Karyotypic normalcy and quasi-normalcy of developmentally totipotent mouse teratocarcinoma cells

Malignant mouse teratocarcinoma cells are, in some cases, able to undergo normal, complete differentiation after injection into blastocysts. Thus far, only three lines—of unrelated origin—have been found (all in this laboratory) to be developmentally totipotent in blastocyst tests. The karyotypes of these lines, and their somatic- and germ-cell derivatives, were investigated by G-banding methods, as a possible clue to their developmental superiority. The first, OTT 6050 (129 strain), is an embryo-derived induced tumor maintained as an ascites transplant line. Its stem cells (from embryoid body “cores”) have 40 chromosomes in the modal class, which comprises two subclasses: one all normal and one with a metacentric chromosome (isochromosome-8). However, mosaic animals from injected blastocysts have only the normal subclass in their teratocarcinomaderived cells; all are of $\text{X}\text{Y}$ male sex chromosome type. Presence of the Y chromosome was verified after transmission through the germ line of two fertile mosaic males, in their F₁ male progeny. The second teratocarcinoma line, 72484-395 (LT strain), is a spontaneous ovarian solid tumor maintained by subcutaneous transplantation. Karyotypes of cells from the tumor, and also of teratocarcinoma-derived cells in mosaic animals, were normal and of $\text{X}\text{X}$ female sex chromosome type. Karyotypes of the F₁ progeny, from tumor-strain germ cells of a fertile mosaic female, were also normal. The third line, NG 2 (129 strain), is a mutant clonal in vitro line deficient in hypoxanthine phosphoribosyltransferase. It originated from an embryo-derived experimental tumor (OTT 5568) that was established in culture (PSA1 line); the culture was then mutagenized and selected for 6-thioguanine resistance. The NG 2 line proved to be quasi-normal, with only two karyotypic anomalies: trisomy of chromosome 6 and $\text{X}\text{O}$ female sex chromosome constitution. Thus, developmental totipotency in all three lines, including one maintained in vitro, is accompanied by karyotypic normalcy or near-normalcy. Other culture lines reported to be aneuploid have not yet given evidence of totipotency. Karyotypic normalcy may therefore have predictive value useful in choosing teratocarcinoma lines with relatively high developmental prospects. This is of importance in identifying those mutant lines that would be promising candidates for introduction, via blastocyst injection, of specific mutant genes into mice.     

Genetic alterations of ribonuclease-sensitive glycoprotein subunits of amino acid transport systems in Neurospora crassa conidia.

Neurospora crassa conidia have multiple and constitutive amino acid transport systems. Extraction by KCl releases amino acid-binding glycoproteins which have been purified by arginine affinity chromatography. Disappearance of certain fractions is coordinate with genetic lesions which reduce amino acid transport. Two such affinity fractions contain radioactivity when cells are grown on l-[¹⁴C]phenylalanine or on [¹⁴C]uridine, but not when cells are grown on [¹⁴C ]glucosamine. One purified arginine-binding fraction (B) contains 113 amino acid residues per minimum molecular weight. This glycoprotein also contains eight types of neutral sugar residues. No amino sugars were detected. Electrophoresis of crude extracts reveals five major Coomassie blue-staining species. The number of species is reduced, and the electrophoretic pattern is altered in extracts from transport-deficient strains. Tryptic “fingerprints” of these extracts indicate that mutations that reduce transport result in amino acid substitutions in the extractable glycoproteins. Nondialyzable material which absorbs light in the 260-nm region becomes dialyzable after digestion with RNase. Digestion of conidia with RNase reduces the amount of l-phenylalanine accumulated by the cells after 10 min of incubation with the amino acid.     

