A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002). Published: December 9, 2002     

A study of the antiresorptive activity of salmon calcitonin microspheres using cultured osteoclastic cells

The purpose of this study was to evaluate salmon calcitonin (sCT) microspheres in vitro for their antiresorptive activity using cultured osteoclastic cells. The antiresorptive activity of sCT-loaded microspheres, prepared from a low molecular weight hydrophilic poly (lactide-co-glycolide) polymer (PLGA), was studied using bone marrow culture cells harvested from juvenile rats and cultured on silces of devitalized bone for up to 4 weeks. The resorptive activity of osteoclastic cells was quantified in terms of number and type of resorption pits and total area of resorption. Microspheres containing 5.1% sCT released 70% peptide in 2 weeks and 88% in 4 weeks. All sCT treatments inhibited total resorptive activity. A dose-dependent decrease in resorption was observed with sCT microspheres at 2 weeks. The high dose (10 mg of microspheres) produced a 99.5% decrease in resorption at 3 weeks, while the low dose (1 mg) produced an 80% reduction. Exposure of cultures to soluble sCT and sCT-loaded microspheres caused a decrease in the number of large pits, which were the predominant type formed in control cultures. Thus, this system could serve as an in vitro method to evaluate the antiresorptive effect of PLGA-sCT microspheres.     

Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis

A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome. A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen. One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations. We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches. To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV. Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes. We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV. In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines. 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes. For comparison, coding sequences account for 44% of the Arabidopsis genome. Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions. However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.     

High-performance liquid chromatographic method for the quantification of unbound evernimicin in human plasma ultrafiltrate

A rapid HPLC method was developed for quantification of unbound evernimicin in human plasma. Protein-free samples prepared by ultrafiltration were injected directly onto a polymeric reversed-phase column and the eluent monitored at 302 nm. Evernimicin that eluted within 3.5 min was well resolved from endogenous components. Linearity was established between peak height and evernimicin concentration from 25 to 2500 ng/ml. Assay precision (C.V.) was within 5% while bias was no greater than 3%. This method has been used for the ex vivo assessment of evernimicin protein binding in human plasma from safety and tolerance as well as liver dysfunction and renal insufficiency studies.     

Post-translational modification detection using metastable ions in reflector matrix-assisted laser desorption/ionization-time of flight mass spectrometry

In addition to protein identification, characterization of post-translational modifications (PTMs) is an essential task in proteomics. PTMs represent the major reason for the variety of protein isoforms and they can influence protein structure and function. Upon matrix-assisted laser desorption/ionization (MALDI) most post-translationally modified peptides form a fraction of labile molecular ions, which lose PTM-specific residues only after acceleration. Compared to fully accelerated ions these fragment ions are defocused and show in reflector mass spectra reduced resolution. A short time Fourier transform using a Hanning window function now uses this difference in resolution to detect the metastable fragments. Its application over the whole mass range yields frequency distributions and amplitudes as a function of mass, where an increased low frequency proportion is highly indicative for metastable fragments. Applications on the detection of metastable losses originating from carboxamidomethylated cysteines, oxidized methionines, phosphorylated and glycosylated amino acid residues are presented. The metastable loss of mercaptoacetamide detected with this procedure represents a new feature and its integration in search algorithms will improve the specificity of MALDI peptide mass fingerprinting.     

Analysis of the collapsibility of the upper airway in a spectrum of sleep-disordered breathing: a modelling approach

Using a simplified model of the upper airways with two independent collapsible elements (nostrils and hypo-pharynx), we calculated the cross-sectional area of these two elements, taking into account pressure drops. We experimentally measured flow and pressure in the fossa and hypo-pharynx in various syndromes. This allowed us to compare the behaviour of the area supplied by our model with the aerodynamic resistance that is often used to analyse upper airway flow limitation events. We showed that nostril and hypo-pharyngeal areas are better correlated than the resistance values and thus concluded that the pressure divided by the square of the flow is a better parameter for analysing flow limitation in upper airways than resistance. Owing to its simplicity, our model is able to supply the area of the collapsible element in real time, which is impossible with more sophisticated models.     

