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Biomedical subjects

A R Shaw

Publications and source records attributed to A R Shaw.

At least 73 records · Page 4Linked to original sources
PubMed1984

Human tumor cell membrane glycoprotein associated with protein kinase activity.

A mouse MAb3 50H.19 raised against the human melanoma cell line MEL-T binds to carcinoma cell lines, carcinoma biopsy material, and certain epithelia of normal tissues. It immunoprecipitates two components from carcinoma cell lines, a major component of 22 kd which is O-glycosylated and a minor one of 24 kd which is additionally N-glycosylated. The immunocomplexed 50H.19 antigen exhibits protein kinase activity with substrate-specificity for casein and phosvitin, but not for histones. It phosphorylates on serine and threonine, but not tyrosine residues. Enzyme activity is cyclic AMP-independent.

Antibodies, Monoclonal↗
PubMed1984

Biochemical characterization of human carcinoma surface antigen associated with protein kinase activity.

MAb 50H.19 immunoprecipitates two proteins from lysates of human carcinoma cell lines, and embryonic fibroblasts intrinsically labelled with 3H-leucine, 35S-methionine, or a 3H-amino acid mixture; a major component of Mr = 22,000 (22 kd component) and a minor component of Mr = 24,000 (24 kd component). Oligomeric forms of the proteins are not observed under reducing or non-reducing conditions. Both proteins are expressed on the plasma membrane, and are glycoproteins. We investigated the relationship between the proteins in terms of their glycosylation and derivation from precursors. The 22 kd component is O-glycosylated as demonstrated by 3H-galactose incorporation, insensitivity to tunicamycin (TM), and its stepwise generation from a 20.5 kd precursor. The 24 kd protein is N-glycosylated, as shown by 3H-mannose incorporation, and by the total inhibition of its synthesis in the presence of TM. Further evidence for its N-glycosylation is provided by the appearance of a 23 kd precursor in lysates from the osteogenic sarcoma cell line SKOSC pulse-labelled for 5 min, a time preceding O-glycosylation of the 20.5 kd protein. Furthermore, mild alkali treatment of the immune complex leads to a loss of approximately 1,000 daltons in each glycoprotein confirming the O-glycosylated nature of the 22 kd component, and suggesting that the 24 kd component is additionally O-glycosylated. Both glycoproteins undergo an apparent increase of molecular weight of about 500 daltons when run in the non-reduced form on SDS polyacrylamide gels under standard electrophoretic conditions, suggesting they contain a similar degree of intra-chain disulphide bonding. Confirmatory evidence that the two components share a common polypeptide backbone is provided by the appearance of only the 20.5 kd component in lysates from SKOSC cells pulse-labelled for 5 min in the presence of TM.

Antibodies, Monoclonal↗
PubMed1984

Monoclonal antibody specific for granulocytic-lineage cells and reactive with human pluripotent and committed haematopoietic progenitor cells.

A murine monoclonal antibody (82H5, IgM class) has been developed that detects an antigenic determinant expressed by greater than 90% of normal granulocytes and 60-80% of light-density normal bone-marrow cells, including human pluripotential progenitors (colony-forming-unit-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GEMM) and committed progenitors: granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and megakaryocytic (CFU-MK). This antibody did not react with erythrocytes, monocytes, platelets, lymphocytes from normal peripheral blood, lymphoblasts from patients with acute lymphoblastic leukaemia, or with lymphoid cells lines. The 82H5-defined antigenic determinant was expressed on greater than 90% of leukaemic cells of promyelocytic, myelomonocytic and monocytic morphology, and cell lines KG.1, ML.1, HL.60, K562 and U.937. Cortical thymocytes were unreactive with 82H5. Treatment of human bone-marrow cells with granulocytic-specific monoclonal antibody 82H5 plus complement significantly inhibited colony formation (48-74%; P less than 0.05) of CFU-GEMM, CFU-GM, BFU-E, CFU-MK, whereas treatment with control monoclonal anti-Ia antibody plus complement caused 79-89% inhibition. This antibody reacted strongly with 3-fuc-NAc lactosamine when tested with a panel of synthetic carbohydrate structures. We conclude that 82H5 may be a useful probe for phenotypic analysis of leukaemic cells and investigation of haematopoiesis.

Animals↗
PubMed1983

Translation of lymphocyte mRNA for mouse colony stimulating factor.