Water transport in a cluster of closely packed erythrocytes at subzero temperatures

A one-dimensional model has been developed to describe the kinetics of water transport in a cluster of closely packed cells. For the case of human red blood cells, the intracellular medium has been treated as an ideal, hydrated, nondilute multicomponent electrolyte solution. Results show that the volume flux of water out of the interior cells of the cluster lags behind that of the exterior cells. At any given temperature (or time), the amount of water retained within a cluster of closely packed cells of a given type exceeds (on an overall percentage basis) the amount of water retained within a single isolated cell of the same type. For a given cooling rate the probability of intracellular ice nucleation at any given temperature will therefore be greater for cells in the interior of a cluster, and the survival signature for a cell cluster should peak at a cooling rate which is less than the corresponding optimal value for a single, isolated cell. These results are consistent with experimental observations.     

Membrane Studies of Streptococcus pyogenes and its L-form growing in hypertonic and physiologically isotonic media. An electron spin resonance spectroscopy approach

Electron spin resonance spectroscopy (ESR) was used to compare the lipid organization, thermal stability and the physical state of the membrane of a human pathogen, Streptococcus pyogenes and its osmotically fragile L-form with this same L-form now adapted to grow under physiologically isotonic conditions (physiological L-form). Comparison of the hyperfine splittings of a derivative of 5-ketostearic acid spin label, I(1 2, 3), after incorporation into the membrane, revealed that the lipid chain rigidity of these membranes is in the order physiological L-form > osmotically fragile L-form > streptococcus. The signal intensity (of the center magnetic field line) versus temperature analysis showed two transitions for these membranes. The first with melting points of 45, 26 and 36 °C and second transition at 70, 63 and 60 °C for the physiological L-form, osmotically fragile L-form and streptococcal membranes, respectively. This same order of membrane lipid chain rigidity was seen from the cooperativities obtained for each of these systems from analysis based on the expression for an $\text{n-}\text{order}$ reaction. The I(12,3) and other probes with the paramagnetic group close to the methyl end of the molecule suggested that this difference in lipid chain rigidity between these organisms resides in the environment closer to the lipid head group region rather than in the hydrophobic lipid core. Another major finding was the binding of I(12, 3) at two or more different sites in each of the membranes examined. This change in lipid chain rigidity now provides an explanation to account for the survival of a previously osmotically fragile L-form in physiologically isotonic media by focusing on changes in the physical nature of its membrane. In so doing, it adds to and reinforces the speculation of the potential survival in vivo and involvement in pathogenesis of osmotically fragile aberrant forms of bacteria.     

Identification of external membrane proteins of the T lymphoblast cell line CCRF-CEM

The 70 membrane proteins of the T lymphoblast cell line CCRF-CEM were characterized by

1. 1. [³⁵S]methionine internal radiolabeling;

2. 2. [¹²⁵I]iodine labeling by a lactoperoxidase-mediated method;

3. 3. [³H]fucose internal labeling;

4. 4. binding to a lentil lectin adsorbant column;

5. 5. susceptibility to digestion with limited amounts of papain.

Of the three methods of radiolabeling membrane proteins, [³⁵S]methionine best displayed all proteins although some individual proteins were heavily iodinated or fucosylated. Thirty proteins were externally exposed as defined by susceptibility to lactoperoxidase-mediated radio-iodination and to digestion with minute amounts of papain. Thirtyfive proteins were bound to a lentil lectin absorbant column. p44 (HLA-A and -B antigens) were iodinated, fucosylated, susceptible to papain digestion and bound to the lectin column. β₂-Microglobulin was iodinated and bound to the lectin column. The identifications and functions of other membrane proteins were not known. In general, proteins of high molecular weight (100 000 to 250 000 D) were more heavily radio-iodinated and fucosylated than were proteins of lower molecular weights. p95 was the most heavily fucosylated protein, p110, which had been identified only on T lymphoblasts, was fucosylated and was iodinated. p65, which was found only on the T lymphoblast line CCRF-CEM and could represent a lymphocyte subpopulation-specific molecule, was iodinated and fucosylated. p15 and p18 were equally and densely labeled with [³⁵S]methionine but only p18 was fucosylated and it was heavily radio-iodinated. These experiments help to define the external membrane proteins of a T lymphoblast cell line in part for the selection of proteins for isolation in order to raise antisera for immunodiagnostic and functional studies.     

