A consideration of biomarkers to be used for evaluation of inflammation in human nutritional studies.

To monitor inflammation in a meaningful way, the markers used must be valid: they must reflect the inflammatory process under study and they must be predictive of future health status. In 2009, the Nutrition and Immunity Task Force of the International Life Sciences Institute, European Branch, organized an expert group to attempt to identify robust and predictive markers, or patterns or clusters of markers, which can be used to assess inflammation in human nutrition studies in the general population. Inflammation is a normal process and there are a number of cells and mediators involved. These markers are involved in, or are produced as a result of, the inflammatory process irrespective of its trigger and its location and are common to all inflammatory situations. Currently, there is no consensus as to which markers of inflammation best represent low-grade inflammation or differentiate between acute and chronic inflammation or between the various phases of inflammatory responses. There are a number of modifying factors that affect the concentration of an inflammatory marker at a given time, including age, diet and body fatness, among others. Measuring the concentration of inflammatory markers in the bloodstream under basal conditions is probably less informative compared with data related to the concentration change in response to a challenge. A number of inflammatory challenges have been described. However, many of these challenges are poorly standardised. Patterns and clusters may be important as robust biomarkers of inflammation. Therefore, it is likely that a combination of multiple inflammatory markers and integrated readouts based upon kinetic analysis following defined challenges will be the most informative biomarker of inflammation.

Understanding the complex phase diagram of uranium: the role of electron-phonon coupling.

We report an experimental determination of the dispersion of the soft phonon mode along [100] in uranium as a function of pressure. The energies of these phonons increase rapidly, with conventional behavior found by 20 GPa, as predicted by recent theory. New calculations demonstrate the strong pressure (and momentum) dependence of the electron-phonon coupling, whereas the Fermi-surface nesting is surprisingly independent of pressure. This allows a full understanding of the complex phase diagram of uranium and the interplay between the charge-density wave and superconductivity.

Inflammatory disease processes and interactions with nutrition.

Inflammation is a stereotypical physiological response to infections and tissue injury; it initiates pathogen killing as well as tissue repair processes and helps to restore homeostasis at infected or damaged sites. Acute inflammatory reactions are usually self-limiting and resolve rapidly, due to the involvement of negative feedback mechanisms. Thus, regulated inflammatory responses are essential to remain healthy and maintain homeostasis. However, inflammatory responses that fail to regulate themselves can become chronic and contribute to the perpetuation and progression of disease. Characteristics typical of chronic inflammatory responses underlying the pathophysiology of several disorders include loss of barrier function, responsiveness to a normally benign stimulus, infiltration of inflammatory cells into compartments where they are not normally found in such high numbers, and overproduction of oxidants, cytokines, chemokines, eicosanoids and matrix metalloproteinases. The levels of these mediators amplify the inflammatory response, are destructive and contribute to the clinical symptoms. Various dietary components including long chain omega-3 fatty acids, antioxidant vitamins, plant flavonoids, prebiotics and probiotics have the potential to modulate predisposition to chronic inflammatory conditions and may have a role in their therapy. These components act through a variety of mechanisms including decreasing inflammatory mediator production through effects on cell signaling and gene expression (omega-3 fatty acids, vitamin E, plant flavonoids), reducing the production of damaging oxidants (vitamin E and other antioxidants), and promoting gut barrier function and anti-inflammatory responses (prebiotics and probiotics). However, in general really strong evidence of benefit to human health through anti-inflammatory actions is lacking for most of these dietary components. Thus, further studies addressing efficacy in humans linked to studies providing greater understanding of the mechanisms of action involved are required.

Calculations for displacive ω-phase transformations in Ti-Al alloys with Nb additions at finite temperature.

