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M J Atkinson

Publications and source records attributed to M J Atkinson.

At least 55 records · Page 3Linked to original sources
PubMed1994

[Quantification of messenger RNA expression in tumors: which standard should be used for best RNA normalization?].

Alterations in gene expression are frequently encountered in malignant tissues. Quantification of the often quite subtle changes in mRNA content is routinely performed by northern blot hybridization. Practical experience shows that the methodology is subject to considerable error, necessitating the use of an internal mRNA standard for quantification. Many different control mRNAs have been employed over the years, but no systematic study has proven their applicability to comparisons made between tumor cells and normal tissues. The validity of these control RNAs, usually housekeeping genes, must be established to exclude the possibility that they themselves are expressed at different levels in tumor and non-tumor cells. Our experience has revealed that serious discrepancies may arise when incorrect standard genes are used to compare tumor and normal tissues.

Actins↗
PubMed1993

Map position and usage of 3' VH family members: usage is not position dependent.

The primary repertoire in mice, in large part, is determined by the Ig gene segments joined to form the variable region genes-VDJ for the heavy chain genes and VJ for the light chain genes. However, the mechanisms that determine which VH gene, of several hundred available, is joined to a DJH structure remain unexplained. One theory proposes that the VH gene segments closest to the 3' end of the VH locus are chosen because of their location, i.e. that proximity to a DJH structure is a prime determinant for VH selection. Alternatively, the content of the DNA may be the determining factor regardless of chromosomal position. According to this hypothesis 3' preference is due to the coincidence of preferred content and proximity to DJH structures. Since the 3' VH families span more than a centimorgan of DNA we were able to distinguish between these concepts by determining VH usage of gene segments within this region. The BALB/c strain is ideal for this study since it uses 3' VH families at a very high frequency. We therefore mapped the positions of the 3' VH gene families and used this map to investigate the usage of these gene segments in BALB/c fetal liver derived A-MuLV transformed cell lines. We found that while VH gene segments throughout the Ig locus, both 3' and 5' were used, there was a clear preference for 3' VH families. However, within the 3' VH segments the most proximal were not preferred, indicating that proximity to DJH structure was not a strict determinant of VH utilization.

Animals↗
PubMed1992

Expression of stromelysin 3 in the stromal elements of human basal cell carcinoma.

In basal cell carcinoma, release of proteolytic activity is implicated in extracellular matrix degradation and tumor infiltration. The stromelysin metalloproteinase family is a major candidate for the matrix proteolytic activity in infiltrative tumors. However, in murine models of basal cell carcinoma, neither stromelysin 1 nor 2 appears to play a role in tumor infiltration. We have analyzed the expression of the newly described stromelysin 3 in human basal cell carcinoma using Northern blot analysis and in situ hybridization. In 12 of 14 cases, levels of stromelysin 3 expression were more than tenfold above those observed in normal skin. In one of five cases of squamous cell carcinoma, stromelysin 3 expression was tenfold above levels seen in normal skin. Stromelysin 3 expression was either undetectable or extremely weak in all five cases of infiltrative malignant melanoma. In basal cell carcinoma, stromelysin 3 transcripts were localized by in situ hybridization to the stromal tissue immediately adjacent to basal cell carcinoma, the tumor cells themselves being negative. Therefore, expression of stromelysin 3 in stromal cells may be expected to play a significant role in destruction of the basal membrane zone and extracellular matrix in basal cell carcinoma invasion.

Blotting, Northern↗
PubMed1991

Ig gene rearrangements on individual alleles of Abelson murine leukemia cell lines from (C57BL/6 x BALB/c) F1 fetal livers.

We have previously shown that selection of Ig H chain V region genes used by colonies obtained from splenic B cells and fetal liver pre-B cells was dependent on strain-specific factors. Moreover, by examining the V gene usage in strains congenic at the Igh locus, we also determined that the strain-specific factor was encoded by sequences lying outside of the Igh locus. We decided to examine whether there are differences in Vh gene rearrangement between alleles in an F1 strain. To do this analysis we chose to examine the relative Ig H chain V region gene usage of pre-B cell lines derived from (C57BL/6 x BALB/c)F1 fetal liver cells by Southern blot analysis. We found a high frequency of Vh-gene rearrangements (77% of the alleles had VDJ rearrangements) and these rearrangements occurred to Vh-genes throughout the Vh locus and were not confined to the D-proximal Vh-genes as has been previously observed with lines from other mouse strains. The Vh-gene usage pattern is similar on both alleles indicating that at least one of the determinants of which Vh-gene is used is trans-acting and acts similarly on each allele. Furthermore, one allele, Ighb (donated by the C57BL/6 parent), rearranged Vh-genes more frequently than the other allele, Igha (donated by the BALB/c parent) suggesting that one of the determinants of Vh-gene rearrangement may be acting in an allele-specific manner.

