Early appearance in neural crest and crest-derived cells of an antigenic determinant present in avian neurons

A monoclonal antibody (“$\text{E}\text{C8}$”) against chicken dorsal root ganglion cells has been produced. The epitope (antigenic determinant) to which this antibody binds appears in neuronal cells—of both the peripheral and central nervous systems—and in a limited number of nonneuronal cell types in avian embryos. The epitope is intracellular and is probably part of a protein as judged by its susceptibility to proteases. This epitope appears very early in neuronal development. It may be detected in brain, spinal cord, and ventral root nerve fibers of Hamburger-Hamilton stage 16 chicken embryos (51–56 hr of incubation). At this same age, $\text{E}\text{C8}\text{-}\text{immunoreactive}$ cells can be found in the neural crest migratory space between the neural tube and the somite about a day before dorsal root ganglia begin to coalesce. Since some cultured neural crest cells (but not somitic mesenchymal cells) also express this epitope, we propose that the $\text{E}\text{C8}$ monoclonal antibody identifies an early differentiating subpopulation of neural crest cells which express this putative neuronal trait soon after the time of cessation of migration in vivo.     

Quantitative changes in the expression of histone H1 and H2B subtypes and their relationship to the differentiation of mouse embryonal carcinoma cells

The pattern of histones from several mouse embryonal carcinoma cell (ECC) lines, differentiated cell lines, and adult organs was analyzed using acid-urea gels containing Triton X-100 and long SDS-gel electrophoresis. All cell lines had comparable histone types except for a unique H2B-like component that was found only in the ECC line PCC4. The mouse histone H1 has four different subtypes (H1a, H1b, H1c, and H1d), as resolved in SDS-gel electrophoresis. The expression of the four subtypes was shown to be cell line specific. Subtypes H1a and H1d are present in approximately the same relative amounts in all cell lines investigated. Subtype H1b is found in higher relative amounts than subtype H1c in ECC lines and testis. The ratio of H1b and H1c is reversed in differentiated cell lines and in kidney, white blood cells, liver and spleen. All four subtypes of H1 are phosphorylated although to a different extent in different cell lines. In ECC lines, subtypes H1b and especially H1d incorporate most of a ³²P label, whereas H1c is predominately phosphorylated in differentiated parietal endoderm cell lines. These data indicate that H1 subtypes differ depending on the stage of cell differentiation. Difference in ratio between H1 subtypes and in phosphorylation might influence the chromatin configuration and thus gene expression in these cells.     

A glycoprotein involved in aggregation of D. discoideum is distributed on the cell surface in a nonrandom fashion favoring cell junctions

We studied the distribution on the cell surface of a glycoprotein (gp150) involved in the aggregation process of Dictyostelium discoideum. Using immunohemocyanin labeling of intact aggregates and visualization by scanning electron microscopy (SEM), we found a distribution gradient of gp150 wherein the concentration was enriched at or near sites of cell contact. When the distribution of gp150 on the cell surface was examined with immunoferritin and transmission electron microscopy (TEM), we found that gp150 was closely associated with the plasma membrane.     

Persistence of exogenous, inserted ganglioside GM1 on the cell surface of cultured cells

Ganglioside GM₁, which can insert spontaneously into the membrane of intact cells, has been measured after insertion into transformed fibroblasts by cholera toxin (choleragen) binding, for which ganglioside GM₁ is the natural receptor. Choleragen binding is not altered in starved, quiescent cells over a four-day period. Dividing cells show decreased binding in proportion to cell division. Thus, neither dividing nor quiescent cells appear to metabolize or otherwise degrade this membrane component.     

The determination of purine levels in human and mouse plasma

Variable levels of acetic anhydride have been recommended for addition to one of two reagents used in the glyoxylic acid method for the determination of tryptophan. For use of this reagent immediately after preparation it was shown that a minimum of 16% (v/v) of acetic anhydride should be included in the formulation to obtain near-maximum sensitivity. It was further demonstrated that reagent formulations with and without acetic anhydride changed with exposure to light. The observed changes are manifest as changes in the relative sensitivities of the assay. Several modifications are recommended to improve the sensitivity and stability of the acetic anhydride-containing reagent in this assay.     