We examine by means of first-principles calculations the bcc-like (bcc: body centered cubic) to ω-like phase transformations in Ti-Al alloys with Nb additions at finite temperature. To simulate the alloy we use different discrete atomic configurations in a six atom unit cell of the stoichiometry Ti(3)Al(2)Nb. Calculated ground state energies show an instability in the ternary Ti(3)Al(2)Nb alloy against the ω structure type atomic displacement. To better understand the role of entropy in the stability/instability of these systems, the first-principles calculations are extended to finite temperature by including various contributions to the free energy. In particular, the vibrational free energy is calculated within a quasiharmonic approximation. It is shown that the bcc structure is stabilized by the contribution of the low energy modes to the lattice entropy against ω type atomic displacements. We find that configurational entropy plays a major role in the ω to B8(2) transformation. Calculated lattice parameters and transition temperatures are found to be in excellent agreement with experiment.

Effect of undermethylation on mRNA cytoplasmic appearance and half-life.

S-Tubercidinylhomocysteine (STH) is a structural analog of S-adenosylhomocysteine and a potent inhibitor of S-adenosylmethionine-dependent methyltransferase reactions. We investigated the effects of STH on HeLa cell mRNA metabolism. Dual labeling studies reveal that STH dramatically inhibits the methylation of HeLa mRNA in a dose-dependent manner. Analysis of the modified nucleosides and 5'-terminal cap structures in radiolabeled mRNA by high-pressure liquid chromatography indicated that internal N6-methylation of adenosine was reduced by 65% at 50 microM STH and by 83% at 500 microM STH. The N6-methylation of adenosine contained in cap structures was similarly reduced at both concentrations of STH. Substantial amounts of cap structures lacking 2'-O-methylated nucleosides (m7GpppN, cap zero) were detected at the higher level of STH. To test the possibility that methylation affects mRNA stability, cytoplasmic mRNA half-life was measured in a pulse-chase experiment. The half-life of undermethylated mRNA, produced as a consequence of STH treatment, was unchanged compared with the control. To determine whether mRNA methylation is coupled to nuclear processing or transport, the time of cytoplasmic appearance of polyadenylated RNA in STH-treated HeLa cells was compared with untreated cells. STH caused a significant lag in the time of appearance of the polyadenylated RNA, suggesting that mRNA methylation may be required for efficient processing or transport.

Sodium and potassium ion-dependent adenosine triphosphatase of mammalian brain. Interactions of magnesium ions with the phosphatase site.

Kinetic parameters are reported for Mg2+, Na+ and K+ as activators of the p-nitrophenylphosphatase activity associated with (Na+ + K+)-ATPase (ATP-phosphohydrolase, EC 3.6.1.3) of beef brain. In each case the phosphatase reaction is activated at low concentrations of the cation and inhibited by higher concentrations. The concentrations of cation that produced half-maximal activation and half-maximal inhibition are increased as the concentration of either of the other two cations is increased. These second ligand effects are all saturable functions. The apparent binding constant that characterizes the effect on activation is closely similar to that acting upon the inhibitory phase in each case.

Temperature effects on cation affinities of the (Na+, K+)-ATPase of mammalian brain.

Effects of temperature on the Na+-dependent ADP-ATP exchange and the p-nitrophenylphosphatase reactions catalysed by (Na+, K+)-ATPase were examined. Apparent Mg2+ affinity decreased with decreasing temperature. Arrhenius plots of p-nitrophenylphosphatase in the presence of Na+ and ATP had discontinuities similar to those previously reported for (Na+ + K+)-ATPase, while those of p-nitrophenylphosphatase measured without Na+ or ATP did not. The apparent activation energy for p-nitrophenylphosphatase was a function of the physical characteristics of the cation acting at the K+ site.

Immunochemical studies on the large polypeptide of electrophorus electroplax (Na+ + K+)-ATPase.