Poly A+ RNA has been purified from phorbol myristate acetate stimulated mouse EL4 cells. Translation, by microinjection into frog oocytes, gave biologically active interleukin 2 (IL2) and colony stimulating factor for granulocytes and macrophages (GM-CSF). These two mRNAs were separated by centrifugation through linear sucrose gradients. The sedimentation coefficients for IL2 and GM-CSF mRNAs were found to be 11.5S and 8S respectively. The clonal and cellular morphologies induced by the oocyte material corresponded to low concentrations of authentic GM-CSF.

Animals↗
PubMed1983

Stimulation of [3H]thymidine uptake in mouse marrow by granulocyte-macrophage colony stimulating factor from mouse lung conditioned medium.

Stimulation of [3H]thymidine uptake in mouse marrow cells by a haematopoietic factor, granulocyte-macrophage colony stimulating factor from mouse lung conditioned medium, was used to follow the activity of the factor in the medium, and during its partial purification from the medium. The assay was performed in microtitre plates and found to be much easier and faster than conventional colony counting. The marrow cells were incubated in flat-bottomed plates in the presence of the factor for 5 days before labelling with [3H]thymidine (2 muCi/well, 5 Ci/mmole) for 6 h. Stimulation of the [3H]thymidine uptake was compared with the development of granulocyte-macrophage colonies in semisolid methylcellulose culture. The activity followed by both assays showed identical behaviour when subjected to ammonium sulphate precipitation, Sephadex gel filtration, concanavalin-A-Sepharose chromatography and polyacrylamide gel electrophoresis.

Animals↗
PubMed1982

Characterization and cross-reactivity of human and mouse oncofetal antigens. Use of a new solid phase assay for detection of cell surface antigens.

Oncofetal (OF) antigens have been isolated from mouse F9 teratocarcinoma cells, mouse testicular cells, and human molar tissue by detergent extraction followed by dialysis. The soluble antigens have been used in solid phase radioimmunoassay (SPRIA) and enzyme-linked immunosorbent (ELISA) assay. Specific antibodies have been raised to these antigens in mice. By using these antisera, extensive cross-reactivity was found between mouse and human OF antigens. A human trophoblastic tumor cell line BEWO absorbed out mouse anti-F9 reactivity. Patients with tumors of germinal origin were found to have antibodies which cross-react with mouse and human OF antigens. This new assay is a rapid and sensitive method for the screening of monoclonal antibodies against these antigens as well as for detecting antibodies to tumors bearing these antigens in patients.

Absorption↗
PubMed1982

Selective inhibition of adenovirus type 2 early region II and III transcription by an anisomycin block of protein synthesis.

The transcription of adenovirus type 2 genes proceeds through a broad three-phase program. From 1 to 4 h postinfection six early transcription units (EIa, EIb, EII, EIII, EIV, and the promoter-proximal segment of the late transcription unit) are activated. From 4 to 6 h postinfection transcription of the early genes is depressed. After the onset of viral DNA replication at approximately 6 h postinfection, the transcript from the late promoter is antiterminated, and this transcript dominates viral RNA synthesis. The early activation period also proceeds through a series of stages; early regions EIa and EIV are activated first, followed by early region EII. We show that in the presence of anisomycin, a stringent inhibitor of protein synthesis, nuclear RNA and cytoplasmic polyadenylated RNA from regions EIa and EIV accumulate at normal rates, whereas RNAs from regions EII and EIII do not accumulate. We also show that failure to accumulate RNAs from regions EII and EIII is due to reduction of the rate of transcription by greater than 90%. We conclude that the regulation of the promoters for early regions EII and EIII is distinct from the mechanism that operates on the other early transcription units. The promoters for early regions EII and EIII diverge and lie approximately 500 nucleotides apart. We examined the structure of viral chromatin in this region early during infection by mild DNase I digestion in isolated nuclei and indirect end labeling with a DNA probe near these promoters. In control, drug-free cells where EII and EIII are transcribed and in anisomycin-treated cells where EII and EIII are not transcribed we detect the same regular DNase I pattern. We suggest that regulation of EII and EIII is not mediated through a change in gross chromatin structure, but rather by a viral effector, possibly a product of region EIa.

Adenovirus Early Proteins↗
PubMed1982

Antigenic heterogeneity of human colorectal cancer cell lines analyzed by a panel of monoclonal antibodies. I. Heterogeneous expression of Ia-like and HLA-like antigenic determinants.

