Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR

Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two-dimensional total correlation spectroscopy. Sequence-specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single-site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild-type molecule. Spin-labeled oligonucleotide-binding studies of wild-type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single-stranded DNA. The degree of aggregation of wild-type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein-protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild-type protein. The discovery of the so-called 'solubility' mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single-stranded DNA by means of two-dimensional NMR.     

Genetic variation determines mast cell functions in experimental asthma

Mast cell-deficient mice are a key for investigating the function of mast cells in health and disease. Allergic airway disease induced as a Th2-type immune response in mice is employed as a model to unravel the mechanisms underlying inception and progression of human allergic asthma. Previous work done in mast cell-deficient mouse strains that otherwise typically mount Th1-dominated immune responses revealed contradictory results as to whether mast cells contribute to the development of airway hyperresponsiveness and airway inflammation. However, a major contribution of mast cells was shown using adjuvant-free protocols to achieve sensitization. The identification of a traceable genetic polymorphism closely linked to the Kit(W-sh) allele allowed us to generate congenic mast cell-deficient mice on a Th2-prone BALB/c background, termed C.B6-Kit(W-sh). In accordance with the expectations, C.B6-Kit(W-sh) mice do not develop IgE- and mast cell-dependent passive cutaneous anaphylaxis. Yet, unexpectedly, C.B6-Kit(W-sh) mice develop full-blown airway inflammation, airway hyperresponsiveness, and mucus production despite the absence of mast cells. Thus, our findings demonstrate a major influence of genetic background on the contribution of mast cells in an important disease model and introduce a novel strain of mast cell-deficient mice.     

Effects of prenatal exposure to diclofenac sodium and saline on the optic nerve of 4- and 20-week-old male rats: a stereological and histological study

We investigated the effects of diclofenac sodium (DS) on development of the optic nerve in utero. Pregnant female rats were separated into three groups: control, saline treated and DS treated. Offspring of these animals were divided into 4-week-old and 20-week-old groups. At the end of the 4th and 20th weeks of postnatal life, the animals were sacrificed, and right optic nerves were excised and sectioned for ultrastructural and stereological analyses. We demonstrated that both DS and saline produced structural and morphometric changes in the total axon number and density of axons, but decreased the myelin sheath thickness in male optic nerves. All ultrastructural and morphometric features were well developed in 20-week-old rats. We showed that development of the optic nerve continues during the early postnatal period and that some compensation for exposure to deleterious agents in utero may occur during early postnatal life.     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> aggravates vein graft intimal hyperplasia in a rat model

Background  

Along with angioplasty, autologus vein grafts are commonly used for artery bypass grafting in patients with advanced arterial stenosis and drug-resistant angina pectoris. Although initially a successful procedure, long-term functionality is limited due to proliferation and migration of smooth muscle cells. Like in atherosclerosis, common chronic infections caused by viruses and bacteria may contribute to this process of vein graft failure. Here we investigated the possible role of Chlamydia pneumoniae (Cpn) in the pathogenesis of venous graft failure in an experimental animal model. In 2 groups (n = 10 rats/group), an epigastric vein-to-common femoral artery interposition graft was placed. Immediately thereafter, rats were infected with Cpn (5*10⁸ IFU) or injected with control solutions. Rats were sacrificed three weeks after surgery and the grafts were harvested for morphometrical and immunohistochemical analysis.     

Network analysis of temporal functionalities of the gut induced by perturbations in new-born piglets

Background

Evidence is accumulating that perturbation of early life microbial colonization of the gut induces long-lasting adverse health effects in individuals. Understanding the mechanisms behind these effects will facilitate modulation of intestinal health. The objective of this study was to identify biological processes involved in these long lasting effects and the (molecular) factors that regulate them. We used an antibiotic and the same antibiotic in combination with stress on piglets as an early life perturbation. Then we used host gene expression data from the gut (jejunum) tissue and community-scale analysis of gut microbiota from the same location of the gut, at three different time-points to gauge the reaction to the perturbation. We analysed the data by a new combination of existing tools. First, we analysed the data in two dimensions, treatment and time, with quadratic regression analysis. Then we applied network-based data integration approaches to find correlations between host gene expression and the resident microbial species.

Results

The use of a new combination of data analysis tools allowed us to identify significant long-lasting differences in jejunal gene expression patterns resulting from the early life perturbations. In addition, we were able to identify potential key gene regulators (hubs) for these long-lasting effects. Furthermore, data integration also showed that there are a handful of bacterial groups that were associated with temporal changes in gene expression.

Conclusion

The applied systems-biology approach allowed us to take the first steps in unravelling biological processes involved in long lasting effects in the gut due to early life perturbations. The observed data are consistent with the hypothesis that these long lasting effects are due to differences in the programming of the gut immune system as induced by the temporary early life changes in the composition and/or diversity of microbiota in the gut.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1733-8) contains supplementary material, which is available to authorized users.     

Heterocyclic thrombin inhibitors. Part 2: quinoxalinone derivatives as novel,potent antithrombotic agents

Quinoxalinone derivatives as prototypes of dual thrombin and factor Xa inhibitors have been discovered. Nanomolar inhibition of both coagulation enzymes resulted in very potent antithrombotic activity in vitro.     

1,000 structures and more from the MCSG

Background  

The Midwest Center for Structural Genomics (MCSG) is one of the large-scale centres of the Protein Structure Initiative (PSI). During the first two phases of the PSI the MCSG has solved over a thousand protein structures. A criticism of structural genomics is that target selection strategies mean that some structures are solved without having a known function and thus are of little biomedical significance. Structures of unknown function have stimulated the development of methods for function prediction from structure.