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J Milner

Publications and source records attributed to J Milner.

At least 73 records · Page 4Linked to original sources
PubMed1990

p53 is associated with p34cdc2 in transformed cells.

The normal functioning of p53 is thought to involve p53 target proteins. We have previously identified a cellular 35 kd protein associated with p53 and now report evidence identifying this 35 kd protein as p34cdc2, product of the cell cycle control cdc2 gene. The association between p53 and p34cdc2 was detected in SV3T3 and T3T3 cell lines, both expressing the wild-type p53 phenotype, and in 3T3tx cells, expressing 'mutant' p53 phenotype. Binding of the mutant p53 phenotype with p34cdc2 was greatly reduced relative to wild-type. Complexes of p53-p34cdc2 may represent inactivation or activation of either component. The p34cdc2 kinase functions at cell cycle control points and is necessary for entry and passage through mitosis. It also operates in G1 and is involved in the commitment of cells into the proliferative cycle. Since we were unable to detect p53-p34cdc2 complexes in mitotic cells we propose that the interaction between p53 and p34cdc2 may be functional in cell growth control, possibly to promote or to suppress cell proliferation.

Animals↗
PubMed1990

Phosphatases PP1 and PP2A act after the G0/G1 interface in lymphocyte activation.

An understanding of the progressive events required for lymphocyte activation is a prerequisite to understanding the molecular mechanism of immunosuppressive reagents, and is of central relevance to autoimmunity and immune tolerance. Although reversible phosphorylation is a major control mechanism in T cell activation, few facts are known about phosphatases in T cells. It has recently become possible to block selectively the serine/threonine specific protein phosphatases PP1 and PP2A by okadaic acid, a C38 polyether fatty acid produced by dinoflagellates. Here we have used this new and powerful biochemical probe to identify and quantify the activities of PP1 and PP2A at various times during lymphocyte activation. The selected model was mouse T cell activation by concanavalin A because this gave a reproducible experimental system that allowed large scale experiments in defined, serum-free conditions. Our results show the following: (1) levels of PP1 and PP2A in lymphocytes appear to be unusually low, effects being measured at 10 nM okadaic acid compared to 1 microM reported for other cell types; (2) the okadaic acid effect was readily reversible; (3) okadaic acid stimulated mitogenesis when present in early G1 but inhibited mitogenesis in late G1; (4) levels of PP1 and PP2A remained relatively constant over the first 7 h following stimulation; (5) lymphocytes activated in the presence of okadaic acid became resistant to subsequent treatment with FK506; and (6) combined or consecutive treatment with okadaic acid and FK506 resulted in additive drug effects. We conclude that PP1 and PP2A are not involved in the G0-G1 transition as defined by the FK506-sensitive step.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Evidence for allosteric variants of wild-type p53, a tumour suppressor protein.

A tumour suppressor function for p53 is indicated in human lung cancer and in carcinoma of the colorectum. Loss of suppressor function, by mutation of the p53 gene, is associated with activation of p53 as an oncogene. The suppressor (wild type) and oncogenic (mutant) forms of the murine p53 protein are distinguishable at the molecular level by reactivity with anti-p53 monoclonal antibodies. For example, activated mutant p53 fails to react with PAb246 (p53-246 degrees). We now demonstrate that wild type p53 mRNA can be expressed either as p53-246+ or p53-246 degrees. We propose that p53-246 degrees may represent an allosteric variant of wild type p53 compatible with positive growth control. Thus, for wild type p53 the variants p53-246+ and p53-246 degrees may reflect suppressor and activator functions of p53 in the normal control of cell proliferation. For human p53 we present evidence that the epitope recognised by PAb1620 is analogous to that for PAb246 on murine p53. Thus the epitope for PAb1620 may prove to be of use as a marker for wild type human p53 with anti-oncogenic function.

Animals↗
PubMed1990

Addition of fresh medium induces cell cycle and conformation changes in p53, a tumour suppressor protein.

