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J Craig

Publications and source records attributed to J Craig.

At least 91 records · Page 5Linked to original sources
PubMed1989

The int-1 proto-oncogene is transcriptionally activated during neuroectodermal differentiation of P19 mouse embryonal carcinoma cells.

We have used the P19 line of mouse embryonal carcinoma (EC) cells to initiate studies on the putative role of the int-1 proto-oncogene during neuronal differentiation. P19 cells are induced to differentiate into neurons, astrocytes and fibroblast-like cells following exposure to retinoic acid (RA). Treatment of the same P19 cells with dimethyl sulfoxide (DMSO) leads to differentiation into mesodermal derivatives, including skeletal and cardiac muscle. Northern blot analysis showed that int-1 RNA was not present in the EC cells but appeared 48 h after RA exposure and could be detected for at least the next 8 days. No int-1 RNA was detected in P19 cells induced to differentiate with DMSO. Nuclear run-on transcription assays showed that int-1 expression in RA-treated P19 cells was induced at the transcriptional level. Immunofluorescent staining with an antibody directed against an int-1 peptide identified immunoreactive material in cytoplasmic granules of fibroblast-like cells in RA-treated P19 cultures. Thus the P19 cell line is a suitable experimental system to study int-1 gene expression and function during neuroectodermal development.

Animals↗
PubMed1988

Expression of the human cardiac actin gene in differentiating rat skeletal myoblasts.

The human cardiac-actin (CH-actin) gene was transfected into rat L6 skeletal myoblasts and stable transformants were isolated. The level of the CH-actin transcript varied between clones but changed little during the differentiation of myoblasts into multinucleate myotubes. Chimeric genes were constructed in which the CH-actin promoter, first non-coding exon (44 bp), and first intron (about 700 bp) were linked to the Herpes simplex virus thymidine kinase (tk) coding region. Clones of L6 cells transformed with these chimeric genes contained variable levels of actin-tk mRNA which changed little during differentiation. Thus, the activity of the CH-actin promoter appeared not to be up-regulated upon differentiation of myoblasts into myotubes. In clones of cells expressing the actin-tk mRNA, the TK protein was not detected in myoblasts but appeared in differentiating multinucleate myotubes. We interpret these results as suggesting developmentally regulated translation of the actin-tk mRNA. Since the first 44 nucleotides of the actin-tk mRNA were derived from the 5'-untranslated region of the CH-actin mRNA. These experiments suggest that translation of the actin-tk mRNA may be controlled by this region.

Actins↗
PubMed1988

AIMS ratings--repeatability.

In the present study, two raters, a psychologist and a nurse, each made five independent ratings of 30 different video-recorded patient-examinations. Having thus excluded patient fluctuation, individual-rater consistency and between-rater agreement over the 6 weeks of the study are examined. While between-rater agreement was apparently being maintained, mean AIMS scores steadily increased. In the hands of these raters, AIMS items 2 and 4 emerged as very reliable, while items 1, 6, and 7 showed high variability. Some patients appeared to be hard to rate. Differences between the study raters and the author JB highlight the issue: how reproducible is an AIMS rating?

Dyskinesia, Drug-Induced↗
PubMed1988

Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture.

We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high-affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.

Acetylcholine↗
PubMed1988

Improved separation of multi-locus hypervariable DNA restriction fragments by field inversion gel electrophoresis and fragment detection using a biotinylated probe.

DNA probes to multiple hypervariable human genomic loci are potentially highly informative as regards individual genome identification and parentage studies. However the resultant Southern blot patterns are generally highly complex and their interpretation difficult, with one important limitation being the extent of fragment separation and resolution. The separation of large DNA restriction fragments in agarose gels may be improved, in comparison to separations obtained by conventional electrophoresis, by the use of field inversion techniques. This is demonstrated by the analysis of the complex human Satellite III related DNA polymorphism. Detection of the Satellite III related restriction fragments is achieved either by using a [35S]-labelled probe (228S) or by using the same probe in a convenient non-isotopic form constructed by the photobiotin process. In addition, the probe 228S is useful for sexing the human genome, by the identification of a Y-specific restriction fragment.

Biotin↗
PubMed1987

The role of aggregation in embryonal carcinoma cell differentiation.

Cultures of the P19 line of embryonal carcinoma cells differentiate into various cell types including cardiac muscle when aggregated and exposed to medium containing 1% dimethylsulfoxide (DMSO). DMSO-treated aggregates became completely covered with an epithelial cell type 3 to 4 days following drug exposure. This epithelial cell was tentatively identified as primitive extraembryonic endoderm by its ultrastructural appearance and its possession of cytokeratin intermediate filaments. Muscle cells developed within the interior of DMSO-treated aggregates. They first became apparent 5 to 6 days after DMSO exposure and were characterized by the presence of striated muscle-specific myosin, immature myofibrils, and intercalated discs. We determined the proportion of cells developing into epithelium and muscle in aggregates of various sizes and showed that the proportion of epithelium was highest in small aggregates whereas muscle cells developed only in aggregates of relatively large size. The muscle was usually associated with necrotic areas which developed within the interior of large aggregates. Our results suggest that cardiac muscle differentiation in the aggregates requires both the DMSO-induced formation of an epithelial cell coat and one other condition which may be the proximity to necrotic areas.

Cell Aggregation↗
PubMed1987

Methotrexate (MTX) concentration in tumors following low-dose MTX.

