67 research outputs found
Multibudded tubules formed by COPII on artificial liposomes
COPII-coated vesicles form at the endoplasmic reticulum for cargo transport to the Golgi apparatus. We used in vitro reconstitution to examine the roles of the COPII scaffold in remodeling the shape of a lipid bilayer. Giant Unilamellar Vesicles were examined using fast confocal fluorescence and cryo-electron microscopy in order to avoid separation steps and minimize mechanical manipulation. COPII showed a preference for high curvature structures, but also sufficient flexibility for binding to low curvatures. The COPII proteins induced beads-on-a-string-like constricted tubules, similar to those previously observed in cells. We speculate about a mechanical pathway for vesicle fission from these multibudded COPII-coated tubules, considering the possibility that withdrawal of the Sar1 amphipathic helix upon GTP hydrolysis leads to lipid bilayer destabilization resulting in fission
Coronary risk factors and myocardial blood flow in patients evaluated for coronary artery disease: a quantitative [15O]H2O PET/CT study
Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines
Background: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and
Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and
in combination with EGF.
Methods: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was
conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression
and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene
expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40
K array A.
Results: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this
level of cell cycle distribution after treatment, suggesting a predominantly differentiated state.
Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their
viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important
reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating
microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and
showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane
reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes
upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the
2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment.
In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological
effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes
appeared to be less stimulated than for single drug cases.
Conclusion: This is the first study to have systematically investigated the effect of cetuximab or
gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors
have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an
expression pattern that inversely correlates with EGF treatment. We found interesting cytomorphological
features closely relating to gene expression profile. Both drugs have an effect on
differentiation towards cellular death
Templated growth of calcium phosphate on tyrosine derived microtubules and their biocompatibility.
Microtubular structures were self-assembled in aqueous media from a newly synthesized bolaamphiphile, bis(N-alpha-amido-tyrosyl-tyrosyl-tyrosine)-1,5-pentane dicarboxylate. In order to increase the biocompatibility of the microtubules, they were functionalized with the peptide sequence GRGDSP. Further, calcium phosphate nanocrystals were grown on the microtubules. In some cases, collagen was added in order to mimic the components of natural bone tissue. The biomaterials obtained were characterized via transmission electron microscopy (TEM), atomic force microscopy (AFM), IR, and energy dispersive X-ray spectroscopy (EDX) analyses. The biocompatibility of the calcium phosphate-coated microtubules was studied by conducting in vitro cell-attachment, cell-proliferation and cytotoxicity studies using mouse embryonic fibroblast (MEF) cells. The studies revealed that the biomaterials were found to be non-toxic and biocompatible. The functionalized tubular assemblies coated with calcium phosphate nanocrystals mimic the nanoscale composition of natural bone and may potentially support bone in-growth and osseointegration when used in orthopaedic or dental applications
Myosin II is associated with Golgi membranes: identification of p200 as nonmuscle myosin II on Golgi-derived vesicles
A variety of peripheral membrane proteins associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells, A monoclonal antibody (AD7) raised against Golgi membranes recognizes a peripheral membrane protein, p200, which associates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myosin on Golgi membranes was investigated, Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II, We show that, in addition to being found in the cytoplasm or associated with stress fibres, nonmuscle myosin II is also specifically localized on Golgi membranes, Myosin II was also detected on Golgi membranes by immunoblotting and by immunogold labeling at the electron microscopy level where it was found to be concentrated on Golgi-derived vesicles, The association of myosin II with Golgi membranes is dynamic and was found to be enhanced following activation of G proteins, Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA), Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immunoprecipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extracts of HeLa cells, was sequenced and identified as another actin-binding protein, filamin, These results show that nonmuscle myosin II is associated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II
Integrin-Dependent Interaction of Human Vascular Endothelial Cells on Biomimetic Peptide Surfactant Polymers
Transport of cytoplasmic particles catalysed by an unconventional myosin in living Drosophila embryos
Actin acting at the Golgi
The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in traf- ficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers
Behavior of cholinesterase and liver mitochondrial function in dogs submitted to normothermic ischemia and reperfusion
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