Using analytical flow cytometry we have monitored changes in the conformation of p53 through the cell cycle under different conditions of cell growth. Conformational variants of murine p53 are characterized by reactivity with monoclonal antibodies PAb246, PAb248 and PAb421. In proliferating cells, p53 conformation was independent of the cell cycle. Addition of fresh medium, however, resulted in loss of p53 reactivity with PAb246 (p53-246(0]. Mutant p53, which lacks suppressor function, is also p53-246(0). Thus in SV3T3 cells p53-246(0) may reflect a change in p53 tertiary structure that is induced by growth stimulation and is compatible with the normal cell growth response. We argue that p53-246(0) and p53-246+ may exert positive and negative constraints in cell growth control. By stabilising p53-246(0) activating mutants of p53 would favour cell proliferation with dominant effect.

Animals↗
PubMed1989

Selenium content and glutathione peroxidase activity of milk from vegetarian and nonvegetarian women.

The concentration of selenium (Se) in milk samples obtained from 26 lacto-ovo-vegetarian (vegetarian) women was significantly greater (22.2 +/- 0.8 ng/ml) than from 12 nonvegetarian women (16.8 +/- 1.3). Mean GSH-Px activity (EC 1.11.1.9) in milk from vegetarians was 146% of that in milk from nonvegetarians. A significant correlation between GSH-Px activity and Se concentration was observed (r = 0.76). Likewise a significant correlation was observed between milk linoleic acid content and GSH-Px activity (r = 0.68). In undialyzed samples from vegetarian women, most of the increased content of Se was found in fractions containing proteins of 100 kdaltons (kD) or more. The high glutathione peroxidase activity in milk from vegetarians was associated with selenoproteins in the 90 to 100 kD range. A distinct 40 kD Se peak was also detected in milk from vegetarians. There was no difference in Se intake between the two groups of women. Therefore, although data from the present study show that both the quantity and distribution of Se in human milk can be modified by the maternal diet, the increased Se content and GSH-Px activity in milk from vegetarians cannot be explained by an increased Se intake.

Adult↗
PubMed1989

p53 is associated with a 35 kD protein in cells transformed by simian virus 40.

The p53 protein is functionally implicated in the normal control of cell proliferation and, abnormally, in cell transformation. p53 is believed to function via specific cellular target proteins and activated mutants of p53 are associated with proteins of the hsp/hsc 70 heat shock family. However, cellular target proteins of wild type p53 have not, as yet, been described. With the aim of detecting such targets we have screened for cellular protein(s) that co-precipitate with wt p53. We now describe a 35 kD protein co-precipitated with p53 from SV40-transformed cells (a similar protein is detectable in non-viral transformed cells). The 35 kD protein does not appear to be a degradation product of p53. In sequential immunoprecipitations the 35 kD protein was depleted in parallel with p53, with which it appeared to be physically associated. This was substantiated by dissociation experiments in which the 35 kD protein was dissociated under conditions that also dissociate p53 from SV40 large T antigen. Thus wt p53 appears to interact with a cellular protein of 35 kD, the identity of which is under investigation.

Antigens, Polyomavirus Transforming↗
PubMed1988

Evidence that immunological variants of p53 represent alternative protein conformations.

In normal murine lymphocytes the cellular oncoprotein, p53, exists as two immunologically distinct species which are reciprocally expressed in quiescent (p53-Go) and mitogenically stimulated (p53-G divided by) cells. More recently, we have identified discrete forms of p53, immunologically similar to p53-Go and p53-G divided by, in 3T3 cells transformed by simian virus 40 (SV40). In this report, we demonstrate that immunologically distinct p53 species can also be expressed in vitro, from a single murine p53 cDNA clone. The p53 variants expressed in vitro and in SV40-transformed 3T3 cells have been studied by immunoprecipitation and Western blotting. Immunoprecipitation data indicated that, in their native conformations, the p53 variants react with either the PAb421 or RA3.2C2 monoclonal antibodies, but not with both. When analyzed by Western blotting, however, the denatured proteins were found to react with both monoclonal antibodies. This suggests that the p53 protein is flexible and can fold in alternative conformations so as to expose or mask different epitopes. We propose, therefore, that immunologically distinct p53 species are generated, at least partly, by conformational changes in a single polypeptide species.

Antibodies, Monoclonal↗
PubMed1987

A new anti-p53 monoclonal antibody, previously reported to be directed against the large T antigen of simian virus 40.