Antibodies against Lubrol-solubilized Electrophorus electroplax (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and its 96 000-dalton polypeptide (P96) were raised in rabbits. The P96 antibody does not cross react with the (Na+ + K+)-ATPase from mammalian species and tissues, but it cross reacts with the (Na+ + K+)-ATPase from both Electrophorus electroplax and brain. The combination of enzyme with anti-P96 is found to inhibit phosphoryl enzyme formation to the same extent that it inhibits enzyme activity. The rate of K+-sensitive dephosphorylation of phosphoryl enzyme appears to be unchanged. These are also found to be true with the antibody against the whole enzyme. Upon tryptic digestion of the enzyme-anti-P96 complex only the large polypeptide of the enzyme is protected. In the case of enzyme-anti-Lubrol-solubilized enzyme complex, both the large and small polypeptides are protected, whereas preimmune sera are without any protecting effect. The data indicate that the phosphoryl acceptor polypeptide and the Lubrol-solubilized electroplax (Na+ + K+)-ATPase from which the polypeptide is derived are phylogenetically distinct from those of the mammalian (Na+ + K+)-ATPases. The selective tryptic resistance of the enzyme-anti-P96 complex indicates that the two polypeptides are spatially well separated, possibly on opposite sides of the membrane.

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ.

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to alpha-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate: other lectins had no such effects. Concanavalin A similar amounts partially inhibited (Na+ + K+)-ATPase; this inhibition was reversible by alpha-methylglucoside. There was no corresponding effect of concanavalin A on the potassium p-nitrophenylphosphatase. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na+ and/or ATP binding, but does not interact with a K+ site.

Block poly(Ala)-poly(Lys). A water-soluble model for intrinsic membrane proteins?

Block poly(Ala)16-poly(Lys)13.5 was synthesized by the Leuchs anhydride method. This polypeptide is water soluble in a largely monomeric form, but binds rapidly and spontaneously to unilamellar vesicles of dimyristoyl phosphatidylcholine at pH 7.4. The interaction is evidently of a hydrophobic nature since the complex is not disrupted by salt and no similar reaction is given by polylysine. Evidence for the interaction was obtained by ultrafiltration, chromatography on Sepharose 4B, and sedimentation velocity ultracentrifugation. While direct information on the molecular structure of the complex is still lacking, we propose that this amphipathic block copolymer binds to lipids in a similar manner as intrinsic membrane proteins and hence can be used to study the interactions of intrinsic proteins with lipids.

Dynamical and transport properties of liquid gallium at high pressures.

Quantum molecular dynamics (QMD) simulations are used to calculate the equation of state, structure, and transport properties of liquid gallium along the principal shock Hugoniot. The calculated Hugoniot is in very good agreement with experimental data up to a pressure of 150 GPa as well as with our earlier classical molecular dynamics calculations using a modified embedded atom method (MEAM) potential. The self-diffusion and viscosity calculated using QMD agree with experimental measurements better than the MEAM results, which we attribute to capturing the complexity of the electronic structure at elevated temperatures. Calculations of the DC conductivity were performed around the Hugoniot. Above a density of 7.5 g/cm(3), the temperature increases rapidly along the Hugoniot, and the optical conductivity decreases, indicating simple liquid metal behavior.

Quantification of natural antibody producing B cells in rats by an improved ELISPOT technique using the polyvinylidene difluoride membrane as the solid support.

We describe here a new type of solid support for the ELISPOT assay, the PVDF membrane. In parallel tests, spot yields on this membrane were superior to those obtained with the frequently used nitrocellulose (NC) membrane, coated with the same rat anti-IgM and anti-IgG antibodies, incubated with the same rat spleen cell suspensions, and developed with the same combination of AP-labeled conjugates and substrate. We therefore used the PVDF membrane, coated with anti-rat IgM and IgG antibodies, ssDNA or bromelain-treated mouse erythrocytes (BrMRBC) (exposing phosphatidylcholine (PC) as major autoantigen) to develop ELISPOT assays for the quantification of isotype-specific natural antibody secreting cells (ASC) in rats. We confirmed the isotype specificity of the binding of the anti-rat IgM and anti-rat IgG coating antibodies and conjugates with the secreted rat antibodies in this assay, and, by inhibition of spot formation with soluble antigen, their specificity for ssDNA and BrMRBC. An in-house 18-well culture device for the easy manufacture of PVDF-lined culture wells greatly facilitated coating, blocking, and washing procedures, as compared to the original method in 24 well culture plates. This simple, fast, specific and sensitive ELISPOT assay was used to make an inventory of the numbers of natural splenic ASC in Wistar and Fischer rats.

