ABSTRACT
In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.
Subject(s)
Receptor, Endothelin B/chemistry , Receptor, Endothelin B/isolation & purification , Animals , Baculoviridae/genetics , Biphenyl Compounds/chemistry , Blotting, Western , Dipeptides/chemistry , Endothelin Receptor Antagonists/chemistry , Endothelins/chemistry , Genetic Engineering/methods , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Peptide Fragments/chemistry , Protein Denaturation , Protein Stability , Receptor, Endothelin B/genetics , Receptor, Endothelin B/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sf9 Cells , TemperatureABSTRACT
We studied the localization of transmembrane receptor P185(HER2) in SKOV-3 and BT-474 cancer cells by fluorescence, confocal and electron immunomicroscopy. P185(HER2) is a marker of breast and ovarian tumors, it is considered as a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, first, to study the distribution of P185(HER2) in cancer cells, secondly, to remove P185(HER2) from the cell surface and, thirdly, to eliminate target cells. In this work for visualization of P185HER2 We prOposed immunocytotoxic system, consisting of the monoclonal miniantibody 4D5 scFv to extracellular P185E domain fused with two molecules of barnase (ribonuclease from Bacillus amyloliquefaciens) and of its specific inhibitor barstar. Fluorescence microscopy has showed that the module 4D5 scFv-dibarnase:barstar efficiently identified P185(HER2) on the surface of cancer cells. It was revealed by confocal microscopy that interaction with 4D5 scFv-dibarnase lead to internalization of P185(HER2). The localization of P185(HER) in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185(HER) distributed on the cell surface unequally with preferential localization on protrusions or close to their bases and in contacts between protrusions and cell membrane. At 37 degrees C, P185(HER2) internalized through coated pits and vesicles and concentrated in the endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in cells BT-474.
Subject(s)
Breast Neoplasms/genetics , Gold Colloid/chemistry , Neoplasms, Glandular and Epithelial/genetics , Ovarian Neoplasms/genetics , Receptor, ErbB-2/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Carcinoma, Ovarian Epithelial , Cell Line, Tumor , Female , Humans , Immunoglobulins/genetics , Neoplasms, Glandular and Epithelial/immunology , Neoplasms, Glandular and Epithelial/pathology , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Receptor, ErbB-2/genetics , Receptor, ErbB-2/immunology , Ribonucleases , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunologySubject(s)
Green Fluorescent Proteins/metabolism , Neoplasms/enzymology , Neoplasms/pathology , Ribonucleases/chemistry , Animals , Bacterial Proteins , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Humans , Luminescent Proteins/analysis , Microscopy, Fluorescence/methods , Models, Biological , Models, Genetic , Neoplasms/metabolism , Ribonucleases/metabolism , Tumor Cells, CulturedABSTRACT
This review considers ways to address specificity to therapeutic targeted anticancer agents. These include transcriptional activation of tissue- and tumor-specific promoters in eukaryotic expression vectors and use of antitumor-directed immunoconjugates. The review deals with analysis of strategies used for selection of targeted promoters and examples of antibody fusion proteins exhibiting antitumor activity. A new direction in antitumor treatment pooling together methods of gene therapy and antibody therapy has appeared. This direction is based on the development of vectors encoding secreted forms of immunoconjugates. After vector introduction into a cell, the latter is capable of synthesizing and secreting antibody fusion protein composed of a therapeutic anticancer agent and antibody specifically targeted to cancer cells.
Subject(s)
Genetic Vectors , Immunoconjugates/therapeutic use , Neoplasms/therapy , Transduction, Genetic , Animals , Antibodies/therapeutic use , Antineoplastic Agents/therapeutic use , Eukaryotic Cells , Gene Expression , Gene Targeting , Genetic Therapy , Humans , Promoter Regions, Genetic , Recombinant Fusion Proteins/therapeutic useABSTRACT
A case of chromobacteriosis in a young Brazilian with toxaemia and multiple skin abscesses is described. The infection responded to treatment with chloramphenicol and cotrimoxazole but recurred 18 months later following insect bites received while fishing in a river. Chromobacterium violaceum was subsequently isolated from the river water. This is the first case of this kind to be reported from South America.