Methotrexate (MTX) is a folate analog competitive with reduced folates for cellular transport and metabolism. Since the normal plasma folate concentration is only 10(-8) M, we tested the possibility that there may be a saturable uptake of MTX by proliferating tumor tissue at plasma MTX concentrations of only 10(-7) to 10(-6) M. Patients with advanced malignancies, refractory to accepted therapy, were given low-dose oral MTX (30-60 mg/m2 total dose in four to eight divided doses). Tumor tissue was biopsied 18-24 h after the last oral dose of MTX. The concentrations of MTX and its polyglutamated derivatives were measured in these samples. Forty-eight percent of the drug in the tumor samples was present as a polyglutamated derivative.

Administration, Oral↗
PubMed1987

Restricted replication of herpes simplex virus type 1 in murine embryonal carcinoma cells.

Herpes simplex virus type 1 (HSV-1) has a broad host range but the KOS strain of HSV-1 did not replicate efficiently in murine embryonal carcinoma (EC) cells. The yield of infectious HSV-1 from EC cells was 100- to 1000-fold lower than that from fibroblast cell lines of mouse, monkey or human origin. The thymidine kinase (TK) gene of HSV-1 is expressed early during the infectious cycle. The levels of TK mRNA and of TK activity in infected EC cells were only two- to threefold lower than levels from infected fibroblast cells. Infected EC cells supported replication of about half as much HSV-1 DNA as did fibroblast cells. The reduced yield of infectious virus was consistent with a paucity of virions in infected EC cells examined by electron microscopy, suggesting a major block late during the HSV-1 infectious cycle. We isolated a variant strain of HSV-1, called KOSEC, which replicated as efficiently in EC cells as in mouse fibroblasts. KOSEC infected EC and fibroblast cells, synthesized more TK mRNA, more TK enzyme, and more HSV-1 DNA than did the same cells infected with the KOS stain. Both HSV-1 strains induced similar levels of synthesis of gD, an early viral glycoprotein. By co-infection of EC cells with the KOS and KOSEC virus, both the elevated virus yield and the elevated TK synthesis seen in KOSEC-infected cells appeared to be recessive. Apparently a viral mutation that affects expression of some early viral functions can also overcome the EC cell restriction to HSV-1 replication.

Animals↗
PubMed1987

[Influence of cell-cell interaction upon differentiation of murine embryonal carcinoma cells].

Embryonal carcinoma (EC) cells are the pluripotent stem cells of teratocarcinoma. The aggregates of P19, a mouse EC cell line, undergo differentiation and rapidly lose its colony-forming ability in culture with retinoic acid (RA) or DMSO. Loss of plating efficiency is used to assess the rate of drug-induced differentiation. When exposed respectively to DMSO or RA, the isolated EC cell mutants (D3 and RAC65) did not differentiate but the gap junctions could be formed. In RA-treated co-aggregates of RAC65 and O1A1, each cell line gave responses independent of the presence of the other cell line. However, in DMSO-treated co-aggregates of P19 and D3, D3 cells remained undifferentiated and P19 cell differentiation was much poorer than that in the absence of D3 cells. The results suggest that cell-cell interaction be present in the early stage of DMSO-induced differentiation and absent in the early stage of RA-induced differentiation.

Animals↗
PubMed1985

Cell-cell interaction can influence drug-induced differentiation of murine embryonal carcinoma cells.

When cultured in the presence of either retinoic acid (RA) or dimethyl sulfoxide (DMSO), aggregates of the P19 line of mouse embryonal carcinoma (EC) cells differentiate and the spectrum of cell types formed depends on the drug dose. It is shown here the EC cells rapidly lose their colony-forming ability when cultured as aggregates in the presence of DMSO. This loss of plating efficiency (PE) also occurs rapidly following RA treatment. Loss of PE has been used as a quantitative procedure for assessing the rate of drug-induced differentiation. The relationship between drug dose and loss of PE is much steeper for DMSO than for RA, suggesting that these two drugs affect different stages of the differentiation decision-making apparatus. Mutant EC cell lines (D3 and RAC65) do not differentiate in the presence of drug-inducers (DMSO and RA, respectively). Neither differentiation-deficient mutant has an altered ability to form gap junctions. When D3 and P19 cells were mixed within the same DMSO-treated aggregates, the D3 cells remained undifferentiated and the P19 cells differentiated much less efficiently than if they were cultured in the absence of the D3 cells. When RAC65 and P19 cells were mixed in RA-treated aggregates, each cell responded to the drug as though the other were absent. Thus RA behaves as a cell-autonomous inducer of differentiation, whereas DMSO-induced differentiation seems to be mediated by interactions between neighboring cells.

Animals↗
PubMed1985

Commitment in a murine embryonal carcinoma cell line during differentiation induced by retinoic acid.

Murine embryonal carcinoma (EC) cells are induced to differentiate when cultured in the presence of retinoic acid (RA). Whereas the EC cells have a high plating efficiency, the differentiated cells have little or no colony-forming ability under the same conditions. We have assumed that the loss of colony-forming ability following exposure of EC cells to RA corresponds to the irreversible commitment of EC cells to differentiate. We found that uncommitted EC cells persist in RA-treated aggregates of EC cells and that the proportion of EC cells stabilizes at a level inversely related to the RA concentration. Both experimental evidence and mathematical modelling results are consistent with the interpretation that there is a dynamic equilibrium achieved by a balance between the processes of EC cell proliferation and differentiation. Since different cell types are induced by different RA concentrations, our results suggest that the commitment to differentiate is not related in any simple way to the developmental program which ensues.

Animals↗