Isolation of plasma membrane glycoprotein from bovine thymocytes

Glycoprotein was isolated from a purified thymocyte membrane preparation by two methods: lithium diiodosalicylate-phenol extraction and hot 75% ethanol extraction. A higher yield of membrane sialic acid was obtained by the latter method. The preparations had similar apparent molecular weights on sodium dodecyl sulfate gel electrophoresis. Both had similar receptor activities against a panel of hemagglutinins, although the 75% ethanol extract was more active on a weight basis. However, there were significant differences in carbohydrate and amino acid compositions of the two thymocyte extracts. The lithium diiodosalicylate-extracted material had much more glucose, ribose, and glycine than the ethanol extract. The glycoprotein preparations from thymocytes were quite distinct from the glycoprotein of bovine erythrocytes in both composition and receptor properties.     

Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [35S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.     

The use of short-term tests to measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds

The effect of reducing agents on the nitrosation of methylguanidine (MG) and on the in vitro activation of dimethylnitrosamine (DMN) was examined by measuring DNA-repair synthesis (unscheduled incorporation of [3h]TdR), shifts in alkaline sucrose gradients, frequency of chromosome aberrations, and clone-forming capacity of cultured human fibroblasts. The reducing agents examined were sodium ascorbate, cysteine, cysteamine, and propyl gallate. Since the short-term bioassays used can be quantitated, it has become relatively easy to detect the inhibitory action of reducing compounds on the nitrosation reaction of MG and metabolic activation (with S-9 preparation) of the precarcinogen DMN, to measure their effective dose range, and to establish the most effective ratios between inhibitory agent and reactant. The results indicate that DNA-repair synthesis is a suitable short-term test for studying the numerous combinations and premutations between several carcinogenic or non-carcinogenic agents, and for estimating the capacity of inhibitory agents to affect formation and activation of chemical carcinogens.     

The use of short-term tests to measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds

The effect of reducing agents on the nitrosation of methylguanidine (MG) and on the in vitro activation of dimethylnitrosamine (DMN) was examined by measuring DNA-repair synthesis (unscheduled incorporation of [³H]TdR), shifts in alkaline sucrose gradients, frequency of chromosome aberrations, and clone-forming capacity of cultured human fibroblasts. The reducing agents examined were sodium ascorbate, cysteine, cysteamine, and propyl gallate. Since the short-term bioassays used can be quantitated, it has become relatively easy to detect the inhibitory action of reducing compounds on the nitrosation reaction of MG and metabolic activation (with S-9 preparation) of the precarcinogen DMN, to measure their effective dose range, and to establish the most effective ratios between inhibitory agent and reactant. The results indicate that DNA-repair synthesis is a suitable short-term test for studying the numerous combinations and permutations between several carcinogenic or non-carcinogenic agents, and for estimating the capacity of inhibitory agents to affect formation and activation of chemical carcinogens.     

Interaction of glutathione transferase from horse erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.     

Redox forms of human placenta glutathione transferase

Human placenta glutathione transferase (EC 2.5.1.18) pi undergoes an oxidative inactivation which leads to the formation of an inactive enzymatic form which is homogeneous in several chromatographic and electrophoretic conditions. This process is pH dependent, and it occurs at appreciable rate in alkaline conditions and in the presence of metal ions. Dithiothreitol treatment completely restores the active form. -SH titration data and electrophoretic studies performed both on the oxidized and reduced forms indicate that one intrachain disulfide is formed, probably between the two faster reacting cysteinyl groups of each subunit. By the use of a specific fluorescent thiol reagent the disulfide forming cysteines have been identified as the 47th and 101th residues. The disulfide formation causes changes in the tertiary structure of this transferase as appears by CD, UV, and fluorometric analyses; evidences are provided that one or both tryptophanyl residues of each subunit together with a number of tyrosyl residues are exposed to a more hydrophilic environment in the oxidized form. Moreover, electrophoretic data indicate that the subunit of the oxidized enzyme has an apparent molecular mass lower than that of the reduced transferase, thereby confirming structural differences between these forms.     

Infection of human natural killer (NK) cells with replication-defective human T cell leukemia virus type I provirus. Increased proliferative capacity and prolonged survival of functionally competent NK cells.

Human T-cell leukemia virus type I (HTLV-I) can infect a variety of human cell types, but only T lymphocytes are efficiently immortalized after HTLV-I infection. This study reports an attempt to infect and to immortalize NK cells with HTLV-I. Co-cultivation of freshly isolated NK cells with a HTLV-I-producing T cell line did not result in NK cell infection. However, NK cells activated with an anti-CD16 mAb and co-cultivated with a HTLV-I-producing T cell line were reproducibly infected by HTLV-I. HTLV-I infection was documented in NK cell lines and clones by the detection of defective integrated provirus by both Southern blot and polymerase chain reaction analysis. Although HTLV-I-infected NK cells produced viral proteins, they did not produce infectious viral particles. HTLV-I-infected NK cells were phenotypically indistinguishable from their uninfected counterparts (CD16+, CD2+, CD56+, CD3-). They also retained the ability to mediate both natural and antibody-dependent cell cytotoxicity. The IL-2-dependent proliferation of HTLV-I-infected NK cells was significantly greater than that of uninfected NK cells. The doubling time of this infected population was reduced from 9 days to 3 days, and the overall survival of the culture in the absence of restimulation was extended from 5 wk to 18 wk. Unlike T lymphocytes, HTLV-I-infected NK cells were not immortal, implying a fundamental difference between these two lymphocyte populations.