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Molecular and Cellular Biology Aug 1984A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium...
A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei.
Topics: Animals; Antigens, Protozoan; Antigens, Surface; Base Sequence; Cloning, Molecular; Crosses, Genetic; DNA Restriction Enzymes; Genes; Mutation; Nucleic Acid Hybridization; Paramecium; Protozoan Proteins
PubMed: 6092921
DOI: 10.1128/mcb.4.8.1583-1590.1984 -
Revista Brasileira de Parasitologia... 2017Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia...
Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.
Topics: Animals; Antigens, Surface; Babesia bovis; Babesiosis; Brazil; Cattle; Cattle Diseases; Female; Genetic Variation; Immunity, Humoral; Longitudinal Studies; Merozoites
PubMed: 29211135
DOI: 10.1590/S1984-29612017069 -
Molecular & Cellular Proteomics : MCP Jan 2018Despite recent efforts toward control and elimination, malaria remains a major public health problem worldwide. resistance against artemisinin, used in front line...
Despite recent efforts toward control and elimination, malaria remains a major public health problem worldwide. resistance against artemisinin, used in front line combination drugs, is on the rise, and the only approved vaccine shows limited efficacy. Combinations of novel and tailored drug and vaccine interventions are required to maintain the momentum of the current malaria elimination program. Current evidence suggests that strain-transcendent protection against malaria infection can be achieved using whole organism vaccination or with a polyvalent vaccine covering multiple antigens or epitopes. These approaches have been successfully applied to the human-infective sporozoite stage. Both systemic and tissue-specific pathology during infection with the human malaria parasite is caused by asexual blood stages. Tissue tropism and vascular sequestration are the result of specific binding interactions between antigens on the parasite-infected red blood cell (pRBC) surface and endothelial receptors. The major surface antigen and parasite ligand binding to endothelial receptors, PfEMP1 is encoded by about 60 variants per genome and shows high sequence diversity across strains. Apart from PfEMP1 and three additional variant surface antigen families RIFIN, STEVOR, and SURFIN, systematic analysis of the infected red blood cell surface is lacking. Here we present the most comprehensive proteomic investigation of the parasitized red blood cell surface so far. Apart from the known variant surface antigens, we identified a set of putative single copy surface antigens with low sequence diversity, several of which are validated in a series of complementary experiments. Further functional and immunological investigation is underway to test these novel blood stage proteins as possible vaccine candidates.
Topics: Animals; Antigens, Protozoan; Antigens, Surface; Cell Membrane; Erythrocytes; Female; Malaria Vaccines; Mice, Inbred BALB C; Plasmodium falciparum; Proteome; Proteomics
PubMed: 29162636
DOI: 10.1074/mcp.RA117.000076 -
Analytical Chemistry Mar 2015Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of...
Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size—AP-1060 and NALM-1—based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside.
Topics: Antigens, CD; Antigens, Surface; Biomarkers; Bone Marrow; Humans; Immunophenotyping; Leukemia, Promyelocytic, Acute; Microfluidics; Porosity; Single-Cell Analysis; Tumor Cells, Cultured
PubMed: 25625182
DOI: 10.1021/ac504613b -
Proceedings of the National Academy of... Oct 1996Nematodes can alter their surface coat protein compositions at the molts between developmental stages or in response to environmental changes; such surface alterations...
Nematodes can alter their surface coat protein compositions at the molts between developmental stages or in response to environmental changes; such surface alterations may enable parasitic nematodes to evade host immune defenses during the course of infection. Surface antigen switching mechanisms are presently unknown. In a genetic study of surface antigen switching, we have used a monoclonal antibody, M37, that recognizes a surface antigen on the first larval stage of the free-living nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans can be induced to display the M37 antigen on a later larval stage by altering the growth conditions. Mutations that result in nonconditional display of this antigen on all four larval stages fall into two classes. One class defines the new gene srf-6 II. The other mutations are in previously identified dauer-constitutive genes involved in transducing environmental signals that modulate formation of the dauer larva, a developmentally arrested dispersal stage. Although surface antigen switching is affected by some of the genes that control dauer formation, these two process can be blocked separately by specific mutations or induced separately by environmental factors. Based on these results, the mechanisms of nematode surface antigen switching can now be investigated directly.
Topics: Animals; Antigens, Surface; Caenorhabditis elegans; Chromosome Mapping; Environmental Exposure; Genetic Complementation Test; Larva; Mutagenesis; Temperature
PubMed: 8901591
DOI: 10.1073/pnas.93.22.12388 -
Cellular and Molecular Gastroenterology... 2023Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome...