Abelson murine leukemia virus↗
PubMed1991

Bovine spongiform encephalopathy: epidemiological studies on the origin.

The results of further epidemiological studies of bovine spongiform encephalopathy (BSE) support the previous findings that the onset of exposure of the cattle population to a scrapie-like agent, sufficient to result in clinical disease, occurred in 1981/82. The onset of this exposure was related to the cessation, in all but two rendering plants, of the hydrocarbon solvent extraction of fat from meat and bone meal. A further possible explanation, related to the geographical variation in the reprocessing of greaves to produce meat and bone meal, was identified for the geographical variation in the incidence of BSE.

Age Factors↗
PubMed1991

Bone tumor induction after incorporation of short-lived radionuclides.

Temporally-limited internal irradiation after incorporation of short-lived bone-seeking radionuclides is a useful experimental tool for the investigation of extrinsic and intrinsic factors which modify the dose dependence of bone tumor risk. Here we describe some of the results obtained in experiments with female mice (mainly NMRI). The future aim of such experiments should be the prediction of risk of late effects using early molecular-biological changes. Molecular-biological descriptions in our model are at present very limited.

Animals↗
PubMed1991

Measurement of recombinase activity in a set of related Abelson murine leukemic virus pre-B cell lines: DJ/DJ lines have more recombinase activity than do VDJ/VDJ lines.

The VDJ recombination potential of a number of Abelson murine leukemic virus transformed fetal liver cell lines derived from (C57BL/6 x BALB/c) F1 mice was measured. The specific developmental stage of each line was determined using Southern blot analysis to ascertain their rearrangement status at the immunoglobulin heavy chain locus (DJ/DJ, VDJ/DJ or VDJ/VDJ). It was observed that DNA from DJ/DJ lines gave many more 'subhaploid' bands hybridizing with JH than did DNA from VDJ/VDJ lines. While the lack of appropriate substrate (VDJ/VDJ lines have exhausted the normal IgH substrate) contributes to the decrease in 'subhaploid bands', this result indicates that the rate of ongoing immunoglobulin heavy chain gene rearrangement was higher in the DJ/DJ lines. However, when the lines were examined using the assay developed by Hesse et al. (4) to measure VDJ recombinase activity, it was found that although all lines had recombinase activity, the DJ/DJ lines had four times more VDJ recombinase activity than did the VDJ/VDJ lines.

Abelson murine leukemia virus↗
PubMed1990

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein.

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.

Animals↗
PubMed1990

Thymosin beta 4 is expressed in ROS 17/2.8 osteosarcoma cells in a regulated manner.

The differential expression of mRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype. Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells. Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed. Steady state mRNA levels were severalfold higher in ROS 17/2.8 cells exhibiting an osteoblast-like phenotype, compared with the less osteoblast-like ROS 25/1. Thymosin beta 4 transcripts were also detected in rat UMR 106 osteosarcoma cells and in intact neonatal and fetal rat calvaria. Sequence analysis of the cDNA indicated that thymosin beta 4 transcripts may arise by processing at a more distal polyadenylation signal. Treatment of ROS 17/2.8 cells with dexamethasone increased, while addition of 1,25-dihydroxyvitamin D3 decreased thymosin beta 4 mRNA. The phenotype-dependent expression in the ROS cells and the response to steroid hormone suggest that thymosin beta 4 expression contributes to the osteoblast phenotype.

Amino Acid Sequence↗
PubMed1990

Characterisation of the osteoblastic phenotype by the use of differential hybridization.

To identify genes active in cells of the osteoblast lineage we have begun to characterise the phenotype of the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. We have used the method of differential hybridization to identify cDNA clones encoding mRNA species which are expressed in ROS 17/2.8 cells but not in a non-osteoblast-like osteosarcoma cell line (ROS 25/1). We have identified a number of gene products exhibiting this pattern of expression.

Animals↗
PubMed1990

VH gene repertoire.

