MT1-MMP Is Required for Myeloid Cell Fusion via Regulation of Rac1 Signaling

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Highlights? Myeloid cells fuse during osteoclast and giant cell formation ? MT1-MMP is required for this fusion in vitro and in vivo ? MT1-MMP regulates the GTPase Rac1 through p130Cas binding ? MT1-MMP proteolytic activity is not required     

Transport of liposomal and albumin loaded curcumin to living cells: an absorption and fluorescence spectroscopic study

Curcumin, a lipid soluble antioxidant, exhibits solvent and medium sensitive absorption and fluorescence properties. Using such changes, the average binding constants of curcumin to phosphatidylcholine (PC) liposomes and human serum albumin (HSA) were estimated to be 2.5 x 10(4) M(-1) and 6.1 x 10(4) M(-1) respectively. From the studies on temperature dependent fluorescence anisotropy of liposomal curcumin and its fluorescence quenching by acrylamide and iodide, it was concluded that curcumin is located in the gel phase of the liposomes. Similarly from the studies on quenching of tryptophan fluorescence in HSA by curcumin, it was found to be in the same domain as that of tryptophan. Both liposomal and HSA vehicles were examined for the transfer of curcumin to spleen lymphocyte cells, EL4 lymphoma cell line and compared with aqueous DMSO vehicles. From these studies it was found that liposomal vehicle is capable of loading more curcumin in to cells than HSA or aqueous-DMSO, and lymphoma cells show preferential uptake of curcumin to lymphocytes. The fluorescence of curcumin in EL4 lymphoma cells was found to be significantly higher as compared to the lymphocytes. The present study demonstrates a simple and quantitative method of estimation of curcumin delivered to cells by different vehicles using absorption and fluorescence spectroscopy.     

Stress relaxation of porcine gluteus muscle subjected to sudden transverse deformation as related to pressure sore modeling

Computational studies of deep pressure sores (DPS) in skeletal muscles require information on viscoelastic constitutive behavior of muscles, particularly when muscles are loaded transversally as during bone-muscle interaction in sitting and lying immobilized patients. In this study, we measured transient shear moduli G(t) of fresh porcine muscles in vitro using the indentation method. We employed a custom-made pneumatic device that allowed rapid (2000 mms) 4 mm indentations. We tested 8 gluteus muscles, harvested from 5 adult pigs. Each muscle was indented transversally (perpendicularly to the direction of fibers) at 3 different sites, 7 times per site, to obtain nonpreconditioned (NPC) and preconditioned (PC) G(t) data. Short-term (GS) and long-term (GL) shear moduli were obtained directly from experiments. We further fitted measured G(t) data to a biexponential equation G(t) = G1 x exp(-t/tau1)+ G2 x exp(-t/tau2) + Ginfinity, which provided good fit, visually and in terms of the correlation coefficients. Typically, plateau of the stress relaxation curves (defined as 10% difference from final GL) was evident approximately 20 s after indentation. Short-term shear moduli GS (mean NPC: 8509 Pa, PC: 5711 Pa) were greater than long-term moduli GL (NPC: 609 Pa, PC: 807 Pa) by about an order of magnitude. Statistical analysis of parameters showed that only G2 was affected by preconditioning, while GL, GS, Ginfinity, tau1, tau2, and G1 properties were unaffected. Since DPS develop over time scales of minutes to hours, but most stress relaxation occurs within approximately 20 s, the most relevant property for computational modeling is GL (mean approximately 700 Pa), which is, conveniently, unaffected by preconditioning.     

Morphine modulates monocyte-macrophage conversion phase

Monocyte migration and their activation into the macrophage phenotype play a role in the modulation of tissue injury. We studied the effect of morphine on the monocyte-macrophage conversion phase (MMCP). Phorbol 12-myristate 13-acetate (PMA) activated THP-1 cells and promoted their adhesion to the substrate. Morphine inhibited PMA-induced MMCP. However, opiate receptor antagonists attenuated this effect of morphine. Interestingly, PMA as well as morphine-stimulated superoxide production by monocytes. Superoxide dismutase (SOD) not only inhibited PMA-mediated MMCP but also attenuated the inhibitory effect of morphine. PMA not only enhanced adhesion of monocytes to a filter but also promoted their migration. These findings suggest that the PMA-induced macrophage phenotype conversion may be accelerating their migration; whereas, morphine may be preventing the migration of monocytes by inhibiting MMCP.     

Host protein interactions with enteropathogenic Escherichia coli (EPEC): 14-3-3tau binds Tir and has a role in EPEC-induced actin polymerization

Enteropathogenic Escherichia coli (EPEC) cause infantile diarrhoea and are characterized by their ability to produce attaching and effacing lesions on the surface of intestinal epithelial cells. EPEC employ a filamentous type III secretion system to deliver effector molecules that subvert mammalian cell function to generate actin- and cytokeratin-rich pedestals beneath adherent bacteria. Tir is a major effector protein that is delivered to the plasma membrane of the eukaryotic cell where it acts as the receptor for the bacterial adhesin intimin. Host cell proteins that are recruited to the site of intimate attachment include focal adhesion and cytoskeletal proteins that contribute to pedestal formation. We have used Tir as bait in a yeast two-hybrid screen to identify the protein 14-3-3tau as a binding partner. 14-3-3 proteins are a family of adaptor proteins that modulate protein function in all eukaryotic cells. Here we demonstrate that the tau isoform (also known as theta) of 14-3-3 can bind specifically to Tir in a phosphorylation-independent manner, and that the interaction occurs during the infection process by co-immunoprecipitation of the partners from infected HeLa cell extracts. 14-3-3tau is recruited to the site of the pedestal (3 h after infection) and can decorate attached EPEC in the later stages of the infection process (5-7 h). Pedestal formation can be impaired by depletion of cellular 14-3-3tau using small interfering RNAs. This study indicates a direct functional role for the 14-3-3tau:Tir interaction and is the first to demonstrate the association of a host protein with the surface of EPEC.     