Purification of bovine liver microsomal NADH-cytochrome b5 reductase using affinity chromatography

Microsomal NADH-cytochrome $\text{b}{}_5$ reductase has been purified from bovine liver by an improved procedure which employs affinity chromatography on ADP-agarose in combination with anion exchange chromatography. The reductase was extracted from a 105,000 × $\text{g}$ microsomal pellet with Triton X-100. The overall purification from isolated microsomes was 98-fold and the yield was 10%. The preparation was nearly homogeneous on SDS-PAGE. This procedure requires less time and effort than previously described procedures. Partially purified cytochrome $\text{b}{}_5$ is also obtained.     

Stable heterogeneity of mitochondrial DNA in grande and petite strains of S. cerevisiae.

Several instances of mitochondrial DNA heterogeneity in grande and petite strains of Saccharomyces cerevisiae were examined. We have detected heterogeneity in the mtDNA from some of the progeny strains of a cross between two grande strains (D273-10B, MH41-7B) which differ in genome size and restriction cleavage pattern of their mtDNA. The progeny strains transmit restriction fragments characteristic of both parental strains from homologous regions of the mitochondrial genome, and this sequence heterogeneity is not eliminated by additional subcloning. Sequence diversity is more common in the mtDNA of petite than of grande strains of yeast. We have examined subclones of one petite strain to identify the origin of this variability. Many of the submolar restriction fragments persist in independent subclones of this petite after 15 and 30 cell divisions; some submolar fragments disappear, and some new fragments appear. We conclude that the observed sequence heterogeneity is due to molecular heterogeneity, i.e., to differences in the multiple copies of the petite mitochondrial genome, as well as to clonal heterogeneity. It is likely that tandem repeats on the same mtDNA molecule also differ, i.e., that there is intramolecular heterogeneity, and that this accounts for the stability of the heterogeneity. Continuing deletion is probably responsible for the appearance of “new” fragments in petite subclones.     

Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation

The position of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and γ-glutamyltransferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.     

Circadian periodicity of tissue glutathione and its relationship with lipid peroxidation in rats

Circadian fluctuations in tissue glutathione (GSH) concentrations and lipid peroxidation in male Sprague-Dawley rats were investigated. Blood and all the organs studied exhibited distinct circadian variation both in GSH concentrations and peroxidation of polyunsaturated fatty acids. There was a great variation among organs in the periodicity and amplitude of the fluctuations in GSH concentrations. Liver displayed the highest variation (approximately 50%) followed by stomach (approximately 37%), heart (approximately 25%) and kidney (approximately 19%). The changes in other organs were significant but of less magnitude. Implications of such variations and caution in interpretation of experimental results in response to the exposure of animals to xenobiotics are discussed.     

A secreted glycoprotein induced by estrogen in human breast cancer cell lines

Estrogen induces the synthesis of a glycoprotein of molecular weight 46,000 daltons in three estrogen receptor-positive breast cancer cell lines (MCF₇, ZR₇₅ and T47D), but not in an estrogen receptor-negative cell line (BT 20) or a nonmalignant cell line (HBL 100). The 46K protein, which accounts for 40% of ³⁵S-methionine incorporation into secreted proteins, is only induced by steroids able to interact with the estrogen receptor. The anti-estrogens tamoxifen and hydroxytamoxifen, which by themselves were inactive, suppressed the induction of this protein by estradiol. In MCF₇ cells, estradiol also induces three intracellular proteins which are resolved in two-dimensional electrophoresis. The induction of the 46K secreted protein(s) makes these cell lines excellent in vitro systems for studying the mechanism of estrogen and anti-estrogen action. This protein may also be a useful probe for studying the action of estrogen on breast cancer growth, and may be a useful marker for predicting the hormonal responsiveness of breast cancer in vivo.     

