Programmed cell death (PCD), or apoptosis, plays an important role in embryonic development, metamorphsis, hormone-dependent atrophy and tumor growth, as a physiological event regulating the cell number or eliminating damaged cells. Currently hybridoma cell production, resulting from the fusion of myeloma cells with antibody producing spleen cells, is widely used in various fields of life science. This technique requires a hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient mutant myeloma cell line as a fusion partner. When these rnutant cells are treated with aminopterin plus hypoxanthine-thymidine (HAT) after the cell fusion they are selectively and efficiently eliminated so that one could get only fused hybridoma cells. But there hasn't been any report regarding this selective elimination mechanism of HGPRT mutant myeloma cell. By using HGPRT myeloma V653 as a model system this study demonstrated that PCD was induced by aminopterin treatment of this V653 cell. And this PCD was further characterized by cotreatement of cycloheximide, actinomycin-D, and calcium ionophore A23187 together with aminopterin. The apoptotic endonuclease involved in this PCD process was also detected and characterized. When V653 cells were incubated for the various periods of time in the presence of 0.4 uM aminopterin, the viability was continued to decrease until 48 hours of aminopterin treatment and there was no viable cell affer 36 hours of incubation. DNA fragmentation was detectable 3 hours of incubation and peaked between 12 and 18 hours of aminopterin treatment. The induction of cell death and DNA fragmentation of V653 cells by aminopterin were inhibited by protein synthesis inhibitor, cycloheximide, and RNA synthesis inhibitor, actinomycin-D and maximal inhibitory effects on cell death were seen at concentrations of 2 ug/ml and 0.5 uM, respectively. Ca2+ ionophore A23187 promoted aminopterin-induced cell death of V653. When the cells were coincubated with A23187 in the presence of aminopterin, cell viability was remarkably decreased at concentrations of more than 2 uM and DNA fragmentation was increased at concentrations of more than 0.2 uM. A23187 also induced cell death when the cells were treated with A23187 alone. When endogenous endonuclease activities of nuclei isolated from intact healthy cells and aminopterin-treated cells were compared for four different conditions, there were notable increases in the Ca2+/Mg2+ -independent and the Mg2+ -dependent endonuclease activity after incubation with aminopterin for 12 hours. In northern blot analysis, induction of c-myc gene was observed in aminopterin-treated V653 cell reached peak at 2 hours and thern decreased drastically.
Aminopterin* ; Animals ; Apoptosis* ; Atrophy ; Biological Science Disciplines ; Blotting, Northern ; Calcimycin ; Calcium ; Cell Count ; Cell Death ; Cell Fusion ; Cell Line ; Cell Survival ; Cycloheximide ; DNA Fragmentation ; Embryonic Development ; Female ; Genes, myc ; Guanine ; Hybridomas ; Hypoxanthine ; Hypoxanthine Phosphoribosyltransferase ; Mice* ; Pregnancy ; RNA ; Spleen ; Transferases

No abstract available.
Splints

Congenital pseudarthroses and non-unions have been recognized as some of the most challenging problems in orthopaedic surgery. with a standard surgical procedure, such as bone grafting, nailing, plating or a combination of these, it was frequently failed to unit. After repeated surgical failures, amputation has been the main course. With the advent of an electrical control of osteogenesis, however, this dismal outlook is brightening. The earliest report of the use of electrical energy to directly stimulate bone healing seems to be in 19th century, but it was not reliable. In this century, the electrical properties of bone were first described by Yasuda et al in 1953. After then, several investigators have shown that the application of small amounts of the electrical current to bone stimulates osteogenesis at the site of the cathode. Clinical trials using various froms in the treatment of delayed union, non-union, and congenital-pseudarthrosis began early in the 1970's. Constant direct current, pulsed current, and electromagnetically induced current have all been used clinically to heal bone defects with varying degrees of success. But, to-this date it is unknown what is the mechanism of stimulating bone healiag with electricity, and which from of electricity is most efficient in stimulating osteogenesis. We have experienced direct current stimulation to promote osteogenesis in 9 cases of non-union and 4 cases of congenital pseudarthses of the tibia from august, 1978 to december, 1980. Of 9 non-unions, 7 (77.8%) achieved solid bony union. We had obtained bony union in 4cases of non-union only with the electrical stimulation. In 4 cases of congenital pseudarthses of the tibia, all cases achieved solid bony union with the electrical stimulation and bone graft, but in 3 cases, refractures were occurred. At this moment, our conclusions from this study are as followa. I. Direct current stimulation is one of the reliable methods inducing ostengenesis. 2. Regular follows-up and determination of the stimulator integrity are essential steps in the electrical stimulation. 3. Combined treatment with the electrical stimulation and bone graft have markedly improved the success rate. 4. In direct current stimulation of congenital pseudarthsis, the mechanically sound bony alignment, massive bone graft and protection using long leg brace seem to be mandatory procedures.
Amputation ; Bone Transplantation ; Braces ; Electric Stimulation ; Electricity ; Electrodes ; Humans ; Leg ; Magnets ; Osteogenesis ; Pseudarthrosis ; Research Personnel ; Tibia ; Transplants

