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1.
Methods Mol Biol ; 1785: 53-63, 2018.
Article in English | MEDLINE | ID: mdl-29714011

ABSTRACT

We present a method to study the interaction between biomolecules and receptors present on the cell surface. This enables studies of molecular interactions in a natural biological context. As the analyte interacts with the receptors still intact on the cell surface, the experimental data provides complete dynamics and complexity of the interaction, thereby generating highly informative data. Attana's cell-based biosensor platform can be used to obtain this information from a diverse range of interactions as described in these protocols, which detail how to grow or capture cells on a surface, how to stabilize and visualize the cells on the surface, and how to set up assays to measure detailed interaction kinetics directly on the cell surface.


Subject(s)
Biosensing Techniques/methods , Cell Membrane/chemistry , Proteins/isolation & purification , Quartz Crystal Microbalance Techniques/methods , Cell Membrane/genetics , Humans , Kinetics , Protein Binding , Proteins/chemistry
2.
Int J Biol Markers ; 28(2): 141-50, 2013.
Article in English | MEDLINE | ID: mdl-23564623

ABSTRACT

Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential as a versatile biomarker tool for screening, diagnosis, prognosis and monitoring of breast cancer. Standardization of methods and biomarker panels will be required to fully exploit this clinical potential.


Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , DNA Methylation/genetics , Epigenesis, Genetic/genetics , Breast Neoplasms/diagnosis , Breast Neoplasms/metabolism , Carcinogenesis/genetics , Female , Humans , Prognosis , Promoter Regions, Genetic
3.
J Biol Chem ; 286(18): 15908-17, 2011 May 06.
Article in English | MEDLINE | ID: mdl-21398524

ABSTRACT

Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.


Subject(s)
Chondroitin Sulfates/chemistry , Peptide Mapping , Plasmodium falciparum/chemistry , Animals , Antigens, Protozoan , Biosensing Techniques/methods , Chondroitin Sulfates/genetics , Chondroitin Sulfates/immunology , Chondroitin Sulfates/metabolism , Erythrocytes/immunology , Erythrocytes/metabolism , Erythrocytes/parasitology , Female , Humans , Malaria Vaccines/chemistry , Malaria Vaccines/genetics , Malaria Vaccines/immunology , Malaria Vaccines/metabolism , Malaria, Falciparum/epidemiology , Malaria, Falciparum/genetics , Malaria, Falciparum/immunology , Malaria, Falciparum/metabolism , Malaria, Falciparum/prevention & control , Placenta/immunology , Placenta/metabolism , Placenta/parasitology , Plasmodium falciparum/genetics , Plasmodium falciparum/immunology , Plasmodium falciparum/metabolism , Pregnancy , Pregnancy Complications, Parasitic/epidemiology , Pregnancy Complications, Parasitic/genetics , Pregnancy Complications, Parasitic/immunology , Pregnancy Complications, Parasitic/metabolism , Pregnancy Complications, Parasitic/prevention & control , Protein Binding , Protein Structure, Tertiary , Rats , Rats, Wistar , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism
4.
Biochem Biophys Res Commun ; 340(2): 696-701, 2006 Feb 10.
Article in English | MEDLINE | ID: mdl-16378592

ABSTRACT

SIFamide is the short name and also the C terminus of the Drosophila neuropeptide AYRKPPFNGSIFamide. SIFamide has been isolated or predicted from various insects and crustaceans, and appears to be extremely well conserved among these arthropods. However, the function of this neuropeptide is still enigmatic. Here, we have identified the Drosophila gene (CG10823) coding for the SIFamide receptor. When expressed in Chinese hamster ovary cells, the receptor is only activated by Drosophila SIFamide (EC(50), 2x10(-8)M) and not by a library of 32 other insect neuropeptides and eight biogenic amines. Database searches revealed SIFamide receptor orthologues in the genomes from the malaria mosquito Anopheles gambiae, the silkworm Bombyx mori, the red flour beetle Tribolium castaneum, and the honey bee Apis mellifera. An alignment of the five insect SIFamide or SIFamide-like receptors showed, again, an impressive sequence conservation (67-77% amino acid sequence identities between the seven-transmembrane areas; 82-87% sequence similarities). The identification of well-conserved SIFamide receptor orthologues in all other insects with a sequenced genome, suggests that the SIFamide/receptor couple must have an essential function in arthropods. This paper is the first report on the identification of a SIFamide receptor.


Subject(s)
Drosophila Proteins/genetics , Drosophila Proteins/isolation & purification , Neuropeptides/metabolism , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cricetinae , Cricetulus , DNA, Complementary , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Molecular Sequence Data , Receptors, G-Protein-Coupled
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