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Biomedical subjects

P N Goodfellow

Publications and source records attributed to P N Goodfellow.

At least 199 records · Page 11Linked to original sources

Introduction of a human X-6 translocation chromosome into a mouse teratocarcinoma: investigation of control of HLA-A, B, C expression.

We have developed an approach to human developmental biology which exploits somatic cell genetics. With this system we have examined the production of the HLA-A,B,C antigens, A human-mouse somatic cell hybrid was constructed which contained a human X-7 chromosome translocation carrying the HLA region; this hybrid was used as a donor of the X-6 translocation in the technique of microcell transfer. The X-6 chromosome recipient was the mouse embryonal carcinoma cell line PCC4. The microcell hybrid MCP-6 retained the embryonal carcinoma phenotype as judged by shape and absence of H-2 expression. Nonetheless, the expression of the HLA-A,B,C genes was not extinguished. HLA-A,B,C antigen production of the cell surface, however, was not detected because this hybrid apparently could not make beta 2-microglobulin.

Animals↗

A human quantitative polymorphism related to Xg blood groups.

A monoclonal antibody, 12E7, raised against lymphocytes from a patient with a T-cell acute lymphocytic leukaemia reacts strongly with cortical thymocytes and, to a lesser extent, with other human cells. The antigen defined by 12E7 is not expressed on mouse or hamster cells; this species specificity allowed us to investigate the genetics of the expression of the 12E7 antigen using human--rodent somatic-cell hybrids. Hybrid cells which contain the human X chromosome as the only human genetic contribution react with 12E7. The X-linkage of the gene, or genes, controlling 12E7 antigen expression and the fact that 12E7 antigen is also expressed on red blood cells led us to investigate the possibility of a relationship with the X-linked Xg blood group system. Although the 12E7 and Xga antigens seem to be different antigenic determinants specified by different loci, we report here a sex-limited effect involving the Xg phenotype on the expression of 12E7 antigen.

Antigens, Surface↗

A human cell-surface antigen defined by a monoclonal antibody and controlled by a gene on chromosome 12.

A murine monoclonal antibody 602-29, subclass IgG1, that recognizes an antigenic determinant expressed by most human cells is described. Immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis analysis indicate that the antigenic determinant is carried by a protein with an apparent molecular weight of 21,000. The antigen is expressed by human-mouse somatic cell hybrids, and analysis of segregants that have lost human chromosomes indicates that the gene controlling expression of the 602-29 antigen is on chromosome 12.

Animals↗

Human--rat muscle somatic cell hybrids form myotubes and express human muscle gene products.

Somatic cell hybrids have been prepared at high frequency between the rat muscle cell line L6 and human fetal muscle cells. The hybrid cells express several human gene products including an antigen, 12E7, controlled by the human X chromosome, Thy-1, and several human isoenzymes. In addition, one clone (37-11) expresses a human muscle-specific surface antigen (5.1 H11) and, under appropriate conditions, can be induced to form myotubes. Upon myotube formation, this clone switches on the production of the human muscle-specific creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) M subunit. This is an example of developmental regulation of human muscle-specific genes in somatic cell hybrids.

Animals↗

The expression of MHC antigens by human teratocarcinoma derived cell lines.

We have used flow microfluorimetry to investigate quantitatively the expression of HLA-A, B and C antigens, and beta 2 microglobulin, by cell lines derived from human teratocarcinomas, Although low levels of these cell surface molecules were expressed by all the lines examined, there was no evidence of discrete HLA-A, B, C/beta 2-microglobulin positive and negative subpopulations in any cultures. In contrast, using similar techniques, no murine embryonal carcinoma cell line was found to express the homologous H-2 antigens, It is suggested that human embryonal carcinoma cells may differ from their mouse counterparts by expressing low levels of major histocompatibility antigens

Animals↗

Deficiency of malic enzyme: a possible marker for malignancy in lymphoid cells.

Soluble malic enzyme (MEs) has been examined in long-term human lymphoid cell lines cultured from 101 individuals. In 65 out of 66 lines derived from people without lymphoreticular malignancy the enzyme was very active. Lines established from 35 individuals with various forms of lymphoreticular malignancy were also examined, including in some cases more than 1 line derived from the same patient. In all cases where the cell line was thought to be derived from normal cells MEs was active, but in 27 out of 29 lines thought to be derived from malignant cells (from 25 patients) MEs was not detected. In the case of two patients with chronic lymphatic leukaemia 'normal' lines active for malic enzyme, and 'leukaemic' lines lacking malic enzyme, had been cultured from the same individual. Preliminary investigations of the lack of malic enzyme in somatic cell hybrids derived from lymphoma and leukaemia cell lines are compatible with an alteration at the level of the structural locus MEs on chromosome 6. However, the restoration of MEs activity in one line by fusion with mouse teratocarcinoma cells suggests that the alteration may be of a regulatory nature.

Animals↗

Antigen expression by somatic cell hybrids of a murine embryonal carcinoma cell with thymocytes and L cells.

