Stereoselective Binding of Flurbiprofen Enantiomers and their Methyl Esters to Human Serum Albumin Studied by Time-Resolved Phosphorescence

The interaction of the nonsteroidal anti‐inflammatory drug flurbiprofen (FBP) with human serum albumin (HSA) hardly influences the fluorescence of the protein's single tryptophan (Trp). Therefore, in addition to fluorescence, heavy atom‐induced room‐temperature phosphorescence is used to study the stereoselective binding of FBP enantiomers and their methyl esters to HSA. Maximal HSA phosphorescence intensities were obtained at a KI concentration of 0.2 M. The quenching of the Trp phosphorescence by FBP is mainly dynamic and based on Dexter energy transfer. The Stern–Volmer plots based on the phosphorescence lifetimes indicate that (R)‐FBP causes a stronger Trp quenching than (S)‐FBP. For the methyl esters of FBP, the opposite is observed: (S)‐(FBPMe) quenches more than (R)‐FBPMe. The Stern–Volmer plots of (R)‐FBP and (R)‐FBPMe are similar although their high‐affinity binding sites are different. The methylation of (S)‐FBP causes a large change in its effect on the HSA phosphorescence lifetime. Furthermore, the quenching constants of 3.0 × 10⁷ M^?1 s^?1 of the R‐enantiomers and 2.5 × 10⁷ M^?1 s^?1 for the S‐enantiomers are not influenced by the methylation and indicate a stereoselectivity in the accessibility of the HSA Trp to these drugs. Chirality 24:840–846, 2012. © 2012 Wiley Periodicals, Inc.     

Thermography imaging during static and controlled thermoregulation in complex regional pain syndrome type 1: diagnostic value and involvement of the central sympathetic system

Background  

Complex Regional Pain Syndrome type 1 (CRPS1) is a clinical diagnosis based on criteria describing symptoms of the disease.     

Circular spectropolarimetric sensing of higher plant and algal chloroplast structural variations

Photosynthetic eukaryotes show a remarkable variability in photosynthesis, including large differences in light-harvesting proteins and pigment composition. In vivo circular spectropolarimetry enables us to probe the molecular architecture of photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological and structural information. In the present study, we have measured the circular polarizance of several multicellular green, red, and brown algae and higher plants, which show large variations in circular spectropolarimetric signals with differences in both spectral shape and magnitude. Many of the algae display spectral characteristics not previously reported, indicating a larger variation in molecular organization than previously assumed. As the strengths of these signals vary by three orders of magnitude, these results also have important implications in terms of detectability for the use of circular polarization as a signature of life.

     

Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum.

In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans. One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it. On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only. All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads. All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed. We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics.     

DNA damage in plant herbarium tissue

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.     

Efficiency of nitrate uptake in spinach: impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with 13N- and 15N-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration. Plants were grown at different nitrate concentrations (0.8 and 4 mM), with mineral composition of growth and uptake solutions identical. Nitrate influx, efflux and net nitrate uptake rate (NNUR) were independent of the external nitrate concentration, despite differences in internal nitrate concentration. At both N regimes, NNUR was adequate to meet the N demand for growth. RGR-related signals predominantly determined the nitrate fluxes. At high RGR (0.25 g g-1 day-1), nitrate influx was 20 to 40% lower and nitrate efflux was 50 to 70% lower than at lower RGR (0.17 g g-1 day-1); efflux:influx ratio (E:I) declined from 0.5 at low RGR to 0.2 at higher RGR. Thus, the efficiency of NNUR substantially increased with increasing RGR. Differences in nitrate translocation between morning and afternoon coincided with differences in nitrate efflux, which is in accordance with the suggested regulation of nitrate efflux by the root cytoplasmic nitrate concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Adherence to a treat-to-target strategy in early rheumatoid arthritis: results of the DREAM remission induction cohort

Introduction

Clinical trials have demonstrated that treatment-to-target (T2T) is effective in achieving remission in early rheumatoid arthritis (RA). However, the concept of T2T has not been fully implemented yet and the question is whether a T2T strategy is feasible in daily clinical practice. The objective of the study was to evaluate the adherence to a T2T strategy aiming at remission (Disease Activity Score in 28 joints (DAS28) < 2.6) in early RA in daily practice. The recommendations regarding T2T included regular assessment of the DAS28 and advice regarding DAS28-driven treatment adjustments.

Methods

A medical chart review was performed among a random sample of 100 RA patients of the DREAM remission induction cohort. At all scheduled visits, it was determined whether the clinical decisions were compliant to the T2T recommendations.

Results

The 100 patients contributed to a total of 1,115 visits. The DAS28 was available in 97.9% (1,092/1,115) of the visits, of which the DAS28 was assessed at a frequency of at least every three months in 88.3% (964/1,092). Adherence to the treatment advice was observed in 69.3% (757/1,092) of the visits. In case of non-adherence when remission was present (19.5%, 108/553), most frequently medication was tapered off or discontinued when it should have been continued (7.2%, 40/553) or treatment was continued when it should have been tapered off or discontinued (6.2%, 34/553). In case of non-adherence when remission was absent (42.1%, 227/539), most frequently medication was not intensified when an intensification step should have been taken (34.9%, 188/539). The main reason for non-adherence was discordance between disease activity status according to the rheumatologist and DAS28.

Conclusions

The recommendations regarding T2T were successfully implemented and high adherence was observed. This demonstrates that a T2T strategy is feasible in RA in daily clinical practice.     

Germination of Penicillium paneum Conidia Is Regulated by 1-Octen-3-ol, a Volatile Self-Inhibitor

Penicillium paneum is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions. P. paneum produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 10⁹ conidia ml⁻¹. By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25°C and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the P. paneum life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development.     

Quantification of an C-labelled β-adrenoceptor ligand, S-(−)CGP 12177, in plasma of humans and rats

β-Adrenoceptors in human lungs and heart can be imaged with the radioligand 4-[3-[(1,1-dimethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-¹¹C-one (CGP 12177, [¹¹C]I). For quantification of receptor density with compartment models by adjustment of rate constants, an ‘input function’ is required which consists of the integral of the concentration of unmodified ligand in arterial plasma over time. A discrepancy in the literature regarding metabolic stability of [¹¹C]I prompted us to study metabolism in rats by reversed-phase HPLC (RP-HPLC) of trichloroacetic acid extracts of arterial plasma after i.v. injection of [¹¹C]I (> 11.1 TBq/mmol, 11 MBq/kg). Some plasma samples were also directly applied to an internal-surface reversed-phase (ISRP) column. In parallel experiments, tritiated [¹¹C]I was employed and methanol extracts of arterial plasma were analyzed by straight-phase TLC. The three methods were in excellent agreement. Unmodified [¹¹C]I decreased from > 98.5% (³H) or > 99.9% (¹¹C) initially to 57 ± 7% at 80 min post injection to formation of two polar metabolites. Using the RP-HPLC method, no metabolism was detectable in humans up to 30 min after injection of [¹¹C]I (1851 MBq). Deproteinization of plasma with acetonitrile resulted in the formation of a radioactive species (artifact) which eluted immediately after the void volume in RP-HPLC and which could be mistakenly interpreted as a metabolite. Plasma protein binding was low (ca. 30%) in both humans and rats. Association of the radioligand to blood cells suggested that equilibrium between receptor-bound and free radioligand was reached within 15 min after high-specific-activity injections, but only after more than 30 min after low-specific-activity injections.     

Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells

This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are alpha-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.