Accumulation of alpha-mannosidase-1 in Dictyostelium discoideum requires many developmentally essential genes

alpha-Mannosidase-1, one of the earliest known developmentally controlled gene products in the cellular slime mold Dictyostelium discoideum, accumulates intracellularly during both axenic growth and development. The accumulation of alpha-mannosidase-1 activity prematurely ceases in all of 125 randomly isolated aggregation-deficient mutants at discrete times in development resulting in significantly reduced levels of cellular enzyme activity. This suggests that, unlike other developmentally controlled enzymes in this organism, the continued accumulation of alpha-mannosidase-1 activity is controlled by a large number of genes essential for early development. alpha-Mannosidase-1 misregulation and the aggregation-deficient phenotype are caused by the same mutation since (1) morphological revertants exhibit a coreversion to both fruiting ability and wild-type alpha-mannosidase-1 accumulation and (2) normal enzyme accumulation depends on the ability to aggregate and ultimately fruit in a conditional aggregation-deficient mutant. This type of regulation does not appear to be due to differences in enzyme secretion or changes in the overall rate of total protein synthesis. Aggregation-deficient mutants continue to synthesize protein beyond the time in development at which alpha-mannosidase-1 accumulation ceases. Our studies indicate that most of the 50-125 genes required for aggregation in Dictyostelium are also required for the normal accumulation of alpha-mannosidase-1 activity.     

Separation and identification of pteroylpolyglutamates by polyacrylamide gel chromatography.

A simplified procedure for the determination of the glutamate chain lengths of labeled and endogenous tissue folate is described. Pteroylpoly-γ-glutamates in tissue extracts were reductively cleaved at the C,9N,10 bond to p-aminobenzoylpolyglutamates, which were converted to azo dyes by coupling their diazonium salts with naphthylethylene diamine. The azo dyes were well resolved, according to glutamate chain length, by gel chromatography on Bio-Gel P4. Unlabeled tissue folates were detected by the absorbance of their azo dye derivatives. The major endogenous pteroylpolyglutamate in rat liver, identified colorimetrically using 0,5 g tissue, was the pentaglutamate. The major labeled folates in Lactobacillus casei and Streptococcus faecalis, after incubating these bacteria with labeled folic acid, were identified as the octa- and tetraglutamates, respectively. Reductive cleavage of 10-formylfolate and 5.10-methenyltetrahydrofolate resulted in a mixture of N-substituted and unsubstituted p-amino-benzoylpolyglutamates. Methods are described for the complete cleavage of these formyl derivatives to unsubstituted p-aminobenzoylpolyglutamates.     

Isolation of phospholipase A2 from sheep erythrocyte membranes in the presence of detergents

Isolation of phospholipase A₂ (EC 3.1.1.4) from sheep erythrocyte membranes was carried out by a combination of (1) extraction of membranes at low ionic strength, (2) solubilization of extracted membranes with sodium dodecyl sulfate, (3) replacement of dodecyl sulfate with cholate by means of gel exclusion chromatography and (4) affinity chromatography on dialkyl-phosphatidylcholine-Sepharose in the presence of cholate. The phospholipase was prepared with good yield and purified to near homogeneity, as judged by sodium dodecyl sulfate gel electrophoresis. The protein is a minor component of the sheep erythrocyte membrane and has an apparent molecular weight of 18 500.     