Cell surface antigens of totipotent mouse teratocarcinoma cells grown in vivo: their relation to embryo, adult, and tumor antigens.

Cell surface antigens on mouse embryonal carcinoma (or teratocarcinoma) cells were investigated by means of a syngeneic antiserum prepared against small-size embryoid bodies from the ascites form of the OTT 6050 transplantable teratoma. These embryoid bodies consist of embryonal carcinoma cells which are usually covered by a yolk-sac-like epithelium. The choice of immunogen was based on the previous demonstration [Mintz, B., and Illmensee, K. (1975) Proc. Nat. Acad. Sci. USA72, 3585–3589] that embryonal carcinoma cells from this specific source are euploid, developmentally totipotent, and completely reversible to normalcy. In indirect immunofluorescence tests, anti-embryoid-body serum reacted with both cell types of the immunogen and with two in vitro lines of embryonal carcinoma cells. Absorption of antiserum with a pure yolk sac carcinoma derived from the epithelial component of the embryoid bodies enabled assessment of reactivity with the embryonal carcinoma component of the immunogen: The absorption revealed that some antigens recognized on the embryonal carcinoma cells were shared by the yolk sac epithelial cells but that some antigens were present only on the embryonal carcinoma cells. The antigens were not shared by sperm, which failed to fluoresce with unabsorbed antiserum and were ineffective when tested as absorbents of antiserum reactivity against embryoid body target cells. Unfertilized eggs also failed to fluoresce. Preimplantation embryos gave immunofluorescence evidence of some antigens shared with embryonal carcinoma cells (and some with yolk sac cells) during cleavage, and in the blastocyst on both inner cell mass and trophoblast. Postimplantation embryos were also antigen-positive (at least through Day 6) in immunofluorescence tests on endoderm as well as ectoderm cells. Absorption of the antiserum with various normal adult tissues showed substantial cross-reactivity, especially with ovary and testis. Other tumors were tested, but only hepatoma cells grown in vitro were reactive, thereby indicating lack of any general tumor recognition in the antiserum. The above results with syngeneic immunizations demonstrate that known totipotent teratocarcinoma cells possess surface molecules which, while not universal on normal cells or tumors, are shared with many other tissues, including developmentally plastic cells of early embryos, developmentally restricted cells of later embryos, and various adult tissues. Immunofluorescence tests of cleavage-stage (Day 2) embryos from matings of $\text{+}\text{t}{}^{12}\text{×}\text{+}\text{t}{}^{12}$ heterozygotes, yielding 40% mutant $\text{t}{}^{12}\text{t}{}^{12}$ homozygotes lethal on Day 3, were uniformly positive on all the embryos, including mutants and normals. Therefore, under these conditions, no evidence was adduced to support the hypothesis that surface components required for normal early development might be coded by the wild-type allele of t¹².     

A reappraisal of the relationship of phosphate-acceptor protein to parvalbumins.

A phosphate-acceptor protein thought to be related to parvalbumins was described from dogfish muscle (Blum, H.E. $\text{et al.}$, 1974 Proc. Nat. Acad. Sci. USA $\text{71}$, 2198–2202). Further examination of this material indicated that the fraction obtained contained mainly classical parvalbumin, contaminated by less than 5% of a true phosphate-acceptor protein of MW ca 18 000 that accompanies parvalbumin throughout its purification. No such acceptor could be found in hake subjected to identical purification procedures. It is concluded that a phosphate-acceptor protein such as found in dogfish muscle bears no relation to parvalbumins.     

The relationship between the osmotic fragility of human erythrocytes and cell age

Erythrocytes in a normal blood sample are hemolyzed over a range of hypotonic salt concentrations. In order to investigate the relationship between the distribution of osmotic fragilities and the distribution of cellular ages, the osmotic fragility has been compared with three indices of cellular age. The activity of glutamic-oxalacetic transaminase (GOT) and the percentage hemoglobin A_1C were measured in samples hemolyzed in different hypotonic salt concentrations. The osmotic fragility curve was also obtained for cells of different density separated by centrifugation. These experiments indicate that the fragility distribution is not an accurate reflection of the distribution of cellular ages. The mean fragility for older cells is higher than that of younger cells. However, cellular aging does not produce a gradual increase in osmotic fragility. Instead, it seems to produce changes which can both increase and decrease the fragility, resulting in a broader distribution of fragilities with some of the older cells actually less fragile than the younger ones.     