The pattern of reactivity of 10 monoclonal antibodies (MCA) developed against human lymphoid leukemia cells and tested with human T-cells, B-cells, as well T-lymphoma and B-lymphoma cell lines suggested that six of them detect la-like determinants, three detect HLA-like determinants, and the remaining one detects a non-Ia B-cell antigen. With the use of three binding assays, the six MCA that appeared to detect Ia-like determinants reacted strongly with the human colorectal cancer cell (CCC) line LoVo, and none of the three MCA that reacted with HLA-like determinants reacted with this cell line. Immunoprecipitation and polyacrylamide gel electrophoresis analysis confirmed the apparent specificities of the MCA for Ia-like and HLA molecules, demonstrated the presence of Ia-like molecules in LoVo, and failed to detect HLA in these cells. The cellular enzyme-linked immunosorbent assay was used for the testing of our anti-Ia and anti-HLA MCA with 15 other CCC lines. Marked heterogeneity was found in the expression of different Ia-like and HLA determinants defined by different or overlapping subsets of MCA, which suggested that these determinants might be present on different molecules or that different conformations of the same molecules exist in various CCC lines. Analysis of the surface phenotype of subclones of LoVo cells revealed the presence of stable variant cell subpopulations, which lost reactivity with four out of six of the anti-Ia-like MCA but retained at least one Ia-like molecule recognized by two of our MCA. All of the subclones maintained the HLA-negative phenotype. The possible immunologic and diagnostic consequences of the presence or absence of Ia-like or HLA markers on nonlymphoid tumor cells are discussed.

Antibodies, Monoclonal↗
PubMed1980

Transcripts from the adenovirus-2 major late promoter yield a single early family of 3' coterminal mRNAs and five late families.

The major late promoter of adeovirus-2 is located at coordinate 16.45 and initiates synthesis of nuclear precursors that are processed into mRNAs which fall into five 3- co-terminal families, L1-L5. These mRNAs all share a common tripartite 5' leader with a capped terminus encoded at th RNA initiation site. We show that the coorindate 16.45 RNA initiation site is also an early promoter, and yields transcripts detectable as early as 1 hr post-infection, prior to DNA replication and the early-late switch at 6-8 hr. Polyadenylated cytoplasmic RNA from the first 3- co-terminal family, L1, is also produced from the earliest stages of infection. L1 mRNA accumulates in the cytoplasm in the presence of cycloheximide, which blocks DNA replication and the onset of the late phase. Early nuclear RNA contains the same capped 5' terminal RNAase T-1 undecanucleotide and promoter proximal oligonucleotides present in late transcripts. This implies that precisely the same transcription start site is utilized for early L1 mRNA synthesis as is used during the late stage for L1-L5 late mRNA synthesis. In contrast to the early apperance of L1 mRNA, neither L2 nor L3 mRNAs are detected until 5-6 hr post infection. We cnclude that a major event in the Ad-2 early-late switch is a novel form of control which activates L2-L5 mRNA production.

Adenoviridae↗
PubMed1979

Terminally redundant deletion mutants of bacteriophage BF23.

Deletion mutants of bacteriophage BF23 were isolated and the positions of the deletions were determined. Two different deletable regions were detected: one in the same region as previously reported for bacteriophage T5, which is closely related to BF23; and the other within both terminal repetitions. The former deletable region lay between positions 0.31 and 0.36, which represented the fractional lengths of the BF23 ( + ) DNA as measured from its left end. The latter deletion was evenly divided between the two terminal repetitions. The deletion in the left terminal repetition lay between positions 0.044 and 0.078 and was repeated in the corresponding region of the right terminal repetition between positions 0.966 and 1.0. The size of the DNA transferred to host cells during the first step of DNA transfer by BF23 carrying deletions in the terminal repetitions of its DNA was less than the size of DNA transferred during the first step by wild-type BF23 by an amount equal to the size of the deletion in each terminal repetition. This finding suggests the existence of a specific mechanism for delineating the position at which the first step of DNA transfer is stopped.

Coliphages↗
PubMed1979

Is abortive infection by bacteriophage BF23 of Escherichia coli harboring ColIb plasmids due to cell killing by internally liberated colicin Ib?

Infection of Escherichia coli harboring ColIb+ plasmids with bacteriophage BF23+ is abortive and resulted in changes of membrane permeability as measured by efflux of nucleotides and K+. A single pre-early gene product of BF23+ was necessary and sufficient to elicit the abortive response. Appropriate mutations in this pre-early gene allowed a productive infection in ColIb+ cells. Appropriate mutations in the ColIb plasmid also allowed a productive infection with BF23+. A comparison of changes occurring during abortive infection and during killing of sensitive cells by external colicin Ib or Ia, together with certain genetic data, has led to the conclusion that membrane changes accompanying the two phenomena are the result of a common mechanism, namely, the interaction of free colicin with the cytoplasmic membrane.

Bacteriocin Plasmids↗