δ-Aminolevulinic acid synthetase: Regulation of activity in various tissues of the aging rat

The basal and ethanol-induced activities of the rate-limiting enzyme of heme biosynthesis, δ-aminolevulinic acid (ALA) synthetase were measured in the liver, heart, kidney, and brain of young, adult, and aged Sprague-Dawley rats. When assayed in whole mitochondria derived from either fed or 24-h fasted animals, the basal levels of hepatic ALA synthetase activity decreased dramatically as a function of age. An equivalent decrease was seen in the ethanol-induced activity although the ratio of induced to basal activities did not change with age. In the heart, ALA synthetase activity also decreased significantly during aging. The activity was not induced by ethanol and was decreased markedly by fasting. By contrast, kidney ALA synthetase activity showed no age-related changes. The activity was unaffected by fasting and showed a variable induction response to ethanol. Brain ALA synthetase activity displayed a significant age-dependent decrease in its activity which was neither affected by fasting nor sensitive to induction by ethanol. The data presented are consistent with the hypothesis that ALA synthetase activity is subject to metabolic regulation. Further, they indicate that while the enzyme activity is regulated in a tissuespecific manner, a time-dependent decrease is a general feature of the aging animal.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. II. Metabolism and binding of histones.

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.     

An immunocytotoxicity assay for neural cells in monolayer cultures

The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell susptnsions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with 10- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.     

Carbonic anhydrase activity in developing sea urchin embryos

A 13-fold increase in carbonic anhydrase specific activity was found during the first 24 h in developing embryos of the sea urchin, Strongylocentrotus purpuratus. Carbonic anhydrase activity was sensitive to inhibition by 10⁻⁴ M acetazolamide. Roles for carbonic anhydrase activity in intracellular pH regulation and spicule formation are discussed.     

DNA repair in UV-irradiated heteroploid cells at different phases of the cell cycle

The variation of DNA repair activity during the cell cycle was studied by analysing the UV-stimulated DNA synthesis in cells synchronized in mitosis. This activity was detected both by autoradiography and by directly measuring the incorporation of tritiated thymidine in cells irradiated and incubated in the presence of hydroxyurea. Cells in all phases were found to be able to perform repair. However the activity appeared to be considerably lower in mitotic cells than in cell in other phases. Increasing values of repair capacity were observed in G1 cells, in mixed G2, S and M cells and in asynchronous cells. The relationship between these findings and data on survival rates in the same synchronized cells is discussed.     

Separation of primitive and definitive erythroid cells of the chick embryo

The primitive and definitive erythroid cells of the chick embryo are separated preparatively by means of velocity sedimentation at unit gravity in BSA gradients. Analyses of the hemoglobins contained by the fractionated cells show a segregation of different hemoglobins between the primitive and definitive cells. Studies of the incorporation of [³H]leucine show that the fractionated cells are normal with respect to their protein synthetic activities and that their relative rates of incorporation are markedly different.     

Reconstitution of calcium transporters from Azotobacter vinelandii membranes

Plasma membranes from Azotobacter vinelandii contain two Ca²⁺ transport activities: an electrophoretic uniporter and an electroneutral $\text{Ca}{}^{\mathrm{2+}}\text{2}\text{H}{}^{\mathrm{+}}$ exchanger (P. Zimniak and E. M. Barnes, Jr. J. Biol. Chem.255, 10,140 (1980)). Both activities were reconstituted by the freeze-thaw technique of M. Kasahara and P. C. Hinkle (J. Biol. Chem.252, 7384 (1977)) using phosphatidylcholine/phosphatidylethanolamine (1:1) at a lipid-to-protein ratio of 40. Reconstitution was evidenced both by expansion of the intravesicular volume accessible to Ca²⁺ and by transfer of the transport activities to vesicles with a buoyant density less than that of native membranes. The Ca²⁺ transporters, reconstituted into K⁺-filled proteoliposomes, retained their dependence on the membrane potential or ΔpH induced by the addition of valinomycin or nigericin, respectively. The kinetic parameters of the reconstituted activities were similar to those in native membranes, as was their sensitivity to inhibitors. The sensitivities of the electrophoretic Ca²⁺ transporter to ruthenium red, morpholinoethanesulfonate, and external K⁺ and of the $\text{Ca}{}^{\mathrm{2+}}\text{2}\text{H}{}^{\mathrm{+}}$ antiporter to Sr²⁺ and heat treatment were also retained by the reconstituted system.