For the purpose of morphologic analysis of human cytomegalovirus (HCMV) antigens reacting with specific monoclonal antibodies, we observed HCMV particles after immunogold staining. HCMV was cultured in human fetal lung fibroblasts to be concentrated by polyethylene glycol 6,000. The HCMV stock was dropped onto Formva-coated grids and was fixed by 2% glutaraldehyde. The grids were reacted with MCMVA57, 93, 135 or with SCMVM1, 6, 14, 49 monoclonal antibodies (MoAbs) follwed by gold (10 nm)-conjugated goat anti-mouse IgG. Then the grids were stained with 2.5% uranyl acetate to be observed under Hitachi 500 or Jeol 1,200 electron microscope. When HCMV was reacted with SCMVM14 and SCMVM49 MoAbs, gold particles were adsorbed to virion envelopes, suggesting that the reactive antigens were envelope proteins. In cases of MCMVA135 and SCMVM6 MoAbs, gold particles were adsorbed to dense bodies as well as to virion envelope. These results, together with the previous results of immunologic and genetic characterization, suggested that the reactive antigens of MCMVA135 and SCMVM6 MoAbs were gB homologue and structural protein, respectively. In case of SCMVM1 MoAb, gold particles were adsorbed to capsids, envelopes, and dense bodies, suggesting that the reactive antigen was structural protein. In case of SCMVM8 MoAb, gold particles were observed between the envelopes and capsids, which space was supposed to be the tegument, suggesting that the reactive antigen was carbohydrate moiety of glycoprotein or its polymer. In cases of MCMVA57 and MCMVA93 MoAbs, gold particles were adsorbed to only dense bodies, suggesting that the reactive antigens were precursors of structural proteins.
Antibodies, Monoclonal* ; Capsid ; Cytomegalovirus* ; Fibroblasts ; Glutaral ; Glycoproteins ; Goats ; Humans* ; Immunoglobulin G ; Lung ; Polyethylene Glycols ; Polymers ; Virion

No abstract available.
Epithelial Cells* ; Salmonella typhimurium* ; Salmonella*

No abstract available.
Antibodies, Monoclonal* ; Glycoproteins* ; Humans*

No abstract available.
Cytomegalovirus* ; Humans*

No abstract available.
Antibodies, Monoclonal* ; Herpesvirus 3, Human*

No abstract available.
Clone Cells* ; Humans* ; Lymphocytes*

BACKGROUND: Thrombopoietin (TPO) has been currently used for ex vivo expansion of hematopoietic progenitor cells. Previously, we have reported that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+cells. In the present study, we have investigated on the relationship between the TPO-induced apoptosis and megakaryocytic differentiation. METHODS: CD34+cells, purified from human CBs, were expanded in serum-free conditions stimulated with TPO. Multidimensional flow cytometry and TUNEL assay as well as electron microscopy were applied for analysis of apoptosis. Asociation of megakaryocytic differentiation and apoptosis was studied by FACS-sorting and immunocytochemistry. Clonogenic potential was studied by CFU-MK assay. RESULTS: The TPO-induced apoptotic cells appeared in CD61+fractions. Immunocytochemical analysis of the FACS-sorted fractions showed that the apoptosis-associated CD44low fraction expressed CD61. Clonogenic assay revealed 7.4+-0.50-fold increase of total CFU-MKs during the initial 9 days. Thereafter, the number of CFU-MKs decreased, which was parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular from day 6, small colonies became predominant. CONCLUSION: These results suggested that the MK progenitors matured as they were expanded during ex vivo expansion with TPO, and then proceeded to apoptosis.
Apoptosis ; Fetal Blood* ; Flow Cytometry ; Hematopoietic Stem Cells ; Humans* ; Immunohistochemistry ; In Situ Nick-End Labeling ; Megakaryocytes ; Microscopy, Electron ; Thrombopoietin

No abstract available.
Humans*

Human cytomegalovirus (HCMV)-specific monoclonal antibody, SCMVM 34, recognizes early antigen confined to the nucleus of HCMV-infected cells. This study was performed to identify the antigen reactive to SCMVM 34 with purification and amino acid sequencing. The nuclear and cytoskeletal fraction of HCMV-infected cells was subjected to 0.4 M NaCl extraction, DEAE-Sephacel ion exchange chromatography, DNA-cellulose chromatography and SDS-PAGE. The molecular weight of the reactive proteins was 52 kD, 40 kD and 34 kD. The modified or blocked amino termini of 52 kD and 40 kD showed resistance to Edman degradation. The internal peptide fragments were isolated by tryptic digeytion and reverse-phase HPLC. The internal amino acid sequence analysis of the peptides from HPLC profile revealed that the antigens recognized by SCMVM 34 was ppUIA4.
Chromatography ; Chromatography, High Pressure Liquid ; Chromatography, Ion Exchange ; Cytomegalovirus* ; Electrophoresis, Polyacrylamide Gel ; Humans* ; Molecular Weight ; Peptide Fragments ; Peptides ; Sequence Analysis, Protein

No abstract available.