BACKGROUND & AIMS
Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome biogenesis. In light of this, it was studied whether intact virions wrapped in exosomes are released by HBV-producing cells.
METHODS
Robust methods for efficient separation of exosomes from virions were established. Exosomes were subjected to limited detergent treatment for release of viral particles. Electron microscopy of immunogold labeled ultrathin sections of purified exosomes was performed for characterization of exosomal HBV. Exosome formation/release was affected by inhibitors or Crispr/Cas-mediated gene silencing. Infectivity/uptake of exosomal HBV was investigated in susceptible and non-susceptible cells.
RESULTS
Exosomes could be isolated from supernatants of HBV-producing cells, which are characterized by the presence of exosomal and HBV markers. These exosomal fractions could be separated from the fractions containing free virions. Limited detergent treatment of exosomes causes stepwise release of intact HBV virions and naked capsids. Inhibition of exosome morphogenesis impairs the release of exosome-wrapped HBV. Electron microscopy confirmed the presence of intact virions in exosomes. Moreover, the presence of large hepatitis B virus surface antigen on the surface of exosomes derived from HBV expressing cells was observed, which conferred exosome-encapsulated HBV initiating infection in susceptible cells in a , large hepatitis B virus surface antigen/Na-taurocholate co-transporting polypeptide-dependent manner. The uptake of exosomal HBV with low efficiency was also observed in non-permissive cells.
CONCLUSION
These data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV.
Topics: Humans; Exosomes; Detergents; Virion; Hepatitis B; Hepatitis B virus; Antigens, Surface
PubMed: 36184032
DOI: 10.1016/j.jcmgh.2022.09.012 -
Frontiers in Bioscience (Elite Edition) Jun 2009Establishment of a system that allows selective gene transfer to a tumor is expected to enable targeted therapy. Using a combination of fiber-modified adenovirus and... (Review)
Review
Establishment of a system that allows selective gene transfer to a tumor is expected to enable targeted therapy. Using a combination of fiber-modified adenovirus and antibody to a cell surface antigen, we have explored methods to enhance the selectivity of gene transfer. In addition, we aimed to establish a systematic screening method to search for antibody and cell surface target candidates for providing highly selective gene transfer to a variety of malignant tumors.
Topics: Adenoviridae; Amino Acid Sequence; Antigens, Surface; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Molecular Sequence Data; Neoplasms
PubMed: 19482650
DOI: 10.2741/E32 -
Biosensors Feb 2022Silicon nanowire field effect transistor (NWFET) sensors have been demonstrated to have high sensitivity, are label free, and offer specific detection. This study...
Silicon nanowire field effect transistor (NWFET) sensors have been demonstrated to have high sensitivity, are label free, and offer specific detection. This study explored the effect of nanowire dimensions on sensors' sensitivity. We used sidewall spacer etching to fabricate polycrystalline silicon NWFET sensors. This method does not require expensive nanoscale exposure systems and reduces fabrication costs. We designed transistor sensors with nanowires of various lengths and numbers. Hepatitis B surface antigen (HBsAg) was used as the sensing target to explore the relationships of nanowire length and number with biomolecule detection. The experimental results revealed that the sensor with a 3 µm nanowire exhibited high sensitivity in detecting low concentrations of HBsAg. However, the sensor reached saturation when the biomolecule concentration exceeded 800 fg/mL. Sensors with 1.6 and 5 µm nanowires exhibited favorable linear sensing ranges at concentrations from 800 ag/mL to 800 pg/mL. The results regarding the number of nanowires revealed that the use of few nanowires in transistor sensors increases sensitivity. The results demonstrate the effects of nanowire dimensions on the silicon NWFET biosensors.
Topics: Antigens, Surface; Biosensing Techniques; Hepatitis B Surface Antigens; Hepatitis B virus; Nanowires; Silicon
PubMed: 35200375
DOI: 10.3390/bios12020115 -
Proceedings of the National Academy of... Jul 1981Monoclonal antibodies (MAb) have been used to probe the relationship of cytolytic T lymphocyte (CTL) surface molecules to CTL function. Rat MAb to mouse CTL were...
