Time-resolved tryptophan fluorescence anisotropy investigation of bacteriophage M13 coat protein in micelles and mixed bilayers

Coat protein of bacteriophage M13 is examined in micelles and vesicles by time-resolved tryptophan fluorescence and anisotropy decay measurements and circular dichroism experiments. Circular dichroism indicates that the coat protein has alpha-helix (60%) and beta-structure (28%) in 700 mM sodium dodecyl sulfate micelles and predominantly beta-structure (94%) in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidic acid (80/20 w/w) small unilamellar vesicles. The fluorescence decay at 344 nm of the single tryptophan in the coat protein after excitation at 295 or 300 nm is a triple exponential. In the micelles the anisotropy decay is a double exponential. A short, temperature-independent correlation time of 0.5 +/- 0.2 ns reflects a rapid depolarization process within the coat protein. The overall rotation of the coat protein-detergent complex is observed in the decay as a longer correlation time of 9.8 +/- 0.5 ns (at 20 degrees C) and has a temperature dependence that satisfies the Stokes-Einstein relation. In vesicles at all lipid to protein molar ratios in the range from 20 to 410, the calculated order parameter is constant with a value of 0.7 +/- 0.1 from 10 to 40 degrees C, although the lipids undergo the gel to liquid-crystalline phase transition. The longer correlation time decreases gradually on increasing temperature. This effect probably arises from an increasing segmental mobility within the coat protein. The results are consistent with a model in which the coat protein has a beta-structure and the tryptophan indole rings do not experience the motion of the lipids in the bilayer because of protein-protein aggregation.     

The colliculus superior modulates ACTH-induced excessive grooming

In previous studies the involvement of nigrostriatal dopaminergic activity in ACTH(1-24)-induced grooming has been established. It was suggested that the dopaminergic modulation of ACTH(1-24)-induced excessive grooming is exerted through the striato-nigro-collicular pathway. To obtain further evidence it was investigated, whether local application of GABAergic agents into the colliculus superior modulates excessive grooming occurring after an intraventricular injection with ACTH(1-24). It appeared that intra-collicular picrotoxin (a GABAergic antagonist) suppressed ACTH-induced grooming, whereas muscimol (a GABAergic agonist) enhanced the grooming response. The picrotoxin-induced R(unning) F(it) B(ehavior), elicited from the colliculus superior was also seen after intraventricular administration of picrotoxin. A detailed comparison of this behavioral response seen after both routes of administration of picrotoxin suggests that intraventricularly injected picrotoxin may well induce the RFB via a direct effect on the colliculus superior. Lesions placed in the colliculus superior completely abolished picrotoxin-induced RFB, exploration and orientation behavior. Yet, these lesions did not reduce excessive grooming suggesting that although this region may be involved in the modulation of ACTH-induced grooming it is not the primary site of peptide action.     

The carbohydrate chains of the beta subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo. Novel O-linked and novel bisecting-GlcNAc-containing N-linked carbohydrates.

The N-linked carbohydrate chains of the beta subunit of human chorionic gonadotropin (hCG-beta) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Subsequently, the O-linked oligosaccharides were split off from the N-deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion-exchange chromatography on Mono Q, HPLC on Lichrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. 1H-NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N-linked oligosaccharides. Residues not written in bold letters are variably present. [formula: see text] The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG-beta (26% vs 2%). The same holds for the alpha 1-6-fucosylation of the asparagine-bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of alpha 2-6-linked Neu5Ac in the most abundant N-glycans, are new features for hCG-beta. The major O-linked carbohydrate chains identified are the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol and the hexasaccharide Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6(Neu5Ac alpha 2-3Gal beta 1-3)GalNAc-ol, both also found in normal urinary hCG. In addition, two novel O-glycans were characterized: [formula: see text]     

ACTH1-4 potentiates alpha-MSH-induced melanophore dispersion and excessive grooming

The biological activity and a possible modulatory role of the N-terminal tetrapeptide Ser-Tyr-Ser-Met from alpha-MSH/ACTH was tested in the Anolis melanophore assay, the Xenopus melanophore assay, tyrosinase stimulation in mouse melanoma cells and in excessive grooming in the rat. ACTH1-4 did not exhibit biological activity in any of these four assays nor did it have modulatory properties in the Xenopus and the melanoma cell assay. However, in the Anolis assay ACTH1-4 potentiated pigment dispersion induced by alpha-MSH, alpha-MSH5-13 and ACTH1-24 by a factor of about 2. In the grooming assay ACTH1-4 potentiated the effects of alpha-MSH, alpha-MSH5-13, ACTH1-16 and ACTH5-16, but not those of ACTH1-24. Oxidized ACTH1-4 was without biological activity and potentiating properties in all four assays. This study shows that small fragments of the pro-opiomelanocortin precursor, which are devoid of biological activity, can modulate peripheral and central actions of alpha-MSH/ACTH.     

Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial–mesenchymal transition

This study aimed to examine whether lung tissue extracellular matrix (ECM) hydrogels have protective effects on radiation-induced lung injury (RILI). The cytocompatibility and histocompatibility were tested for the obtained ECM-derived hydrogel. Sprague–Dawley rats were randomly divided into three groups (n = 18): control group (control); rats receiving irradiation and intratracheal injection of normal saline (IR + NS); and rats receiving irradiation and intratracheal injection of lung ECM-derived hydrogel (IR + ECM). The wet/dry weight ratio was used to evaluate the congestion and edema of the lungs. Histopathological analysis of lung tissues was performed using hemotoxylin and eosin staining and Masson's trichrome staining. Immunohistochemical staining and western blot analyses were carried out to determine the expression of epithelial–mesenchymal transition (EMT)-related proteins in lung tissues (E-cadherin, α-smooth muscle actin [α-SMA], and vimentin). In addition, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6), hydroxyproline, malondialdehyde (MDA), and superoxide dismutase (SOD) levels were also evaluated. The ECM-derived hydrogels had good cytocompatibility and histocompatibility. ECM-derived hydrogel treatment improved lung histopathology injury and pulmonary edema. Higher expression of E-cadherin and lower expression of vimentin and α-SMA were found in the IR + ECM group compared with those in the IR + NS group. Hydroxyproline levels were reduced by ECM-derived hydrogel treatment compared with those in the IR + NS group. Obvious increases of TNF-α, IL-6, and TGF-β1 were identified following irradiation. Marked reductions in MDA content and increases in SOD were induced by ECM-derived hydrogel treatment in rats after radiation. ECM-derived hydrogels were shown to protect against RILI, potentially by reducing EMT, inflammation, and oxidative damage.     

Is it possible to replace stimulus animals by scent-filled cups in the social discrimination test?

A study in which the rat social discrimination test was refined is described. This test measures social memory by using, in general, juvenile rats as stimulus animals. Rats are offered a first juvenile to investigate (learning trial), and after a specified interval, the rats are offered the same rat and a second juvenile rat to investigate again (retrieval trial). When the rats sniff the second juvenile in the retrieval trial more than the first, social memory for the second juvenile is said to be present. This test is mainly based on scents from the juvenile. Attempts were made to refine the test to reduce the number of animals used, to enhance the scope of the test, and to improve its validity. Firstly, the stimulus animals were replaced by the scent of juveniles, in the form of cups filled with sawdust taken from cages of juvenile rats. Similar results to those in the original test were obtained when using these scents. Furthermore, male and female scents were tested, and showed the same results as for the juvenile scents. Secondly, rats were also given two cups (one scent-filled and one filled with plain sawdust) in the learning trial, to determine which allowed a more-precise delineation of motivational, discriminatory and memory components. Overall, it is possible to replace stimulus animals by scent-filled cups in the social discrimination test, to enhance the scope of the test, and to draw more-valid conclusions with respect to social memory.     

Membrane assembly of the bacteriophage Pf3 major coat protein

The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Chimpanzees (Pan troglodytes) fail a what-where-when task but find rewards by using a location-based association strategy

Recollecting the what-where-when of an episode, or episodic-like memory, has been established in corvids and rodents. In humans, a linkage between remembering the past and imagining the future has been recognised. While chimpanzees can plan for the future, their episodic-like memory has hardly been investigated. We tested chimpanzees (Pan troglodytes) with an adapted food-catching paradigm. They observed the baiting of two locations amongst four and chose one after a given delay (15 min, 1 h or 5 h). We used two combinations of food types, a preferred and a less preferred food that disappeared at different rates. The subjects had to base their choices on the time elapsed since baiting, and on their memory of which food was where. They could recover either their preferred food or the one that remained present. All animals failed to obtain the preferred or present foods above chance levels. They were like-wise unsuccessful at choosing baited cups above chance levels. The subjects, thus, failed to use any feature of the baiting events to guide their choices. Nonetheless, their choices were not random, but the result of a developed location-based association strategy. Choices in the second half of the study correlated with the rewards obtained at each location in the first half of the study, independent from the choices made for each location in the first half of the study. This simple location-based strategy yielded a fair amount of food. The animals' failure to remember the what-where-when in the presented set-up may be due to the complexity of the task, rather than an inability to form episodic-like memories, as they even failed to remember what was where after 15 minutes.