Selective high-performance liquid chromatographic assay for itraconazole and hydroxyitraconazole in plasma from human immunodeficiency virus-infected patients

A sensitive and selective reversed-phase liquid chromatographic assay for itraconazole and hydroxyitraconazole in human plasma has been developed and validated. Itraconazole and hydroxyitraconazole were extracted from the matrix using solid-phase extraction on a strong cation-exchange sorbent. All compounds were detected using fluorescence at 265 and 363 nm for excitation and emission, respectively. The assay has been validated over the range 10-1,000 ng/ml for both compounds, 10 ng/ml being the lower limit of quantification. Accuracies ranged from 104 to 113% for itraconazole and from 91 to 103% for hydroxyitraconazole. The intra-assay precisions were all below 9% for itraconazole and below 8% for hydroxyitraconazole. The selectivity has been evaluated with respect to all registered anti-human immunodeficiency virus (HIV) drugs and other potential co-medications and a few of their metabolites, commonly used by HIV-infected individuals. Both itraconazole and hydroxyitraconazole were stable under relevant conditions for HIV-inactivation and storage of samples. The applicability of the assay was demonstrated for samples collected from a treated HIV-infected patient.     

Generic solid phase extraction-liquid chromatography-tandem mass spectrometry method for fast determination of drugs in biological fluids

A generic method was developed for the fast determination of a wide range of drugs in serum or plasma. The methodology comprises generic solid-phase extraction, on-line coupled to gradient HPLC with tandem mass spectrometric detection (SPE-LC-MS/MS). The individual components of the SPE-LC-MS/MS system were optimized in an integrated approach to maximize the application range and minimize the method development time. The optimized generic SPE-LC-MS/MS protocol was evaluated for 11 drugs with different physicochemical properties. Good quantification for 10 out of 11 of the pharmaceuticals in serum or plasma could be readily achieved. The quantitative assays gave recoveries better than 95%, lower quantification limits of 0.2-2.0 ng/ml, acceptable precision and accuracy and good linearity over 2-4 orders of magnitude. Carry-over was determined to be in the range of 0.02-0.10%, without optimization.     

Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full-length VirD2, VirC2, or VirE2 leads to insertion of aberrant T-DNA structures

Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.     

Fc receptor gamma chain residues at the interface of the cytoplasmic and transmembrane domains affect association with FcalphaRI, surface expression, and function

The assembly of multiple subunit immunoreceptors is dependent on transmembrane interactions. The Fc receptor gamma (FcR-gamma) chain is a ubiquitous immune receptor tyrosine-based activation motif-containing dimeric subunit, gamma(2), which in humans associates with both the activating members of the leukocyte receptor cluster, including the IgA receptor FcalphaRI, and the classical Fc receptors, including the IgE receptor FcepsilonRI. This study identifies a new site in the transmembrane region of FcR-gamma that affects receptor assembly and surface expression with FcalphaRI but not with FcepsilonRI. The wild type complex, FcalphaRI-gamma(2)WT, remains robustly associated in both Brij-96 and Thesit detergent conditions. However, mutation of either Tyr(25) or Cys(26) of FcR-gamma, near the interface of the transmembrane and cytoplasmic regions, resulted in impaired FcR-gamma association with FcalphaRI. This association was disrupted in the presence of the detergent Brij-96 but was preserved in milder conditions using the detergent Thesit. Ligand-mediated cross-linking of the FcalphaRI-gamma(2)Y25F mutant receptor resulted in diminished signal transduction, including an abnormal calcium response, compared with the FcalphaRI-gamma(2)WT receptor. Furthermore, the FcalphaRI-gamma(2)Y25F mutant receptor was expressed at the cell surface at approximately 10% of that of the wild type, whereas the surface expression of FcepsilonRI-gamma(2)Y25F was not significantly different from the wild type. In contrast, although the FcalphaRI-gamma(2)C26S mutant was also less stably associated, it was not reduced in surface expression or function. Thus, these TM residues of FcR-gamma are important for association with FcalphaRI and probably other activating LRC members but not with the classical FcR, FcepsilonRI.     

Opsonization with C1q and mannose-binding lectin targets apoptotic cells to dendritic cells

Deficiencies of early components of the classical complement pathway, particularly C1q, are strongly associated with susceptibility to systemic lupus erythematosus. Recent data link this predisposal to autoimmunity to an inappropriate clearance of apoptotic cells, which could lead to a loss of self-tolerance. In the present study, we demonstrate that opsonization of apoptotic cells with C1q and mannose-binding lectin allows and facilitates their uptake not only by macrophages but also by human immature dendritic cells (DCs). Both C1q and mannose-binding lectin enhance the uptake of apoptotic cells by DCs in a dose-dependent way. The uptake of C1q-opsonized apoptotic cells, but not nonopsonized apoptotic cells, by DCs stimulated the production of IL-6, IL-10, and TNF-alpha, without an effect on IL-12p70. We conclude that these recognition molecules of the complement system do not sequester apoptotic cells from DCs, but rather promote their uptake by immature DCs. Therefore, we propose that early complement components support safe clearance of cellular debris by facilitating phagocytosis and possibly by immunomodulatory mechanisms, thus preventing autoimmunity.     

Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces pombe

The RAD52 gene of Saccharomyces cerevisiae is essential for repair of DNA double-strand breaks (DSBs) by homologous recombination. Inactivation of this gene confers hypersensitivity to DSB-inducing agents and defects in most forms of recombination. The rad22+ gene in Schizosaccharomyces pombe (here referred to as rad22A+) has been characterized as a homolog of RAD52 in fission yeast. Here, we report the identification of a second RAD52 homolog in Schizosaccharomyces pombe, called rad22B+. The amino acid sequences of Rad22A and Rad22B show significant conservation (38% identity). Deletion mutants of respectively, rad22A and rad22B, show different phenotypes with respect to sensitivity to X-rays and the ability to perform homologous recombination as measured by the integration of plasmid DNA. Inactivation of rad22A+ leads to a severe sensitivity to X-rays and a strong decrease in recombination (13-fold), while the rad22B mutation does not result in a decrease in homologous recombination or a change in radiation sensitivity. In a rad22A-rad22B double mutant the radiation sensitivity is further enhanced in comparison with the rad22A single mutant. Overexpression of the rad22B+ gene results in partial suppression of the DNA repair defects of the rad22A mutant strain. Meiotic recombination and spore viability are only slightly affected in either single mutant, but outgrowth of viable spores is almost 31-fold reduced in the rad22A-rad22B double mutant. The results obtained imply a crucial role for rad22A+ in repair and recombination in vegetative cells just like RAD52 in S. cerevisiae. The rad22B+ gene presumably has an auxiliary role in the repair of DSBs. The drastic reduced spore viability in the double mutant suggests that meiosis in S. pombe is dependent on the presence of either rad22A+ or rad22B+.     

A lipidomic analysis approach to evaluate the response to cholesterol-lowering food intake

Plant sterols (PS) are well known to reduce serum levels of total cholesterol and LDL-cholesterol. Lipidomics potentially provides detailed information on a wide range of individual serum lipid metabolites, which may further add to our understanding of the biological effects of PS. In this study, lipidomics analysis was applied to serum samples from a placebo-controlled, parallel human intervention study (n?=?97) of 4-week consumption of two PS-enriched, yoghurt drinks differing in fat content (based on 0.1% vs. 1.5% dairy fat). A comprehensive data analysis strategy was developed and implemented to assess and compare effects of two different PS-treatments and placebo treatment. The combination of univariate and multivariate data analysis approaches allowed to show significant effects of PS intake on the serum lipidome, and helped to distinguish them from fat content and non-specific effects. The PS-enriched 0.1% dairy fat yoghurt drink had a stronger impact on the lipidome than the 1.5% dairy fat yoghurt drink, despite similar LDL-cholesterol lowering effects. The PS-enriched 0.1% dairy fat yoghurt drink reduced levels of several sphingomyelins which correlated well with the reduction in LDL-cholesterol and can be explained by co-localization of sphingomyelins and cholesterol on the surface of LDL lipoprotein. Statistically significant reductions in serum levels of two lysophosphatidylcholines (LPC(16:1), LPC(20:1)) and cholesteryl arachidonate may suggest reduced inflammation and atherogenic potential.     

The flat-plate plant-microbial fuel cell: the effect of a new design on internal resistances

Due to a growing world population and increasing welfare, energy demand worldwide is increasing. To meet the increasing energy demand in a sustainable way, new technologies are needed. The Plant-Microbial Fuel Cell (P-MFC) is a technology that could produce sustainable bio-electricity and help meeting the increasing energy demand. Power output of the P-MFC, however, needs to be increased to make it attractive as a renewable and sustainable energy source. To increase power output of the P-MFC internal resistances need to be reduced. With a flat-plate P-MFC design we tried to minimize internal resistances compared to the previously used tubular P-MFC design. With the flat-plate design current and power density per geometric planting area were increased (from 0.15 A/m² to 1.6 A/m² and from 0.22 W/m² to and 0.44 W/m²)as were current and power output per volume (from 7.5 A/m³ to 122 A/m³ and from 1.3 W/m³ to 5.8 W/m³). Internal resistances times volume were decreased, even though internal resistances times membrane surface area were not. Since the membrane in the flat-plate design is placed vertically, membrane surface area per geometric planting area is increased, which allows for lower internal resistances times volume while not decreasing internal resistances times membrane surface area. Anode was split into three different sections on different depths of the system, allowing to calculate internal resistances on different depths. Most electricity was produced where internal resistances were lowest and where most roots were present; in the top section of the system. By measuring electricity production on different depths in the system, electricity production could be linked to root growth. This link offers opportunities for material-reduction in new designs. Concurrent reduction in material use and increase in power output brings the P-MFC a step closer to usable energy density and economic feasibility.     

Mechanism of homology recognition in DNA recombination from dual-molecule experiments

In E. coli homologous recombination, a filament of RecA protein formed on DNA searches and pairs a homologous sequence within a second DNA molecule with remarkable speed and fidelity. Here, we directly probe the strength of the two-molecule interactions involved in homology search and recognition using dual-molecule manipulation, combining magnetic and optical tweezers. We find that the filament's secondary DNA-binding site interacts with a single strand of the incoming double-stranded DNA during homology sampling. Recognition requires opening of the helix and is strongly promoted by unwinding torsional stress. Recognition is achieved upon binding of both strands of the incoming dsDNA to each of two ssDNA-binding sites in the filament. The data indicate a physical picture for homology recognition in which the fidelity of the search process is governed by the distance between the DNA-binding sites.     

The inflammasome puts obesity in the danger zone

Obesity-induced inflammation is an important contributor to the induction of insulin resistance. Recently, the cytokine interleukin-1β (IL-1β) has emerged as a prominent instigator of the proinflammatory response in obesity. Several studies over the last year have subsequently deciphered the molecular mechanisms responsible for IL-1β activation in adipose tissue, liver, and macrophages and demonstrated a central role of the processing enzyme caspase-1 and of the protein complex leading to its activation called the inflammasome. These data suggest that activation of the inflammasome represents a crucial step in the road from obesity to insulin resistance and type 2 diabetes.