) increased in warmed plots and in elevated CO₂ plots, but not when these treatments were combined. Furthermore, drought led to an increase in Calluna biomass and total plant nitrogen pool. The full combination of warming, elevated CO₂ and periodic drought did not unambiguously express the ecosystem responses of single factors additively, which complicates predictions of ecosystem responses to multifactor climate change.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

The spotted wolffish (Anarhichas minor Olafsen) complement component C3: isolation,characterisation and tissue distribution

The complement component C3 was isolated from spotted wolffish (Anarhichas minor Olafsen) serum by polyethylene glycol precipitation, anion exchange chromatography and gel filtration. Silver staining in SDS-PAGE and rabbit anti-wolffish C3 antiserum used in Western blotting revealed that spotted wolffish C3 contains two polypeptide chains, M(r)65 and 115kDa, respectively. The high molecular weight alpha-chain of the C3 incorporated 14C-methylamine suggesting that it contained a reactive thioester group. The deduced amino acid sequence, after screening a liver cDNA expression library, showed that the wolffish C3 contained key amino acids for binding C3 convertase, factor H, I and properdin. Also, high degree of homology to other vertebrate C3 was found in the beta-alpha junction site. Phylogenetic tree analysis indicated that the Japanese flounder and spotted wolffish that belong to order pleuronectiformes and perciformes, respectively, are phylogenetically close species. Immunohistochemical experiments showed that liver hepatocytes and blood contained C3, and in situ hybridisation experiments revealed that liver hepatocytes expressed C3.     

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.The evolving maturation of the field of proteomics has, in the same way as in genomics, highlighted the need of better sampling procedures and sample preparation methodologies to minimize the effect of post mortem alterations. The aspect of sample quality is not new in any way and is relevant in most biomedical fields but has only lately started to receive adequate attention. The main factors influencing sample quality is storage temperature of the body until tissue removal (foremost a problem in clinical settings and extraction of less accessible tissue samples from model organisms) and post mortem interval (PMI)¹ (1–3). Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2, 3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3–8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1, 3–12). There are also a few proteomic studies on muscle tissue degradation in livestock (13–16).We and others have previously explored the effect of focused microwave irradiation with regard to sample quality, demonstrating that this method is more reliable than snap freezing in liquid nitrogen, especially with regard to post-translational modification (PTM) stability (2, 3, 17–20). An alternative method based on cryostat dissection with subsequent heat treatment through boiling has also been reported to improve endogenous peptide sample quality (21). Besides focused microwave irradiation, which is specifically used for rodent brain tissue sampling, we have also demonstrated the efficiency of rapid heat stabilization through conductivity with regard to sample degradation (3, 22). Although somewhat constrained by its dependence on how quickly the tissue is harvested from the body, the latter procedure has the added advantage that it can be used on any type of tissue and species, fresh as well as frozen. This study will compare effects of sampling procedures on the liver and pancreas degradome following rapid heat stabilization, the more traditional snap freezing, or the combination of snap freezing with subsequent heat stabilization.To summarize, this study investigated the effects of post mortem degradation in pancreas and liver. Both tissues are well studied because of their multiple functions in the body and their involvement in different diseases such as diabetes or hepatocarcinoma. Pancreas is especially interesting in this context as it displays endocrine secretion of peptides, and exocrine secretion of digestive enzymes, the later making it a protease rich tissue. We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display (2, 18), the later to better investigate changes in small molecular fragment that are not easily detectable by gel-based methods. 2D-DIGE is an unrivaled methodology to characterize alterations in isoform patterns, which is an important aspect considering that post-translational modifications (PTMs) such as phosphorylations are especially sensitive to post mortem influence within a few minutes PMI (3). The peptidomics approach has been used in several studies to point out early post mortem changes and protein degradation that tissue undergo following sampling and is therefore a well-suited method (3, 18, 22).     