Screening tests for autoimmune-related immunotoxicity.

A large number of chemicals induce or exacerbate autoimmune-like diseases in man. Because of the complexity of processes involved, these adverse effects are often if not always missed in standard toxicity testing. To date no validated and generally applicable predictive animal model exists and only a few chemicals have actually been shown to induce adverse autoimmune effects in certain animals. The popliteal lymph node assay (PLNA) is a very promising animal test to (pre)screen for systemic immunosensitizing, including autoimmunogenic potential. This review describes the essentials of the various PLNAs against the background of current understanding of chemically induced systemic immunostimulation. The most simple primary PLNA measures enlargement of the popliteal lymph node 6-8 days after subcutaneous injection of a chemical into the footpad. The primary PLNA can distinguish between immunostimulating (both sensitizers and irritants) and innocent chemicals but does not assess the involvement of T cells or immunosensitization. For this, but also for elucidation of relevant mechanisms, detection of anamnestic responses in secondary PLNAs or responses to reporter antigens in the modified PLNA are suitable. To date over 100 compounds (drugs and environmental pollutants) have been tested, and results show a good correlation with reported immunostimulating (both autoimmunogenic and allergic) potential. Importantly, no false-negative chemicals were detected if metabolism was considered. The various types of the PLNA, but in particular the secondary and modified PLNAs, await extensive validation before they can be recommended as a standard test for autoimmunogenic potential.

Effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid (CLA) isomers on immune function in healthy men.

OBJECTIVES: To study the effects of two different mixtures of the main conjugated linoleic acid (CLA) isomers cis-9, trans-11 CLA and trans-10, cis-12 CLA on human immune function. DESIGN: Double-blind, randomized, parallel, reference-controlled intervention study. SUBJECTS AND INTERVENTION: Seventy-one healthy males aged 31-69 y received one of the following treatments: (1). mixture of 50% c9,t11 CLA and 50% t10,c12 CLA isomers (CLA 50:50); (2). mixture of 80% c9,t11 CLA and 20% t10,c12 CLA isomers (CLA 80:20); and (3). sunflower oil fatty acids (reference). The treatments were given as supplements in softgel capsules providing a total of 1.7 g (c9,t11+t10,c12) CLA fatty acids (50:50) or 1.6 g (c9,t11+t10,c12) CLA glycerides (80:20) per day in treatment groups for 12 weeks. RESULTS: Almost twice as many subjects reached protective antibody levels to hepatitis B when consuming CLA 50:50 fatty acids (15/24, 62%) compared with subjects consuming the reference substance (7/21, 33%, P=0.075). In subjects consuming CLA 80:20 glycerides this was 8/22 (36%). Other aspects of immune function, ie DTH responses, NK cell activity, lymphocyte proliferation and production of TNF-alpha, IL1-beta, IL6, IFN-gamma, IL2, IL4, and PGE(2), were not affected. CONCLUSION: This is the first study that suggests that CLA may beneficially affect the initiation of a specific response to a hepatitis B vaccination. This was seen in the CLA 50:50, but not in the CLA 80:20 group.

Site-selective electronic correlation in α-plutonium metal.

An understanding of the phase diagram of elemental plutonium (Pu) must include both, the effects of the strong directional bonding and the high density of states of the Pu 5f electrons, as well as how that bonding weakens under the influence of strong electronic correlations. Here we present electronic-structure calculations of the full 16-atom per unit cell α-phase structure within the framework of density functional theory together with dynamical mean-field theory. Our calculations demonstrate that Pu atoms sitting on different sites within the α-Pu crystal structure have a strongly varying site dependence of the localization-delocalization correlation effects of their 5f electrons and a corresponding effect on the bonding and electronic properties of this complicated metal. In short, α-Pu has the capacity to simultaneously have multiple degrees of electron localization/delocalization of Pu 5f electrons within a pure single-element material.