No abstract available.
Animals ; Embryonic Structures* ; Mice*

No abstract available.
Animals ; Asian Continental Ancestry Group* ; Cricetinae ; Cricetulus* ; Female ; Humans* ; Ovary*

CTLA-4 (=CD152), a T cell activation antigen, has been known to be homologous to CD28 in its molecular and genomic structure. Both of these two molecules are sharing their counterreceptors, B7 (CDSO) and B7-2 (CD86) and are known to play a crucial role in T cell activation. In previous our study it was reported that there are 2 forms of CTLA-4 antigen in activated human T cells, 30 kD membrane-bound form and 34 kD cytosolic-sequestered form and the former was less than 5 % of total of this antigen induced. Aims of this study are to confirm previous finding by using flow cytometry and to characterize the cytoplasmic form of human CTLA-4 by using ultrafiltration and immunoprecipitation techniques. In PHA stimulated peripheral blood lymphocyte surface expression of CTLA-4 was less than 2.1% of any of CD4+, CD8+ and CD56+ subsets. And the 34 kD form of CTLA-4 was detected in CDS+ subset only. This discrepancy confirms that 34 kD antigen is the cytoplasmic form of human CTLA-4. In ultrafiltration and subsequent Western blot analysis study this 34 kD antigen was detected in >100 kD fraction only. And in non-reducing condition this antigen formed high molecular weght complex (MW > 350 kD). In immunoprecipitation study using anti-peptide A antibody it was found that this high molecular weight complex consists of the 34 kD cytoplasmic form of CTLA-4 and previously unknown 54 kD antigen and 46 kD antigen at 1:1:8-10 ratio. And none of these 3 molecules were identified in membrane fraction of activated human T cell. The result of this study implies that CTLA-4 molecule induced upon T cell activation mainly sequestered in cytoplasrn and another signal is necessary to target this antigen on the activated T cell surface.
Antigens, CD27 ; Blotting, Western ; CTLA-4 Antigen ; Cytoplasm* ; Flow Cytometry ; Humans* ; Immunoprecipitation ; Lymphocytes ; Membranes ; Molecular Weight ; T-Lymphocytes ; Ultrafiltration

No abstract available.
Animals ; Immunohistochemistry* ; Neurons* ; Rats* ; Substantia Nigra*

Antigen-specific T cell activation requires interaction of the T cell with specialized antigen-presenting cells. Signaling through the TCR is necessary but not sufficient to induce antigen-specific T cell activation and cytokine secretion. This first signal, termed signal 1, is both antigen-specific and MHC-restricted. Signal 2, which is neither antigen-specific nor MHC-restricted, is necessary to induce cytokine secretion, cellular proliferation, and effector function. Recently immunological studies in T cell activation area are mainly focused on biological and molecular biological characterization of TCR/CD3 complex and accessary molecules providing costimulatory signal (signal 2). If signal 2 is not delivered, T cell enter a state of long term un-responsiveness to specific antigen-termed anergy. Monoclonal antibody technique has been especially involved in recognizing novel inducible cell surface antigens on T cell activation. This study was aimed to develop monoclonal antibodies recognizing novel cytoplasmic proteins present in activated T cells. We make 6 monoclones involved in changing pattern of T cell activated cytoplasmic proteins. Using these 6 monoclonal antibodies analyze to find novel molecules involved in T cell activation associated response, apoptosis, and/or heat shock response of the T cells in early T cell activation.
Antibodies, Monoclonal* ; Antigen-Presenting Cells ; Antigens, Surface ; Apoptosis ; Cell Proliferation ; Cytoplasm* ; Heat-Shock Proteins ; Heat-Shock Response ; Humans* ; T-Lymphocytes*

