A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis

Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.     

Few alterations in clinical pathology and histopathology observed in a CYP2C18&;19 humanized mice model

Background  

This study was performed to characterize a gene-addition transgenic mouse containing a BAC (bacterial artificial chromosome) clone spanning the human CYP2C18&19 genes (tg-CYP2C18&19).     

Phototherapy: the hospital as risk factor.

In a retrospective study over six years the incidence of phototherapy was examined in two groups of healthy neonates who were born spontaneously and at term in hospital. They were comparable in all respects except that one group was cared for at home and the other group was cared for in hospital. It appeared that the infants in hospital received phototherapy seven times more often than those at home, and surveillance at home was not inferior to that in hospital. There is no reason to assume that neonatal jaundice occurred more often in neonates in hospital than in those at home. Thus the difference in the frequency of treatment with phototherapy between the two groups is ascribed to the influence of the hospital environment, which may encourage intervention.     

Approaches for systematic proteome exploration

With the completion of the human genome project (HUGO) during recent years, gene function, protein abundance and expression patterns in tissues and cell types have emerged as central areas for the scientific community. A mapped human proteome will extend the value of the genome sequence and large-scale efforts aiming at elucidating protein localization, abundance and function are invaluable for biomarker and drug discovery. This research area, termed proteomics, is more demanding than any genome sequencing effort and to perform this on a wide scale is a highly diverse task. Therefore, the proteomics field employs a range of methods to examine different aspects of proteomics including protein localization, protein-protein interactions, posttranslational modifications and alteration of protein composition (e.g. differential expression) in tissues and body fluids. Here, some of the most commonly used methods, including chromatographic separations together with mass spectrometry and a number of affinity proteomics concepts are discussed and exemplified.     

Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea

The planarian Schmidtea mediterranea is a well-studied model organism for developmental research, because of its stem cell system. This characteristic also provides a unique opportunity to study stress management and the effect of stress on stem cells. In this study, we characterised the stress signature at different levels of biological organization. The carcinogenic metal cadmium was used as a model chemical stressor. We focused on stem cell activity and its interaction with other known stress parameters. Here, we have found that S. mediterranea is able to cope with high internal levels of cadmium. At endpoints such as size and mobility, cadmium-related stress effects were detected but all of these responses were transient. Correspondingly, cadmium exposure led to an elevated mitotic activity of the neoblasts, at the same time points when the other responses disappeared. At the molecular level, we observed redox-related responses that can be linked with both repair as well as proliferation mechanisms. Together, our results suggest that these animals have a high plasticity. The induction of stem cell activity may underlie this 'restoring' effect, although a carcinogenic outcome after longer exposure times cannot be excluded.     

Comparative sequence and structure analysis reveal features of cold adaptation of an enzyme in the thermolysin family

Knowledge about the structural features underlying cold adaptation is important for designing enzymes of different industrial relevance. Vibriolysin from Antarctic bacterium strain 643 (VAB) is at present the only enzyme of the thermolysin family from an organism that thrive in extremely cold climate. In this study comparative sequence-structure analysis and molecular dynamics (MD) simulations were used to reveal the molecular features of cold adaptation of VAB. Amino acid sequence analysis of 44 thermolysin enzymes showed that VAB compared to the other enzymes has: (1) fewer arginines, (2) a lower Arg/(Lys + Arg) ratio, (3) a lower fraction of large aliphatic side chains, expressed by the (Ile + Leu)/(Ile + Leu + Val) ratio, (4) more methionines, (5) more serines, and (6) more of the thermolabile amino acid asparagine. A model of the catalytic domain of VAB was constructed based on homology with pseudolysin. MD simulations for 3 ns of VAB, pseudolysin, and thermolysin supported the assumption that cold-adapted enzymes have a more flexible three-dimensional (3D) structure than their thermophilic and mesophilic counterparts, especially in some loop regions. The structural analysis indicated that VAB has fewer intramolecular cation-pi electron interactions and fewer hydrogen bonds than its mesophilic (pseudolysin) and thermophilic (thermolysin) counterparts. Lysine is the dominating cationic amino acids involved in salt bridges in VAB, while arginine is dominating in thermolysin and pseudolysin. VAB has a greater volume of inaccessible cavities than pseudolysin and thermolysin. The electrostatic potentials on the surface of the catalytic domain were also more negative for VAB than for thermolysin and pseudolysin. Thus, the MD simulations, the structural patterns, and the amino acid composition of VAB relative to other enzymes of the thermolysin family suggest that VAB possesses the biophysical properties generally following adaptation to cold climate.     

Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer cells

NKG2D is a surface receptor expressed on NK cells but also on CD8⁺ T cells, γδ T cells, and auto-reactive CD4⁺/CD28⁻ T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (K_D) of 2.7 ± 1.4 × 10⁻¹¹ M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFα, IFNγ and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after seven days in human serum at 37°C, and resisted thermal inactivation up to 70°C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases.Key words: NKG2D, NK cell, T cell, monoclonal antibody, human IgG1, humanization, phage display, autoimmune disease     

The Expression of the Beta Cell-Derived Autoimmune Ligand for the Killer Receptor Nkp46 Is Attenuated in Type 2 Diabetes

NK cells rapidly kill tumor cells, virus infected cells and even self cells. This is mediated via killer receptors, among which NKp46 (NCR1 in mice) is prominent. We have recently demonstrated that in type 1 diabetes (T1D) NK cells accumulate in the diseased pancreas and that they manifest a hyporesponsive phenotype. In addition, we found that NKp46 recognizes an unknown ligand expressed by beta cells derived from humans and mice and that blocking of NKp46 activity prevented diabetes development. Here we investigated the properties of the unknown NKp46 ligand. We show that the NKp46 ligand is mainly located in insulin granules and that it is constitutively secreted. Following glucose stimulation the NKp46 ligand translocates to the cell membrane and its secretion decreases. We further demonstrate by using several modalities that the unknown NKp46 ligand is not insulin. Finally, we studied the expression of the NKp46 ligand in type 2 diabetes (T2D) using 3 different in vivo models and 2 species; mice and gerbils. We demonstrate that the expression of the NKp46 ligand is decreased in all models of T2D studied, suggesting that NKp46 is not involved in T2D.