Regulation of caveolar endocytosis by syntaxin 6-dependent delivery of membrane components to the cell surface

Caveolar endocytosis has an important function in the cellular uptake of some bacterial toxins, viruses and circulating proteins. However, the molecular machinery involved in regulating caveolar uptake is poorly defined. Here, we demonstrate that caveolar endocytosis is regulated by syntaxin 6, a target membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) involved in membrane fusion events along the secretory pathway. When syntaxin 6 function was inhibited, internalization through caveolae was dramatically reduced, whereas other endocytic mechanisms were unaffected. Syntaxin 6 inhibition also reduced the presence of caveolin-1 and caveolae at the plasma membrane. In addition, syntaxin 6 inhibition decreased the delivery of GM1 ganglioside (GM1) and glycosylphosphatidylinositol (GPI)-GFP (but not vesicular stomatitis virus-glycoprotein G; VSV-G) protein from the Golgi complex to the plasma membrane. Addition of GM1 to syntaxin 6-inhibited cells resulted in the reappearance of caveolin-1 and caveolae at the plasma membrane, and restored caveolar uptake. These results suggest that syntaxin 6 regulates the delivery of microdomain-associated lipids and proteins to the cell surface, which are required for caveolar endocytosis.     

The conundrum of CD40 function: host protection or disease promotion?

T cells regulate the immune responses to pathogens and autoantigens. The immune responses are tolerizing or anti-inflammatory against autoantigens but are inflammatory against pathogens and allografts. Such contradictory immune responses have been attributed to two counteracting effector cell types or to two counterregulatory sets of molecules: cell-surface expressed or secreted. By contrast, recent reports suggest that CD40, a co-stimulatory molecule on antigen-presenting cells, is a crucial controller of these counteractive immune responses, and emphasize reciprocal inhibition as an essential feature of biological responses. The molecular mechanism of such reciprocity in CD40 functions is the basis of immunotherapy in many diseases.     

Synthesis of polyvalent inhibitors of controlled molecular weight: structure-activity relationship for inhibitors of anthrax toxin

We describe a novel method to synthesize activated polymers of controlled molecular weight and apply this method to investigate the relationship between the structure and activity of polyvalent inhibitors of anthrax toxin. In particular, we observe an initial sharp increase in potency with increasing ligand density, followed by a plateau where potency is independent of ligand density. Our simple strategy for designing polyvalent inhibitors of controlled molecular weight and ligand density will be broadly applicable for designing inhibitors for a variety of pathogens and toxins, and for elucidating structure-activity relationships in these systems. Our results also demonstrate a role for kinetics in influencing inhibitory potency in polyvalent systems. Finally, our work presents a synthetic route to polyvalent inhibitors that are more structurally defined and effective in vivo. This control over inhibitor composition will be generally useful for the optimization of inhibitor potency and pharmacokinetics, and for the eventual application of these molecules in vivo.     

Fluorescence probe with a pH-sensitive trigger

Acid-catalyzed hydrolysis was used as the mechanism to design a new type of environmentally sensitive fluorescence probe. A mild and selective periodate oxidation of the 2-amino alcohol of serine in the presence of a disulfide bond was developed to prepare dialdehyde peptides. Two identical fluorochrome hydrazide derivatives were then linked to the dialdehyde peptide forming an acid-labile hydrazone linkage. This self-quenched probe is weakly fluorescent at a physiological pH of 7.4 but shows more than 3-fold fluorescence enhancement at pH 4.5.     

Invited review: Transformation of strawberry: The basis for translational genomics in Rosaceae

Summary Translational genomics is defined as the application of molecular-genetic principles derived from model systems to species of experimental or economic interest. The past 20 years of research in plant model systems such as Arabidopsis thaliana have relinquished vast amounts of information regarding gene function, the integration of genetic components into pathways, and the interrelationships between pathways to control form and function in plants and plant-products alike. At present, the challenge is to relate these paradigms to other species of economic or scientific interest. Apart from being an important and valuable crop, strawberry (Fragaria spp.) is a member of the Rosaceae, a plant family containing fruit, nut, ornamental and wood-bearing species. Strawberry is unique within the Rosaceae in that it is a rapidly growing herbaceous perennial with a small genome and the ability to thrive in a laboratory setting. Strawberry species may also be transformed and regenerated in a time scale of weeks or months instead of years. For these reasons, strawberry has been recognized as the translational genomics model for the Rosaceae family. This review summarizes and synthesizes the technical reports of strawberry regeneration and transformation, consolidating the large body of information regarding genetic modification of this important genus.