Oldest known pantherine skull and evolution of the tiger

The tiger is one of the most iconic extant animals, and its origin and evolution have been intensely debated. Fossils attributable to extant pantherine species-lineages are less than 2 MYA and the earliest tiger fossils are from the Calabrian, Lower Pleistocene. Molecular studies predict a much younger age for the divergence of modern tiger subspecies at <100 KYA, although their cranial morphology is readily distinguishable, indicating that early Pleistocene tigers would likely have differed markedly anatomically from extant tigers. Such inferences are hampered by the fact that well-known fossil tiger material is middle to late Pleistocene in age. Here we describe a new species of pantherine cat from Longdan, Gansu Province, China, Panthera zdanskyi sp. nov. With an estimated age of 2.55-2.16 MYA it represents the oldest complete skull of a pantherine cat hitherto found. Although smaller, it appears morphologically to be surprisingly similar to modern tigers considering its age. Morphological, morphometric, and cladistic analyses are congruent in confirming its very close affinity to the tiger, and it may be regarded as the most primitive species of the tiger lineage, demonstrating the first unequivocal presence of a modern pantherine species-lineage in the basal stage of the Pleistocene (Gelasian; traditionally considered to be Late Pliocene). This find supports a north-central Chinese origin of the tiger lineage, and demonstrates that various parts of the cranium, mandible, and dentition evolved at different rates. An increase in size and a reduction in the relative size of parts of the dentition appear to have been prominent features of tiger evolution, whereas the distinctive cranial morphology of modern tigers was established very early in their evolutionary history. The evolutionary trend of increasing size in the tiger lineage is likely coupled to the evolution of its primary prey species.     

Epitope grafting, re-creating a conformational Bet v 1 antibody epitope on the surface of the homologous apple allergen Mal d 1

Birch-allergic patients often experience oral allergy syndrome upon ingestion of vegetables and fruits, most prominently apple, that is caused by antibody cross-reactivity of the IgE antibodies in patients to proteins sharing molecular surface structures with the major birch pollen group 1 allergen from Betula verrucosa (Bet v 1). Still, to what extent two molecular surfaces need to be similar for clinically relevant antibody cross-reactivity to occur is unknown. Here, we describe the grafting of a defined conformational antibody epitope from Bet v 1 onto the surface of the homologous apple allergen Malus domestica group 1 (Mal d 1). Engineering of the epitope was accomplished by genetic engineering substituting amino acid residues in Mal d 1 differing between Bet v 1 and Mal d 1 within the epitope defined by the mAb BV16. The kinetic parameters characterizing the antibody binding interaction to Bet v 1 and to the mutated Mal d 1 variant, respectively, were assessed by Biacore experiments demonstrating indistinguishable binding kinetics. This demonstrates that a conformational epitope defined by a high affinity antibody-allergen interaction can successfully be grafted onto a homologous scaffold molecule without loss of epitope functionality. Furthermore, we show that increasing surface similarity to Bet v 1 of Mal d 1 variants by substitution of 6-8 residues increased the ability to trigger basophil histamine release with blood from birch-allergic patients not responding to natural Mal d 1. Conversely, reducing surface similarity to Bet v 1 of a Mal d 1 variant by substitution of three residues abolished histamine release in one patient reacting to Mal d 1.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

Feedback regulation of ornithine decarboxylase expression. Studies using a polysomal run-off system

The rate-controlling enzyme in polyamine synthesis, ornithine decarboxylase (ODC), is subject to feedback regulation by the polyamines at the level of translation. In the present study we used a cell-free translation system to further investigate the mechanism by which this regulation occurs. Lysates of ODC-overproducing cells were capable of synthesizing large amounts of ODC. The degree of initiation was poor in the lysates and the synthesis of ODC was mainly a result of continued elongation of peptide chains on pre-initiated ribosomes. By determining the amount of ODC produced in the lysate, we obtained an estimate of the number of ribosomes that were actively translating ODC mRNA at the moment of lysis. Using this polysomal run-off assay we demonstrated that the polyamine-mediated regulation of ODC synthesis occurs without any change in the number of ribosomes associated with the message. This finding indicates that the polyamines exert a coordinate effect on initiation and elongation.