Skeletal muscles, heart, and lung are the main sources of oxygen radicals in old rats

The aim of this study was to compare rat tissues with respect to their reactive oxygen and nitrogen species (RONS) generating activities as a function of age. We quantified the RONS generation in vivo in young (6 months) and in old (30 months) male Sprague-Dawley rats using the recently developed spin trap 1-hydroxy-3-carboxy-pyrrolidine, applied intravenously. This spin trap reacts with superoxide radical and peroxynitrite yielding a stable spin adduct which is detectable by means of electron paramagnetic resonance (EPR) spectroscopy in frozen tissues. In old rats RONS generation was significantly increased compared to their young counterparts in the following order: blood相似文献   

The nonequilibrium phase and glass transition behavior of beta-lactoglobulin

Concentrated solutions of bovine beta-lactoglobulin were studied using osmotic stress and rheological techniques. At pH 6.0 and 8.0, the osmotic pressure was largely independent of NaCl concentration and could be described by a hard sphere equation of state. At pH 5.1, close to the isoelectric point, the osmotic pressure was lower at the lower NaCl concentrations (0 mM, 100 mM) and was fitted by an adhesive hard sphere model. Liquid-liquid phase separation was observed at pH 5.1 at ionic strengths of 13 mM and below. Comparison of the liquid-liquid and literature solid-liquid coexistence curves showed these solutions to be supersaturated and the phase separation to be nonequilibrium in nature. In steady shear, the zero shear viscosity of concentrated solutions at pH 5.1 was observed at shear rates above 50 s(-1). With increasing concentration, the solution viscosity showed a progressive increase, a behavior interpreted as the approach to a colloidlike glass transition at approximately 60% w/w. In oscillatory shear experiments, the storage modulus crossed the loss modulus at concentrations of 54% w/w, an indication of the approaching glass transition. Comparison of the viscous behavior with predictions from the Krieger-Dougherty equation indicates the hydrodynamic size of the protein decreases with increasing concentration, resulting in a slower approach to the glass transition than a hard sphere system.     

Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr(32)Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe(94)Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe(94)Ala up to the level of the e-pep affinity to the wild-type scFv.     

Inhibition of keratinocyte apoptosis by IL-15: a new parameter in the pathogenesis of psoriasis?

Keratinocytes (KC) are important source of and targets for several cytokines. Although KC express IL-15 mRNA, the functional effects of IL-15 on these epithelial cells remain to be dissected. Investigating primary human foreskin KC and HaCaT cells, we show here by semiquantitative RT-PCR and flow cytometric analysis that both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Ralpha protein on the cell surface, suggesting that human KC can employ IL-15 for juxtacrine signaling. While IL-15 exerted no significant effect on KC proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is in line with the recognized potent anti-apoptotic effects of IL-15. IL-2, whose receptor shares two components with the IL-15R, failed to inhibit KC apoptosis. Together with the role of IL-15 in sustaining chronic immune reactions, this invited the question of whether a reduction of KC apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low KC apoptosis in the epidermis. Remarkably, compared with nonlesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC apoptosis deserves exploration as a novel therapeutic strategy in psoriasis management.     

Fluorescence clamp: A direct measure of fluxes into and out of the antenna pool of Photosystem II

Fluorescence clamp (FC) is a method of directly measuring the fluxes out of Photosystem II antenna. This is achieved by a feed-back loop which controls the light intensity of light emitting diodes in order to keep the amplitude of modulated chlorophyll fluorescence constant, and by taking the intensity or the current fed into the light emitting diodes as a measure of the fluxes. Saturating flashes serve to distinguish between fluxes into thermal deactivation and into the photosynthetic electron transfer chain (ETC). As FC is only active in the light period of the measuring light, the background signal (induced by actinic light) is compensated by a second feed-back loop in the dark period of the measuring light. Equations are provided for the interpretation of the FC signals. This includes the quenching parameters of chlorophyll fluorescence, the flux into the electron transfer chain and the redox state of Q_A. Experiments are presented which show that traditional fluorescence (LC) and FC measurements yield the same results. However, the FC method provides a better presentation of fluxes as the scaling factor (flux/signal) is constant for all states of Photosystem II. This leads to a simpler analysis of quenching mechanisms. Examples are given which show that the co-existing quenching mechanisms with different effects on photochemical and non-photochemical fluxes can be better identified by FC rather than by LC. This revised version was published online in June 2006 with corrections to the Cover Date.     

Syntheses of alpha-dystroglycan derived glycosyl amino acids carrying a novel mannosyl serine/threonine linkage

-Dystroglycan (-DG) is a membrane-associated, extracellular glycoprotein. It is anchored to the cell-membrane by binding to the transmembrane glycoprotein -dystroglycan (-DG) to form an /-DG-complex. It was discovered that the bovine peripheral nerve -DG possesses the Ser/Thr linked tetrasaccharide as the major constituent of the O-linked carbohydrates, which was proposed to contribute laminin binding activity of this glycoprotein.This structure has a striking feature in terms of the mode of linkage between oligosaccharide and the core protein. It has a mannose residue linked to the core protein through Ser/Thr residue. A similar structure was proposed to exist in brain derived HNK-1 immunoreactive O-glycans. Being interested in the structural novelty and potential biological significance of this type of glycan chains, the chemical synthesis of Ser/Thr linked mannose containing tetrasaccharide was investigated. Tetrasaccharide donor was constructed from monosaccharide blocks and coupled with Ser/Thr derivatives. Subsequent deprotection afforded target tetraosyl serine. Furthermore, synthetic routes to lower homologues, namely Gal--(1,4)-GlcNAc--(1,2)-Man--Ser and GlcNAc--(1,2)-Man--Ser were also provided.     

Transport of resistance-inducing sterols in phloem sap of barley

After root application of [7alpha-3H]-7beta-hydroxysitosterol and [3alpha,6beta-3H2]-6alpha-hydroxylathosterol these sterols could be detected in the leaves and phloem sap feeding aphids. These results imply that the phloem sap is a sterol transport system in barley plants.