Beta-glucosidase-catalyzed hydrolysis of indican from leaves of Polygonum tinctorium

In this article, a HPLC method to identify and quantify the dyes and the indigo precursors produced in Polygonum tinctorium is described. Using this technique, indican has been positively identified in extracts of P. tinctorium. Our work with two cultivars of P. tinctorium has confirmed that the quantity of indican is dependent on the cultivars, harvest period, and age of the leaves. Two enzymes, Novozym 188 (cellobiase) and Novarom G (beta-glucosidase), are compared on the basis of their activities to hydrolyze the indican at several pH values. We observed that Novarom G is more active than Novozym 188 whatever the pH and that optimum pH of both enzymes for indican hydrolysis is 3. Liberated indoxyl can be oxidized in alkaline media and transformed into indigo and indirubin.     

SHIP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts

The hematopoietic-restricted protein Src homology 2-containing inositol-5-phosphatase (SHIP) blunts phosphatidylinositol-3-kinase-initiated signaling by dephosphorylating its major substrate, phosphatidylinositol-3,4,5-trisphosphate. As SHIP(-/-) mice contain increased numbers of osteoclast precursors, that is, macrophages, we examined bones from these animals and found that osteoclast number is increased two-fold. This increased number is due to the prolonged life span of these cells and to hypersensitivity of precursors to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). Similar to pagetic osteoclasts, SHIP(-/-) osteoclasts are enlarged, containing upwards of 100 nuclei, and exhibit enhanced resorptive activity. Moreover, as in Paget disease, serum levels of interleukin-6 are markedly increased in SHIP(-/-) mice. Consistent with accelerated resorptive activity, 3D trabecular volume fraction, trabecular thickness, number and connectivity density of SHIP(-/-) long bones are reduced, resulting in a 22% loss of bone-mineral density and a 49% decrease in fracture energy. Thus, SHIP negatively regulates osteoclast formation and function and the absence of this enzyme results in severe osteoporosis.     

Urotensin II mediates ERK1/2 phosphorylation and proliferation in GPR14-transfected cell lines

Urotensin-II (U-II), a vasoactive cyclic neuropeptide, was recently identified as the natural ligand for the G-protein coupled receptor GPR14. The expression pattern of U-II and GPR14 are consistent with a role as a neurohormonal regulatory system in cardiovascular homeostasis. Urotensin-II induces a rapid and short-lasting rise in intracellular calcium in recombinant GPR14 expressing cells. In the present study we show that U-II induces signal transduction pathways leading to the long-lasting activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in chinese hamster ovary cells expressing human GPR14 (CHO-GPR14). Furthermore, we observed a growth-stimulating and PD98059 sensitive activity of U-II in CHO-GPR14 cells, but not CHO-K1 cells. The investigation of the GPR14 induced signal transduction pathways leading to ERKI/2 phosphorylation revealed a previously unsuspected role for G(i/o)-protein coupling and showed an involvement of phospatidylinositol-3-kinase, phospholipase C and calcium channel mediated mechanisms. Our results suggest that U-II and its receptor GPR14 may be involved in long-lasting physiological effects such as cardiovascular remodeling.     

Dual-specific T cells combine proliferation and antitumor activity

An effective immune response against cancer requires the activation and expansion of specific T cells. Tumor antigens, however, are generally poor immunogens. To achieve expansion of tumor-reactive T cells in vivo, we used a strategy of generating dual-specific T cells that could respond to a powerful immunogen while also possessing tumor reactivity. We generated dual-specific T cells by genetic modification of alloreactive T cells with a chimeric receptor recognizing folate-binding protein, an ovarian cancer-associated antigen. Mouse dual-specific T cells responded in vitro to both allogeneic antigen and tumor cells expressing folate-binding protein, and expanded in number in vivo in response to immunization with allogeneic cells. Most importantly, the combination of dual-specific T cells and immunization had an antitumor effect in vivo. We also generated human dual-specific T cells and characterized the dual-specific nature of individual clones. Assigning the tasks of expansion and tumor reactivity to different receptors within the same lymphocyte may help to overcome the problem of poor immunogenicity of tumor antigens.