The PAb1620 antibody belongs to a group of monoclonal antibodies reported to recognize nuclear large T antigen of simian virus 40 (SV40). In the case of PAb1620 there was some indication that the antibody reacted preferentially with large T when it was complexed with p53, the cellular transformation-related antigen. However, we found that PAb1620 does not appear to react with SV40 large T, either free or in complex with p53. In this paper we demonstrate that PAb1620 is, in fact, directed against the p53 protein and recognizes a conformation-dependent epitope. In addition, PAb1620 recognises human p53 from SV80 cells.

Antibodies, Monoclonal↗
PubMed1986

The cellular tumour antigen p53: evidence for transformation-related, immunological variants of p53.

The protein p53 is involved in the control of normal cell proliferation. However, when expressed abnormally, p53 also contributes to the process of cell transformation. The possibility that multifunctional forms of p53 exist was realised when two cell cycle dependent forms of the protein were identified in normal cells (lymphocytes: J. Milner, 1984, Nature (London) 310, 143-145). Interestingly these two forms of p53 in normal cells lack some of the epitopes recognised by monoclonal antibodies developed against p53 in transformed cells. This suggests that p53 may bear additional transformation-related epitopes in transformed cells. We have now investigated this possibility by screening a variety of cell lines for immunological variants of p53. The monoclonal antibodies PAb421, PAb122, RA3.2C2, PAb248, PAb200.47, and PAb246 were used. Our results indicate the existence of two immunologically distinct variants of p53 expressed in transformed cells. The two p53 variants are distinguished by the presence or absence of the epitope recognised by PAb246. A given cell line generally expressed a single immunological variant of p53. However, 3T6 cells expressed two subsets of p53 and appeared to modulate p53 synthesis in response to cell density.

Animals↗
PubMed1986

Visualisation, by immunocytochemistry, of p53 at the plasma membrane of both nontransformed and SV40-transformed cells.

The cellular protein p53 normally functions in the control of cell proliferation; but, when expressed abnormally p53 also contributes towards the process of cell transformation. The functioning of p53 is thought to involve interaction with specific cellular targets and, in SV40- and other transformed cells, p53 is located in the nucleus: thus p53 may function via interacting with specific nuclear components. In addition, there is indirect evidence that in SV40-transformed cells p53 is also associated with the plasma membrane; however, this is not evident in cells stained by immunofluorescence. We have sought to obtain direct evidence for p53 at the plasma membrane by staining with a very sensitive immunocytochemical procedure. We have compared SV40-transformed BALB 3T3 cells with BALB 3T3, NIH 3T3, and NIH 3T3 transfected with v-mos provirus. The last cell line is tumorogenic. We found direct evidence for p53 at the plasma membrane for each cell line. In nontransformed and SV40-transformed cells the association of p53 with the plasma membrane was restricted to the period of mitosis. These results indicate that association of p53 with the plasma membrane is a normal, rather than a transformation-related phenomenon, and is temporally linked to the period of mitosis.

Animals↗
PubMed1986

Customary diet, anthropometry, and dyslipoproteinemia in selected North American populations. The Lipid Research Clinics Program Prevalence Study.

The intake of nutrients, determined by 24 hr diet recall, and body measurements were obtained in 8250 free-living white study participants divided into 20 to 49 and 50 + age groups for males and female nonusers and users of gonadal hormones. They were classified into dyslipoproteinemia (DLP) phenotypes: hyperHDL, hypoHDL, IIA, hpypoLDL, IV, and normal. The dyslipoproteinemia DLP phenotypes, compared with the normal, had biologically meaningful differences in nutrient intake and indexes of obesity that were most marked for males aged 20 to 49 years as shown in the table (below). Those with the hyperHDL phenotype were thinner and ingested more energy and more alcohol and less carbohydrate as percent kilocalories (%kcal). Individuals classified as hypoHDL were fatter and tended to ingest less energy and less alcohol as %kcal. Persons with the type II phenotype were fatter and ingested less energy. Those with hypoLDL tended to be thinner and ingested more energy. Individuals with the type IV phenotype were fatter, ingested less energy and carbohydrate and more alcohol as %kcal. Similar trends were observed in female nonusers of hormones aged 20 to 49 and to a lesser extent in the 50 + age groups and in female users of hormones. Dietary protein, cholesterol, total fat, and polyunsaturated and saturated fatty acids had no consistent associations with DLP phenotype, and sucrose and starch had no association independent of total carbohydrate. This is the first evidence of an association of customary diet and DLP phenotypes in the free-living population. Equating energy intake with energy expenditure, persons with the high-risk phenotypes, IIA, IV, and hypoHDL, compared with the normal, had decreased energy expenditure and were fatter, whereas those with the low-risk phenotypes, hyperHDL and hypoLDL, had increased energy expenditure and were thinner.

