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J W Littlefield

Publications and source records attributed to J W Littlefield.

At least 37 records · Page 2Linked to original sources
PubMed1983

Calcium-induced compaction and its inhibition in embryonal carcinoma cell aggregates.

H6 embryonal carcinoma cells form aggregates of cells in culture medium which contains 2 mM calcium. These aggregates are described as uncompacted, indicating that the individual cells of the aggregate are spherical and are in limited contact with each other. In contrast, compaction of the aggregate, induced by increasing the calcium concentration, results in a tight mass of cells flattened against one another and connected by intercellular junctions. At least 85-97% of the aggregates undergo compaction in 7 mM calcium and are subsequently decompacted if removed to 2 mM calcium. Since calcium ionophore A23187 does not induce compaction, extracellular rather than intracellular calcium seems to be the limiting factor. We have demonstrated that this calcium-induced morphogenetic change is sensitive to inhibition by agents which also prevent the calcium-dependent compaction of the 8-cell mouse embryo. The cytoskeletal-binding drugs tetracaine HCl, colcemid, vinblastine, colchicine, and cytochalasin B each inhibit compaction of H6 aggregates. Interference at surface molecule sites by exposure to the lectins wheat germ agglutinin or concanavalin A or by interruption of glycosylation with exposure to tunicamycin, or by reaction with anti-H6 Fab or anti-F9, also prevent compaction. Since the mouse embryo and embryonal carcinoma cells share certain processes which are involved in initiating and maintaining compaction, these processes and their subsequent roles in differentiation may be examined using embryonal carcinoma cell aggregates.

Animals↗
PubMed1982

Hypoxanthine-guanine phosphoribosyltransferase in human erythroid cells: posttranslational modification.

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) (HGPRT) of human red blood cells has been separated into three major isoenzymes, the relative quantities of which change as the cell ages. The predominant isoenzyme in the youngest circulating red blood cells, reticulocytes, has the same isoelectric point as the single enzyme of lymphoblasts. This lymphoblast-like enzyme is diminished in older red cells, and the major fraction of HGPRT activity is recovered in the two more acidic isoenzymes. The HGPRT enzymes of human lymphoblasts and red cells have been purified to apparent homogeneity, as evidenced by the criterion of subunit molecular weight in NaDodSO4 gels. The lymphoblast enzyme dissociates to a single subunit (alpha) upon isoelectric focusing in 8 M urea and is presumed to be a homo dimer (alpha alpha). The red cell isoenzymes dissociate to two subunits, one with the same isoelectric point as that in lymphoblasts (alpha) and one more negatively charged (alpha'). We infer that the three major red cell isoenzymes, I-III, correspond to enzyme species with none (alpha alpha), one (alpha alpha'), or both (alpha' alpha') subunits modified. Tryptic peptide maps of these iodo[2-14C]acetamide-labeled enzyme subunits indicate that the one red cell subunit (alpha) is identical with that in lymphoblasts and that the second subunit (alpha') differs from these in only one of the five cysteine-containing tryptic peptides. These results indicate that the HGPRT subunit is subject to at least one covalent and site-specific modification in human erythroid cells.

Erythrocyte Aging↗
PubMed1982

Rescue of terminally differentiating teratocarcinoma cells by fusion to undifferentiated parental cells.

A new clonal CBA mouse teratocarcinoma cell line called H6 grows rapidly without calcium in suspension culture. When attached to a gelatin-coated surface in the presence of calcium, differentiation to large flat cells, which secrete plasminogen activator, occurs after exposure to retinoic acid. Differentiation is terminal after about six cell divisions. H6 variants resistant to 6-thioguanine, 5-bromodeoxyuridine, ouabain, chloramphenicol, retinoic acid, or combinations thereof, have been isolated. Cell hybridizations were made between undifferentiated stem cells and the differentiated derivatives of H6. These experiments indicate that the undifferentiated cells can "rescue" cells in the initial stages of differentiation and that the signs of differentiation are soon lost in such hybrids. Hybridization without "rescue" may also occur, as suggested by small abortive colonies of large cells.

Cell Differentiation↗
PubMed1981

The organization of the dihydrofolate reductase gene of baby hamster kidney fibroblasts.

