A proteomics approach to the study of absorption, distribution, metabolism, excretion, and toxicity.

A proteomics approach was used to identify liver proteins that displayed altered levels in mice following treatment with a candidate drug. Samples from livers of mice treated with candidate drug or untreated were prepared, quantified, labeled with CyDye DIGE Fluors, and subjected to two-dimensional electrophoresis. Following scanning and imaging of gels from three different isoelectric focusing intervals (3-10, 7-11, 6.2-7.5), automated spot handling was performed on a large number of gel spots including those found to differ more than 20% between the treated and untreated condition. Subsequently, differentially regulated proteins were subjected to a three-step approach of mass spectrometry using (a) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting, (b) post-source decay utilizing chemically assisted fragmentation, and (c) liquid chromatography-tandem mass spectrometry. Using this approach we have so far resolved 121 differentially regulated proteins following treatment of mice with the candidate drug and identified 110 of these using mass spectrometry. Such data can potentially give improved molecular insight into the metabolism of drugs as well as the proteins involved in potential toxicity following the treatment. The differentially regulated proteins could be used as targets for metabolic studies or as markers for toxicity.     

Are Sexual Dissatisfaction and Sexual Abuse Associated with Obesity? A Population‐Based Study

Objective: To investigate whether there is any association between obesity and sexual satisfaction and sexual abuse in a normal population. Research Methods and Procedures: A representative sample of 2810 subjects from a population study was interviewed about sexual satisfaction, sexual abuse, and life satisfaction. The answers from normal weight, overweight, and obese participants were compared. Univariate and multivariate analyses were performed. Results: Data were presented separately for two age groups, 18 to 49 and 50 to 74 years, and gender. The older group of obese men reported a greater decrease of sexual desire compared with 5 years prior than normal weight men [odds ratios (OR), 2.44; 95% confidence interval (CI), 1.4 to 4.3]. The older group of overweight men reported involuntary participation in sexual activities more often than normal weight men (OR, 2.06; 95% CI, 1.1 to 3.8). Although older overweight and obese women were diagnosed with a lingering disease (defined as >1 month) more often than normal weight women (overweight: OR, 2.41; 95% CI, 1.3 to 4.4; obese: OR, 4.45; 95% CI, 1.7 to 11.5), there was no difference between BMI groups in satisfaction with physical health. Discussion: Overweight and obese groups seem to be heterogeneous with respect to sexual satisfaction and experiences of sexual abuse. No significant differences were detected between BMI groups, which does not exclude the possibility of significant differences between BMI groups among patients seeking medical attention.     

The use of circular dichroism spectroscopy for studying the chiral molecular self-assembly: an overview

Self-assembly plays an important role in the formation of many chiral biological structures and in the preparation of chiral functional materials. Therefore the control of chirality in synthetic or biological self-assembled systems is important either for the comprehension of recognition phenomena or to obtain materials with predictable and controllable properties. Circular dichroism was developed to study molecular chirality, however, because of its outstanding sensitivity to chiral perturbations of the system under investigation; it has been extended more recently to supramolecular chemistry. In particular, self-assembly processes leading to the formation of chiral supramolecular architectures (and eventually to gels or liquid crystal phases) can be monitored by CD. Furthermore, CD spectroscopy often allows one to obtain structural information on the assembled structures. This review deals with representative contributions to the study of supramolecular chirality by means of circular dichroism.     

Apoptotic neutrophils containing Staphylococcus epidermidis stimulate macrophages to release the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6.

Staphylococcus epidermidis infections are usually nosocomial and involve colonization of biomaterials. The immune defense system cannot efficiently control the bacteria during these infections, which often results in protracted chronic inflammation, in which a key event is disturbed removal of neutrophils by tissue macrophages. While ingesting uninfected apoptotic neutrophils, macrophages release anti-inflammatory cytokines that lead to resolution of inflammation. In clinical studies, we have previously found elevated levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 in synovial fluid from prostheses infected with coagulase negative staphylococci. We show that macrophages phagocytosing apoptotic neutrophils containing S. epidermidis released TNF-alpha and interleukin-6, whereas macrophages phagocytosing spontaneously apoptotic neutrophils did not. This difference was not due to dissimilar phagocytic capacities, because macrophages ingested both types of neutrophils to the same extent. The activation was induced mainly by the apoptotic neutrophils themselves, not by the few remaining extracellular bacteria. Macrophages were not activated by apoptotic neutrophils that contained paraformaldehyde-killed S. epidermidis. Proinflammatory reactions induced by clearance of apoptotic neutrophils containing S. epidermidis might represent an important mechanism to combat the infective agent. This activation of macrophages may contribute to the development of chronic inflammation instead of inflammation resolution.     

Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.     

Metal retention on pine bark and blast furnace slag--on-site experiment for treatment of low strength landfill leachate

Treatment of landfill leachate using blast furnace slag and pine bark as reactive sorbents was studied in an in situ column experiment at the Lilla Nyby landfill site in Eskilstuna, Sweden. The columns were filled with approximately 101 of each sorbent and leachate was supplied at three different flow rates during a period of 4 months. Samples of inflow and outflow were collected three times a week and were analyzed for physical and chemical parameters, including concentrations of some metals, and toxicity. It was found that pine bark removed metals more efficiently than did the blast furnace slags; that Zn was most efficiently retained in the filters and that both retention time and initial concentration played an important role in the sorption process. It was also observed that the pine bark column did not release COD. No toxicity of the untreated or the treated leachate was found with the test organisms and test responses used.     

Active beta-catenin signaling is an inhibitory pathway for human immunodeficiency virus replication in peripheral blood mononuclear cells

The Wnt/β-catenin pathway is involved in cell functions governing development and disease. In modeling postentry restriction of human immunodeficiency virus (HIV) replication in astrocytes, we reported that part of this natural resistance to productive replication of HIV in astrocytes involved expression of proteins of the Wnt/β-catenin signaling pathway. We determined here whether induction of β-catenin signaling in peripheral blood mononuclear cells (PBMCs) can modulate HIV replication. Given that lithium is an inducer of β-catenin signaling, we used it as a tool to determine the impact of β-catenin signaling on HIV replication in PBMCs. We demonstrated that lithium inhibited the replication of T-tropic and primary isolates of HIV by >90% and did so in noncytotoxic/noncytostatic concentrations and in a β-catenin-dependent manner. Specifically, inhibiting β-catenin signaling by transfection of dominant-negative mutant constructs to either T-cell factor 4, the downstream effector of Wnt signaling, or β-catenin, the central mediator of this pathway, abrogated the ability of lithium to inhibit HIV replication. Moreover, when Wnt/β-catenin signaling was inhibited, the level of HIV replication was enhanced by fourfold. To confirm the in vivo relevance of the β-catenin pathway in repressing HIV replication, we evaluated HIV-positive antiretroviral therapy-naive patients who were on lithium therapy. These patients demonstrated a reduction in viral load, which increased as the dose of lithium was reduced. Collectively, these data indicate that β-catenin signaling is an intrinsic molecular pathway restricting HIV replication in PBMCs.     

The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide

Background  

The formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites.     

Differential tyrosine phosphorylation of fibroblast growth factor (FGF) receptor-1 and receptor proximal signal transduction in response to FGF-2 and heparin

The sulfated regions in heparan sulfate and heparin are known to affect fibroblast growth factor (FGF) function. We have studied the mechanism whereby heparin directs FGF-2-induced FGF receptor-1 (FGFR-1) signal transduction. FGF-2 alone stimulated maximal phosphorylation of Src homology domain 2 tyrosine phosphatase (SHP-2) and the adaptor molecule Crk, in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells expressing FGFR-1. In contrast, for phospholipase Cgamma(1) (PLCgamma(1)) and the adaptor molecule Shb to be maximally tyrosine-phosphorylated, cells had to be stimulated with both FGF-2 and heparin (100 ng/ml). Tyrosine residues 463 in the juxtamembrane domain and 766 in the C-terminal tail in FGFR-1 are known to bind Crk and PLCgamma(1), respectively. Analysis of tryptic phosphopeptide maps of FGFR-1 from cells stimulated with FGF-2 alone and FGF-2 together with heparin showed that FGF-2 alone stimulated a several-fold increase in tyrosine 463 in the juxtamembrane domain. In contrast, heparin had to be included in order for tyrosine 766 to be phosphorylated to the same fold level. Our data imply that tyrosine 463 is phosphorylated and able to transduce signals in response to FGF-2 treatment alone; furthermore, we suggest that FGFR-1 dimerization/kinase activation is stabilized by heparin.