Programrned cell death (PCD), or apoptosis, is a process by which cells die in response to specific physiological and toxicological signals. This genetically programmed form of cellular suicide is intirnately involved in many biological processes including growth, metamorphosis, embryogenesis, and oncogenesis. Cells undergoing PCD in normal and neoplasmic tissues display the following biochemical and morphological features: internucleosomal DNA fragmentation, reduced cell volume, condensed chromatin in nucleus, zeiosis and generation of apoptotic bodies containing intact organelles and plasma rnembrane. Hybridoma cell production, resulting from the fusion of myeloma cells with antibody producing spleen cells, is widely used in various fields of life science. This technique requires hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient mutant myeloma cell line as a fusion partner. When these mutants cell is treated with aminopterin plus hypoxanthine-thymidine (HAT) after the cell fusion they are selectively and efficiently eliminated remaining fused hybridoma celis. But there hasn't been any report regarding the selective elimination mechanism of this HGPRT mutant myeloma cell. By using HGPRT myeloma P3-X 63-Ag8.653 (V653) as a model system, this study demonstrated that PCD was induced by aminopterin treatment of this V653 cell line. And the sequential ultrastructural changes during this death process were observed by using electron microscope. When V653 cells were incubated with 0.4 uM aminopterin, DNA fragmentation was detectable after 3 hours and peaked between 12 and 18 hours of aminopterin treatment and the cell viability decreased in a time dependant manner. V653 cells incubated with amiopterin showed following ultrastructural changes during the death process. Dilatation of rough endoplasmic reticulum (RER) and detachment of ribosomes were the earliest ultrastructural changes and first seen after 30 minute incubation. Dilatation of perinuclear cisternae began to appear after 1 hour and deformation of nucleoplasm such as decreased electron density of perinuclear heterochromatin and increased electron density of euchromatin were seen after 3 hours. Increased electron density of cytoplasm, decreased cell volume, condensation of chromatin and apoptotic bodies were observed in many cells after 9 hours but vacuolation by severe dilatation of RER was seen in some cells. 24 hours after incubation with aminopterin, many cells showed typical form of apoptosis characterized by cell shrinkage, increased electron density of cytoplasm and apoptotic bodies. On the contrary, some cells showed different type of cell death characterized by increased cell volume, decreased electron density of cytoplasm, severely dilated RER and apoptotic bodies. In both types of cells, mitochondrial cristae and limiting membrane of mitochondria are comparatively well preserved. In other cells, nuclei did not show significant changes but there were deformations of mitochondria such as markedly increased electron density and formation of lamella bodies. The death process of V653 cell was not synchronized among cells. The results of this study proved that aminopterin-induced selective elimination of fusion partner V653 myeloma cell is due to PCD. The earliest ultrastructural changes observed in this process were dilatation of RER and detachment of ribosomes. And there were two distinct morphological types in the PCD.
Aminopterin* ; Animals ; Apoptosis* ; Biological Processes ; Biological Science Disciplines ; Carcinogenesis ; Cell Death ; Cell Fusion ; Cell Line ; Cell Size ; Cell Survival ; Chromatin ; Cytoplasm ; Dilatation ; DNA Fragmentation ; Embryonic Development ; Endoplasmic Reticulum, Rough ; Euchromatin ; Female ; Guanine ; Heterochromatin ; Hybridomas ; Hypoxanthine ; Hypoxanthine Phosphoribosyltransferase ; Membranes ; Mice* ; Mitochondria ; Organelles ; Plasma ; Pregnancy ; Ribosomes ; Spleen ; Suicide ; Transferases

No abstract available.
Acute Kidney Injury* ; Child* ; Humans