Selective recognition in erythrophagocytosis by mouse peritoneal macrophages. Isolation and purification of a possible self-marker from mouse erythrocyte membranes

The percental participation of exogenous cytidine in liver RNA synthesis was determined after application of ³H-cytidine to rats. The amount of exogenous cytidine was varied by a factor of 5 × 10⁵, between 0.000 02 and 10.0 μg/g rat. With the ³H-cytidine doses and specific activities most frequently reported in the literature, the percental participation of the exogenous precursor is only about 0.1%, with 99.9% of the cytidylic acid incorporated into RNA under these conditions being of endogenous origin.The results show that the upper limit of the tracer dose of exogenous cytidine is about 1.0 μg/g rat. Within this tracer region 1.8% of ³H-activity—and therefore 1.8% of the amount of exogenous cytidine—is incorporated into liver RNA. The dependence of the percental participation on the duration of the experiments is examined.It is shown that autoradiographic grain density and specific activity of RNA can only be regarded as direct measures for the rate of RNA synthesis in different cells and animals if the percental participation of exogenous cytidine in RNA synthesis is generally of equal value.Comparable situations exist in the incorporation of ³H-thymidine into DNA as shown by earlier experimental work.     

X chromosome expression during oogenesis in the mouse.

The activities of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) have been assayed in mouse oocytes at several stages of follicle development isolated from XX and XO female mice. Throughout the entire growth period the activity of G6PD was proportional to the number of X chromosomes present in the oocyte, whereas no difference in LDH activity was detected between XX and XO oocytes. It is concluded, therefore, that both X chromosomes are functional throughout oogenesis.     

Inhibition of cell aggregation by specific carbohydrates.

One approach to investigating the potential role of surface carbohydrates in mediating intercellular adhesion is to study cell reaggregation in the presence of defined concentrations of specific saccharides. Fifteen different exogenously added saccharides were tested for their effect on the reaggregation of 24 h sea urchin embryo cells (Strongylocentrotus purpuratus) dissociated by removal of divalent cations. Aliquots (0.2 ml) of cell suspension were rotated at 68 rpm, 17 °C, pH 8.0, with varying concentrations (0.5 × 1^?1?0.5 × 10^?5 M) of the sugars. Relative percents of cell aggregation were determined using an electronic particle counter assay. In all experiments cell viability using trypan blue was over 95.8%. Among the sugars tested, in 15 separate experiments, d-galactose and N-acetyl-d-galactosamine consistently inhibited aggregation to the greatest extent at early time points. d-Galactose, at all concentrations tested, at 10, 20, 30, 40, and 60 min rotation, showed mean decreases of aggregation over control values in the absence of sugar of 59.3, 53.6, 43.2, 35.0 and 36.4%, respectively. N-Acetyl-d-galactosamine also caused mean decreases in aggregation of 73.5, 54.5, 40.8, 42.2 and 45.6%, respectively. Each difference over the control is significant to the p value of less than 0.01. In three experiments, β-galactosidase substantially inhibited reaggregation of these cells. These results suggest that galactopyranosyl-like groups may be implicated in mediating adhesion of 24 h sea urchin embryo cells to each other.     

Amphetamine inhibition of alpha-bungarotoxin binding at the mouse neuromuscular junction.

Amphetamine inhibited neuromuscular transmission and prevented the irreversible blockade produced by α-bungarotoxin (α-BGT) in the isolated mouse phrenic nerve-diaphragm preparation. Similarly, amphetamine (1.35 × 10^?4 to 3 × 10^?3M) inhibited the binding of ¹²⁵I-α-BGT to mouse hemidiaphragms in a concentration-dependent manner; (+)-amphetamine was found to be twice as potent as its (-)-isomer with respect to inhibition of ¹²⁵I-α-BGT binding. It is suggested that amphetamine binds to the nicotinic, cholinergic receptor of skeletal muscle and may produce weakness and paralysis in amphetamine overdosage.     