Superconducting gap structure of the 115s revisited.

Density functional theory calculations of the electronic structure of Ce- and Pu-based heavy fermion superconductors in the so-called 115 family are performed. The gap equation is used to consider which superconducting order parameters are most favorable assuming a pairing interaction that is peaked at (π, π, qz)­the wavevector for the antiferromagnetic ordering found in close proximity. In addition to the commonly accepted dx2−y2 order parameter, there is evidence that an extended s-wave order parameter with nodes is also plausible. We discuss whether these results are consistent with current observations and possible measurements that could help distinguish between these scenarios.

Elastic properties of the light actinides at high pressure.

Using density-functional perturbation theory, we have calculated the elastic constants for the ground-state crystal structures of the light actinide metals both at their equilibrium volumes (for Th through Np) and as a function of pressure (for Th through U). As necessary, we take into account the effects of atomic relaxation and show that these can be neglected for α-U, but are crucial for α-Np. The elastic constants of Th and U are compared with experimental measurements near ambient conditions and good agreement is found. Studies of the Born stability criteria in pressure reveal that Th and Pa are mechanically unstable while U remains stable up to 85 GPa, as is observed in diamond anvil cell experiments.

First-principles study of stability of the bcc and ω phases of a low Al concentration Nb1-xAlx alloy.

The phase stability and site occupancy of bcc (body centered cubic) Nb(5)Al and slightly rearranged atomic structures have been examined by means of first-principles calculations. In order to use first-principles methods, a periodic cell is required and we used ordered Nb(5)Al compounds as a tractable example of a low Al concentration Nb(1 - x)Al(x) alloy (in this case, for about 17 at.% Al). The instability against an ω-structure atomic displacement was also studied, since this structure is detrimental to ductility. Mulliken population analysis was used to provide an understanding of the hybridization between the atoms and the electronic origin of the site occupancy and instability of the underlying bcc structures. By making calculations for several different configurations of the Nb-Al system we estimated the strengths of the Nb-Nb and Nb-Al bonds. It is shown that the stability of the underlying bcc phases is directly related to Nb-Nb and Nb-Al first-nearest-neighbor interactions. The first-principles calculations were extended to finite temperature by including various contributions to the free energy. In particular, the vibrational free energy was calculated within the quasiharmonic approximation, and it is shown that the contribution of the low energy modes to the lattice entropy helps to stabilize ordered bcc phases against ω-type phase transformations. Semi-quasi-random structures were employed to study the stability of the ordered and disordered bcc phases. Our study showed, in agreement with experiment, that the ω, ordered, and disordered phases can coexist in a nonequilibrium state at finite temperature.

Evaluation of the interaction of cyclin-dependent kinase 5 with activator p25 and with p25-derived inhibitor CIP.

A high-affinity inhibitor protein called CIP, produced by small truncations of p35, was experimentally identified. P35 is a physiological activator of the cyclin-dependent kinase cdk5. P25 is derived from proteolytic truncation of p35 within "stressed" neurons, and it is associated with the hyperphosphorylation of specific neuronal proteins, typically occurring in neurodegenerative diseases such as Alzheimer's. Here, we report a study of the binding mechanisms of the cdk5-p25 and cdk5-CIP complexes. This provides a better understanding of the source of the inhibitory activity of the protein CIP. We use a geometry-based technique to test the hypothesis that p25's truncation increases the flexibility of CIP and thus prevents cdk5 from reaching its active conformation. Our study is based on a geometry-based alignment algorithm, which aligns two given protein conformations with respect to their interfaces. Our results support the flexibility hypothesis and will be used as a basis for targeted molecular dynamics simulations.