Monoclonal antibodies (MAb) have been used to probe the relationship of cytolytic T lymphocyte (CTL) surface molecules to CTL function. Rat MAb to mouse CTL were generated. Twelve MAb so obtained gave preferential binding to T cells as compared to B cells, and three of these recognized previously undescribed surface polypeptides. These Mab and more broadly reactive and previously obtained MAb were tested for their ability to block CTL-mediated killing in the absence of complement. To ensure that any observed blocking was due to binding of MAb to the effector cell rather than the target cell, a xenogeneic mouse CTL anti-rat BN lymphoma target cell system was utilized (MAb and target cells both of rat origin). Of 24 MAb tested here, 21 had little or no effect on CTL function, including those to H-2, Thy-1, Lyt-1, Ly 5, Ly 6, Lgp 100, and at least six other defined antigens. We confirmed inhibition of killing with two MAb to Lyt-2,3. Another MAb, M7/14, gave profound and consistent blockade of CTL function. It was confirmed that M7/14 MAb blocks killing by binding to the mouse CTL and does not bind to the rat lymphoma target cells used for the CTL assay. The findings suggest that the antigen defined by M7/14, termed a lymphocyte function-associated antigen, LFA-1, participates in or is closely associated with the mechanism of CTL-mediated killing. LFA-1 contains two polypeptide chains of 180,000 and 95,000 Mr and is distinct from other described lymphocyte glycoproteins. LFA-1 thus represents both a previously undescribed lymphocyte surface antigen and molecular site for blockade of CTL-mediated killing.
Topics: Animals; Antibodies, Monoclonal; Antigens, Ly; Antigens, Surface; Cytotoxicity, Immunologic; Immunity, Cellular; Lymphocyte Function-Associated Antigen-1; Male; Mice; Molecular Weight; Rats; T-Lymphocytes
PubMed: 7027264
DOI: 10.1073/pnas.78.7.4535 -
The Journal of Experimental Medicine Dec 1985The H8 protein is a surface-exposed antigen that is found, among members of the Neisseria genus, primarily on pathogenic species. In this study, the surface exposure of... (Comparative Study)
Comparative Study
The H8 protein is a surface-exposed antigen that is found, among members of the Neisseria genus, primarily on pathogenic species. In this study, the surface exposure of H8 was reassessed by four techniques. Results of slide agglutination, indirect fluorescent antibody binding, absorption of sera with whole gonococci, and immune electron microscopy all confirmed the presence of H8 in the outer membrane. The degree to which protein A-gold-labeled monoclonal antibodies bound to H8 was marked, and suggested that this antigen was present in abundant amounts in the outer membrane. Also in this study, the electrophoretic heterogeneity of this common surface antigen was examined. Because H8 stains poorly, electrophoretic mobility was assessed using polyclonal antibodies and a monoclonal antibody that recognizes a common H8 epitope. H8 was analyzed with respect to protein I, lipopolysaccharide (LPS), and pilus and opacity phenotypic variation; results confirmed that heterogeneity of Mr was the rule among strains (21 were examined), however, the variability in Mr was independent of protein I or LPS Mr. In one strain (FA1090), the heterogeneity of H8 was examined among 10 piliation/opacity variants; the H8 (and LPS) Mr was identical in all variants; similar data were generated in strains JS3 and JS1. The electrophoretic mobility of H8 was altered in serum-resistant and neutrophil enzyme-resistant gonococci compared to the sensitive gonococci. Some of the unusual electrophoretic migration characteristics of the antigen were also examined. H8 formed a unique mushroom-shaped band in one-dimensional gels; in a two-dimensional electrophoresis system, the antigen migrated aberrantly, very similarly to LPS. Also seen in the two-dimensional electrophoresis profile were multimers of the H8 antigen; in strain JS3 (Mr 23,500), these migrated at 43,600, 86,000, and greater than 150,000. In other strains, the Mr of the multimers differed depending upon the Mr of the monomer. The two-dimensional migration characteristics (as measured by antigenicity) were completely destroyed by proteinase K digestion. Activity of H8 polyclonal antibodies to the antigens in two-dimensional gels was completely removed by adsorption of formalin-fixed whole cells, but was not affected by adsorption with LPS. These electrophoretic characteristics may reflect the close association of some nonprotein constituent, perhaps lipid or carbohydrate or both.
Topics: Animals; Antibodies, Monoclonal; Antigens, Bacterial; Antigens, Surface; Bacterial Outer Membrane Proteins; Blood Bactericidal Activity; Electrophoresis, Polyacrylamide Gel; Epitopes; Fimbriae, Bacterial; Lipopolysaccharides; Mice; Molecular Weight; Neisseria gonorrhoeae; Phenotype; Rabbits; Sodium Dodecyl Sulfate; Species Specificity
PubMed: 2415662
DOI: 10.1084/jem.162.6.2017