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.     

Glucocorticoids down-regulate the number of 1, 25-dihydroxyvitamin D3 receptors in mouse intestine

This study examined the hypothesis that glucocorticoids might regulate intestinal receptors for 1,25(OH)₂vitamin D₃. Mice were treated with saline vehicle or dexamethasone in doses of 50–200 μg/100 gm body weight for 7 days. A dose-dependent fall in the number of receptors was found in dexamethasone treated animals (～30%); there was no change in the receptor affinity for 1,25(OH)₂D₃ (0.2 nM). The decline in receptor number required 5–7 days of dexamethasone treatment depending on dose. Adrenalectomy led to a 30% rise in receptor number which could be prevented by the 50 μg dose of dexamethasone. Receptor changes of equal magnitude were exhibited in vitamin D replete or deplete mice.     

Colchicine and cytochalasin B (CB) effects on random movement, spreading and adhesion of mouse macrophages

The role of microtubules and microfilaments in the control of random movement of mouse peritoneal macrophages was examined by studying the colchicine and cytochalasin B (CB) effects. Colchicine in the concentration range of 10^?8–10^?4 M enhances the random movement of these cells. Enhanced movement of macrophages is observed only at colchicine concentrations which cause inhibition of their spreading and adhesion. CB does not enhance random movement at any concentration; it inhibits movement at 10^?7 M and higher concentrations. Furthermore, though 10^?8 M CB alone has no effect on the migration of macrophages, when present along with colchicine, the two drugs act synergistically and enhance random movement to a greater extent than colchicine alone. These findings suggest a cooperative interaction between microtubules and microfilaments in the control of movement of macrophages. A model is presented to explain the nature of this interaction.     

The relationship between concentration of prostaglandin E and rates of cell replication

Phorbol esters act synergistically with phytohemagglutinin (PHA) and Concanavalin A to promote DNA synthesis in bovine lymphocytes. Studies of this response indicate that phorbol esters are useful tools for elucidating the cellular processes that are related to the action of mitogens.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

The relationship of alpha-L-iduronidase and Hurler corrective factor.

Hurler corrective factor (the protein that normalizes the faulty mucopolysaccharide catabolism of fibroblasts derived from Hurler patients) was previously identified as the enzyme α-l-iduronidase. However, not all α-l-iduronidase functions as Hurler corrective factor. The corrective and noncorrective forms of the human urinary enzyme have been separated from each other by chromatography on Sepharose-bound heparin. They have similar catalytic properties, some difference in ability to bind to the galactose-specific lectin of castor bean, and a significant difference in molecular weight (87,000 and 67,000 for the corrective and noncorrective forms, respectively). Only the corrective form is efficiently internalized by Hurler fibroblasts.     

Effect of histamine and acetylcholine on hypophysial stalk plasma dopamine and peripheral plasma prolactin levels.

To further define the role of dopamine in the regulation of prolactin secretion, we studied the effect on prolactin and hypothalamic dopamine secretion of histamine and acetylcholine (ACh) injected into the lateral ventricle of urethane anesthetized diestrus-1 rats. Histamine (10 μg) caused a 592% increase in plasma prolactin levels and a 26% decrease in stalk plasma dopamine levels. ACh (50 μg) caused a 2090% increase in plasma prolactin levels but no significant change in stalk plasma dopamine concentration.To determine if the 26% fall in stalk plasma dopamine following histamine administration could account for the 6-fold increase in plasma prolactin, we measured the effect on prolactin secretion of a similar decrease in administered dopamine. During an infusion of physiologic levels of dopamine, a 25% decrease in arterial plasma dopamine concentration resulted in only a 2-fold increase in prolactin secretion.The results of these experiments suggest that the effect of histamine on prolactin secretion may be mediated in part by decreased hypothalamic secretion of dopamine but that an additional hypothalamic hormone is probably involved. The stimulatory effect of ACh on prolactin secretion is not mediated by dopamine. These data are consistent with the growing evidence for the participation of multiple hypothalamic factors in the regulation of prolactin secretion.