Using cloned DNA complementary to mouse dihydrofolate reductase (DHFR) mRNA, the organization of the hamster DHFR gene has been determined in two baby hamster kidney (BHK) cell lines, A5 and B1. A5 cells are highly methotrexate-resistant, containing 200-fold more copies of the DHFR gene than do the parental B1 cells. The DHFR gene has the same organization in A5 and B1 cells, suggesting that it has not been altered by the amplification process. The BHK DHFR gene spans a maximum of 10.7 kb and contains at least three introns. Thus the BHK DHFR gene is much smaller than the mouse DHFR gene, which has a minimum size of 42 kb and at least five introns. This striking size difference is probably due to much smaller introns in the BHK DHFR gene.

Animals↗
PubMed1981

The extraembryonic endodermal differentiation and polyploidization of embryonal carcinoma cells in vitro.

Embryonal carcinoma cells (EC cells) can form a wide variety of differentiated cell types and thus resemble the pluripotential stem cells of the normal embryo. Certain EC cell derivatives acquire the biochemical and morphological features of primitive endoderm and have been called 'END' or endodermlike cells. Although these have also been called 'giant' because of their large size, their nuclear DNA contents are not known. Since cell size often corresponds to DNA content and primitive endoderm becomes polyploid during the course of normal development, EC-derived endoderm has been studied cytophotometrically. Thus, EC- and embryo-derived endoderm were found to be similar in that both of these tissues undergo polyploidization. Moreover, the polyploid cells of either EC or embryonic origin do not appear to be terminal cell types, since they can occasionally enter renewed cell division in spite of their large size.

Animals↗
PubMed1980

Human epithelial cells cultured from urine: growth properties and keratin staining.

Epithelial cells can be cultured from the urine of newborn infants, providing a simple, noninvasive biopsy method. We established such cultures by standard techniques from 44% of uncontaminated specimens obtained from newborn infants up to 1 week of age. There was an average of three colonies per milliliter of urine. Many cultures accomplished 15 to 25 population doublings in as many as five subcultures and yielded total potential culture sizes of 10(4) to 6 x 10(8) cells. Plating efficiency was high at each passage. The cultures displayed two morphologically distinct epithelial cell types. Immunofluorescent staining of keratin fibers in most of these cells further identified them as epithelial.

Blood↗
PubMed1980

Isolation of diploid human lymphoblast mutants presumably homozygous for ouabain resistance.

From a mass culture of diploid human lymphoblasts we have isolated a subline resistant to 1 muM ouabain, and from this, a subline resistant to 10 muM ouabain. These sublines occurred spontaneously, but similar mutants were induced with 10-fold increased frequency by treatment with mutagens, in which case they could be selected as clones in soft agarose. Scatchard plot analyses of ouabain binding indicated that the subline resistant to 1 muM ouabain retained an average of 39% of the high-affinity ouabain receptors of the parental lymphoblast line, and the subline resistant to 10 muM ouabain retained an average of 8.4%. The ouabain binding site is known to be located in the Na(+),K(+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) molecule, an essential cell membrane enzyme that mediates ion transport. Studies on Na(+),K(+)-ATPase activity, using (86)Rb in the absence and presence of ouabain, indicated that our parental lymphoblasts contained one population of Na(+),K(+)-ATPase molecules highly sensitive to ouabain inhibition, the subline resistant to 10 muM ouabain contained one population relatively insensitive to ouabain, and the subline resistant to 1 muM ouabain contained both populations. Thus, the moderately resistant subline appears to be heterozygous for ouabain resistance, probably containing a structural mutation in the ouabain receptor region of the Na(+),K(+)-ATPase molecule in one of the two homologous loci for this enzyme, whereas the highly resistant subline derived from it appears to be homozygous, containing an additional mutation in the other Na(+),K(+)-ATPase locus.

Binding Sites↗
PubMed1979

Analysis of HGPRT- CRM+ human lymphoblast mutants.

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.

Cell Line↗
PubMed1977

Mutagen-induced diploid human lymphoblast variants containing altered hypoxanthine guanine phosphoribosyl transferase.

The human lymphoblast line MGL8 was treated with HAT and subsequently "mutagenized" with EMS (200 microgram/ml) to give 15% survival, and 6-thioguanine-resistant cells were selected by cloning in soft agarose containing the drug (1 microgram/ml). Eighteen sublines of independently derived resistant clones were isolated and studied in detail. One subline had a low residual HGPRT activity of about 1% of the parental cells. The HGPRT of this subline had a higher Km for PRPP, was more sensitive to heat, and was degraded faster by trypsin than the enzyme in extracts of MGL8 cells. This resistant subline and three others contained CRM levels of 1--38%, compared to the wild-type, so they probably represent true structural mutants of the HGPRT gene. All the variants maintained the karyotype of the parental line (46, XY, 6p-).

Cell Line↗