Adult↗
PubMed1985

The SV40 large T-p53 complex: evidence for the presence of two immunologically distinct forms of p53.

The transforming protein of SV40 is the large T antigen. Large T binds a cellular protein, p53, which is potentially oncogenic by virtue of its functional involvement in the control of cell proliferation. This raises the possibility that p53 may mediate, in part, the transforming function of SV40 large T. Two immunologically distinct forms of p53 have been identified in normal cells: the forms are cell-cycle dependent, one being restricted to nondividing cells (p53-Go) and the second to dividing cells (p53-G divided by). We have now dissociated and probed the multimeric complex of SV40 large T-p53 for the presence of immunologically distinct forms of p53. Here we present evidence for the presence of p53-Go and p53-G divided by complexed with SV40 large T.

Animals↗
PubMed1984

A new indicator of human malignant tumour.

In haemagglutination tests we have found that the monoclonal antibody B5 discriminates between erythrocytes from patients with malignant cancer (total 386; greater than 80% B5 positive) and individuals with no known tumour (total 247; less than 20% B5 positive). The B5 antigen detected on intact erythrocytes is a tightly bound surface component which does not appear to be derived from the plasma. The B5 antigen is not T, Tn, Ca1, CEA or the Forsmann antigen; nor is it related to any of the major blood group antigens. Abnormal levels of surface B5 are found on erythrocytes from patients with a great diversity of tumours and this fact presents B5 as an indirect marker of malignancy. Successful eradication of tumour is associated with a switch from positive to negative B5 haemagglutination. We believe that B5 is a valuable addition to the few useful tumour markers already employed for monitoring tumour status.

Antibodies, Monoclonal↗
PubMed1983

Source of variance in 24-hour dietary recall data: implications for nutrition study design and interpretation. Carbohydrate sources, vitamins, and minerals.

Beaton et al (Am J Clin Nutr 1979;32:2546-59) reported on the partitioning of variance in 1-day dietary data for the intake of energy, protein, total carbohydrate, total fat, classes of fatty acids, cholesterol, and alcohol. Using the same food intake data and the expanded National Heart, Lung and Blood Institute food composition data base, these analyses of sources of variance have been expanded to include classes of carbohydrate, vitamin A, vitamin C, thiamin, riboflavin, niacin, calcium, iron, total ash, caffeine, and crude fiber. The analyses relate to observed intakes (replicated six times) of 30 adult males and 30 adult females obtained under a paired Graeco-Latin square design with sequence of interview, interviewer, and day of the week as determinants. Neither sequence nor interviewer made consistent contribution to variance. In females, day of the week had a significant effect for several nutrients. The major partitioning of variance was between interindividual variation (between subjects) and intraindividual variation (within subjects) which included both true day-to-day variation in intake and methodological variation. For all except caffeine, the intraindividual variability of 1-day data was larger than the interindividual variability. For vitamin A, almost all of the variance was associated with day-to-day variability. One day data provide a very inadequate estimate of usual intake of individuals. In the design of nutrition studies it is critical that the intended use of dietary data be a major consideration in deciding on methodology. There is no "ideal" dietary method. There may be preferred methods for particular purposes.

Adult↗
PubMed1983

A twin study examining the genetic influence on food selection.

Taste sensitivity, preference and use of 24 foods were studied to identify the extent of genetic influence. Thirteen monozygotic (MZ) and ten dizygotic (DZ) adult, female twin-pairs of comparable family background participated in the research. Heritability was demonstrated for phenylthiocarbamide (PTC) sensitivity and was found in both preference and use for unsweetened grapefruit juice and green beans. In terms of preference and use separately, a heritability component was indicated in one-third and one-eighth of the foods respectively. Environmental adaptation and learning, unmeasured in the present study, would appear to exert a stronger influence than the genetic component on food-related behaviour.

Adult↗