Thymocyte x PCC4azaR (a murine embryonal carcinoma cell) hybrid cells resembled PCC4azaR. They continued to express two embryonic antigens (SSEA-1 and 3C4-10) found on PCC4azaR, but did not express the thymocyte antigens Thy-1 and Ly-2. However, H-2Kk of thymocyte origin continued to be expressed, while H-2Db of PCC4azaR origin was not reexpressed. This is consistent with H-2 expression being subject to cis control. In syngeneic hosts these hybrids formed tumors containing stem cells and differentiated derivatives. In contrast, an L-cell x PCC4azaR hybrid resembled the L-cell parent, did not express SSEA-1 and 3C4-10, but did express both H-2Kk and H-2Db. In syngeneic hosts this hybrid formed undifferentiated sarcomas.

Animals↗

Monoclonal antibodies reacting with murine teratocarcinoma cells.

Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000.

Animals↗

Assay for antibody mediated cytotoxicity of mouse spermatozoa by 86rubidium release.

The analysis of the antigenic structure of murine spermatozoa has been hampered by the lack of a convenient objective serological assay. Spermatozoa, in common with other cells, are capable of concentrating 86rubidium. Antibody mediated complement dependent cytotoxicity of murine spermatozoa has been quantitated by measuring release of 86rubidium from pre-labeled spermatozoa. We have used this assay to study the murine T/t locus.

Animals↗

Serological studies of Cw1 and Cw3 expressed on the same haplotype.

A serological comparison has been made between individuals who are phenotypically Cw1 and Cw3. The first individual is Oriental and carries Cw1 and Cw3 on the same haplotype, the other individuals are Caucasian and carry either Cw1, Cw3 or Cw1 and Cw3 on different haplotypes. No unique specificities could be found on the Oriental Cw1, Cw3 haplotype by reciprocal absorption experiments, inhibition of complement dependent microcytotoxicity by F(ab') 2 fragments prepared from Cw1 and Cw3 antisera or by lysostrip experiments. The blocking and lysostrip experiments also suggested molecular independence of Cw1 and Cw3 determinants coded for by Oriental Cw1 haplotype. The results suggest a recent gene duplication in Oriental populations or two loci in the C region.

Cytotoxicity, Immunologic↗

Cellular distrubtion, purification, and molecular nature of human Ia antigens.

Human Ia antigens were extensively purified (1390-fold increase in specific activity) in 32% yield from BRI 8 cells, a lymphoblastoid B-cell line. Purification was monitored by using allogeneic antisera arising by foetal-maternal stimulation. The product, a glycoprotein fraction, contained the Ia antigens, the HLA-A and -B antigens, and a glycoprotein of unknown function. The glycoprotein fraction was composed of four glycosylated polypeptides with molecular weights of 43,000, 39,000, 33,000, and 28,000, and beta2-microglobulin; no polypeptide was linked to another by disulphide bridges. The A and B antigens only were absorbed by antibody against beta2-microglobulin. The Ia antigens comprised one each of the 33,000 and 28,000 molecular weight glycosylated polypeptides noncovalently linked together. Thus, only these chains were absorbed by xenogeneic anti-Ia antisera and were cross-linked by dimethyl-3-3'-dithiobispropionimidate dihydrochloride. The dimeric molecule bound deoxycholate (0.26 g/g of protein) and, when solubilized in deoxycholate, has a molecular weight of 77,000. The Ia allo- and xeno-antigenic activities were labile to heating and proteolysis and are probably determined by the polypeptide structure. Xenogeneic specific anti-Ia antisera were raised in rabbits and mice by immunizing with the glycoprotein fraction. These antisera reacted with B lymphocytes and monocytes but not T lymphocytes and fibroblasts. Their Fab fragments blocked the cytotoxicity of the allogeneic antisera for B lymphocytes and were potent inhibitors of the mixed lymphocyte reaction.

Antigens↗

Human gene mapping using an X/autosome translocation.

Human fibroblasts containing a translocation between the X chromosome and chromosome 15 were fused with the 6-thioguanine-resistant mouse cell line, IR. Resulting hybrids, selected in HAT medium, retained the X/15 chromosome. Hybrids which were counterselected in 6-thioguanine lost this chromosome. The X-linked markers glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and hypoxanthine phosphoribosyl transferase (HPRT), and the non-X-linked markers pyruvate kinase (PKM2) mannose phosphate isomerase (MPI), N-acetyl hexosaminidase A (HEXA) and beta2-microglubulin (beta2-m) all segregated in concordance with the X/15 translocation chromosome. The latter markers have been assigned to chromosome 15. Selection against the X/15 chromosome was done using antihuman beta2-m serum. Electrophoretic and immunochemical analyses of the N-acetyl hexosaminidases A and B in these hybrids were performed.

Acetylglucosaminidase↗

Expression of HLA system antigens on placenta.

The levels of HLA-A and -B antigens expressed by placenta have been assessed relative to parental and other A and B antigen types that were not shared by the foetus. A purified preparation of placenta plasma membrane was used to estimate the antigen activities. The results indicate that maternally and paternally inherited A and B antigen activities and beta2-microglobulin are expressed to similar extents but at much lower levels than in spleen lymphocytes (less than 5%). The possibility that the amounts detected were caused by contamination with blood or maternal tissue was ruled out. The low levels of A and B antigens may account for the lack of a cellular immune response to the other polymorphic cell surface antigens of the trophoblast. No evidence was obtained for the expression of a significant level of Ia antigens.

Cell Membrane↗