In this review, we have assembled some results on VH gene usage by mouse and human. We conclude that there is an early bias in usage of certain VH genes in both mouse and human. This biased usage has a strain dependent component as evidenced by its continued presence in the adult repertoire of some mouse strains, notably BALB/c, and not in others. The reason for the fetal bias is uncertain. However, the finding that the VH gene segments used in the human fetal repertoire are similar in sequence but not in chromosomal position to those expressed in the mouse fetal-repertoire leads us to suggest that the bias is not due to chromosomal location but rather may be reflecting the functioning of these gene products early in ontogeny.

Animals↗
PubMed1987

Parathyroid hormone stimulation of mitosis in rat thymic lymphocytes is independent of cyclic AMP.

The in vitro mitotic response of rat thymic lymphocytes to hPTH(1-34), hPTH (1-38), and 8,18 Nle hPTH(1-34) exhibits a dependency upon extracellular calcium. Removal of extracellular calcium or the addition of Verapamil (5 micrograms/ml) or trifluoroperazine (10 microM) abrogated the mitotic response. Mitogenic concentrations of 8,18 Nle hPTH(1-34) increased calcium 45 uptake from 4.49 +/- 0.25 to 8.23 +/- 0.75 pMol/10(6) cells/min. The intracellular calcium concentration, measured by Quin 2 fluorescence, also increased after addition of 8,18 Nle hPTH(1-34). Parathyroid hormone-induced activation could not be demonstrated in an otherwise responsive thymocyte membrane adenylate cyclase. In intact cells mitogenic levels of 8,18 Nle hPTH(1-34) decreased intracellular cyclic AMP content. This response was blocked by both 3-isobutyl 1-methyl xanthine and trifluoroperazine, and may indicate activation of calcium-dependent phosphodiesterase. We conclude that PTH stimulates thymic lymphocyte proliferation independently of cyclic AMP, and that changes in cellular calcium homeostasis are intimately involved in the action of PTH. In all of the assays employed, the hitherto antagonistic analogue 8,18 Nle 34 Tyr bPTH(3-34)amide proved to be an agonist. We postulate that the receptor utilized for this PTH action may not exhibit classical PTH structure-activity specificities.

Animals↗
PubMed1985

Immunoreactive parathyroid hormone is present in subretinal fluids of the human eye.

(44-68) human Parathyroid hormone (hPTH) was studied in subretinal fluids (SRF) of 20 human eyes with rhegmatogenous retinal detachments. A (44-68) hPTH carboxyl mid-regional radioimmunoassay was used. Depending on the extent and the duration of the retinal detachments, (44-68) hPTH ranged between normal serum levels (ca. 100-300 pg/ml) up to excessive amounts of 4000 pg/ml with longstanding disease and proliferative vitreoretinopathy. Aqueous humors (AH) of one patient with bilateral iridocyclitis and complicated cataract ranged at 90-132 pg/ml (44-68) hPTH, whereas a control population undergoing surgery for senile cataracts had no detectable (44-68) hPTH in AH. The AH of one diseased eye of heterochromia complicata Fuchs showed excessive amounts of more than 4000 pg/ml (44-68) hPTH.

Adult↗
PubMed1985

A naturally occurring polyclonal B cell activator of normal and autoantibody responses.

Previous studies from this laboratory have demonstrated the presence of a suppressor molecule in B/W spleen cultures. Now we present evidence suggesting the existence of a polyclonal B cell activator (sPBA) spontaneously released by B/W splenocytes. Large sPBA (120,000 to 160,000 daltons) was separable from splenic inhibitory factor(s) (less than 10,000 daltons) by ultrafiltration. Reconstitution experiments demonstrated these two molecules had antagonistic effects on splenocyte mitotic events. sPBA is directly mitogenic for T cell-depleted, B cell-enriched cultures. Furthermore, sPBA augments antibody responses in the presence of antigen, or stimulates antibody responses in the absence of antigen. sPBA appears to stimulate IgG as well as IgM antibody production. sPBA stimulates significant in vivo anti-ssDNA responses in preautoimmune B/W but not DBA mice. Physiochemical studies suggest sPBA is a DNA protein complex. sPBA activity is completely lost by heating to 56 degrees C for 30 min. sPBA activity is precipitated at pH 7.8 to 8.0. Furthermore, sPBA preparations do not display significant endotoxin levels or IL 1 and IL 2 activity.

Adsorption↗