Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

Stimulation by polydeoxyribonucleotide of histone phosphorylation by guanosine 3':5'-monophosphate-dependent protein kinase.

[³H]Dihydroalprenolol bound to a single population of high affinity sites in rat myocardial membranes when the concentration of the radioligand was below 5 nM. These sites displayed characteristics which would be expected of binding to the β-receptor. Kinetic- and Scatchard-derived dissociation constants were 0.6 and 2.0 nM, respectively. Binding was to a limited number of sites, 60 fmols/mg protein. Scatchard analysis using radioligand concentrations in excess of 5 nM resulted in concave upward plots suggestive of more than one population of binding sites. The lower affinity sites (labeled at high radioligand concentration) were non-stereospecific in nature and became a progressively larger fraction of “specific binding” as the concentration of dihydroalprenolol was increased above 5 nM.     

Correlations among the responses of suspensions of Dictyostelium Discoideum to pulses of 3′,5′-cyclic AMP: Transient turbidity decrease,the cyclic AMP signal,and variation in adenine mononucleotide levels

Aggregation-competent myxamoebae of the cellular slime mold Dictyostellium discoideum are known to exhibit two responses to extracellular pulses of 3′5′-cyclic AMP: an immediate chemotactic movement; and a delayed generation of intracellular cyclic AMP which is subsequently released into the medium. The mechanism of the latter, the so-called signalling response, may depend on alterations in intracellular metabolite levels and is the subject of this communication.Myxamoebae of the wild-type strain NC-4 of D. discoideum were suspended in an aerated, stirred 17 mM potassium phosphate buffer. pH 6.0, at a concentration of approx. 6 · 10^?7 cells/ml (8%, v/v) at 25°C and were pulsed with 1. 10^?8—1 · 10^?7 M cyclic AMP at 10–20-min intervals for periods of 3–5 h over incubation of 4–9 h. Suspensions were monitored continuously for transient turbidity decreases following the cyclic AMP pulses as an indication of the magnitude and duration of the cellular response to cyclic AMP. When the pattern of turbidity decrease indicated that a signalling response had developed, samples were withdrawn at 10–15-s intervals from the suspension, inactivated with perchloric acid, and analyzed for cyclic AMP, ATP, ADP, AMP, pyruvate, and glucose 6-phosphate. In separate experiments, steady-state oxygen tension was monitored along with turbidity to detect possible changes in respiratory rate.The following consistent patterns were observed after the added cyclic AMP pulse: a transient increase in the ADP level which reaches maximum between 0.7 and 1.7 min; transient decreases in ATP and pyruvate which concide with and approximately equal the magnitude of the increase in ADP; a later increase in glucose 6-phosphate which reaches maximum approx. 2 min after the ADP     

Multiple forms of cyclic nucleotide phosphodiesterases in normal and goitrous rat thyroid

The stoichiometry of H⁺ ejection coupled to electron flow from succinate to ferricyanide in the electron transport chain of mitochondria from Ehrlich ascites tumor and AS30-D hepatoma cells was determined. Values close to 4.0 for the $\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ ejection ratio were found in both cell lines, with either Ca²⁺ or K⁺ (+ valinomycin) as charge-compensating permeant cation. The 4 H⁺ ejected were compensated by outward movement of two negative charges to reduce 2 Fe(CN)6^3? to 2 Fe(CN)6^4?, and the uptake of two positive charges in the form of the permeant cation. Experiments on (a) omission of rotenone (b) the effect of antimycin A and (c) depletion of endogenous NAD(P)-linked substrates showed that no significant endogenous electron flow or H⁺ ejection occurred, thus eliminating possible overestimation of the H⁺/2e^? ratio from endogenous substrates. These data on mitochondria from two tumor cell lines are fully consistent with earlier measurements of the H⁺/O stoichiometry for succinate and NADH oxidation in tumor mitochondria and with the $\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ stoichiometry for site 2 in normal rat liver mitochondria.     

The correlation between plasminogen activator-stimulated DNA synthesis and cell morphology in 3T3 cells

The internalization of plasma membrane components labelled with ConA and peroxidase was investigated in monolayer cultures of rat liver cells. After the labelling procedure, the cells were reincubated with PBS free of both ConA and peroxidase for different time periods between 5 min and 3 h at 37 °C. Ligand-induced redistribution of ConA-binding sites finally resulted in a cap with uropod formation after 2–3 h of reincubation. Simultaneously with redistribution, the cell surface label disappeared through internalization, and a membrane recycling into the Golgi apparatus could be observed. Besides the lamellar Golgi apparatus which exhibited a labelling of the cisternae as a consequence of the membrane recycling, the hypertrophied unlabelled Golgi apparatus could be detected in the same cell. Furthermore, many vesicles formed by the hypertrophied Golgi apparatus were found between them and the plasma membrane and in close proximity to the plasma membrane. Fusion of the vesicles with the plasma membrane could be observed. These morphological findings indicate the possibility that the membrane internalization and the membrane recycling simultaneously effect an enhancement of membrane biogenesis and exocytosis, thus compensating for the membrane removal by internalization.     

Methionine biosynthesis in normal and transformed fibroblasts.

SV-40 transformed human fibroblasts show a growth requirement for methionine, whereas normal fibroblasts do not. Activities of the N⁵-methyltetrahydrofolate-homocysteine transmethylase and N^5–10-methylenetetrahydrofolate reductase in extracts of both cell lines are similar. These observations indicate that the absolute growth requirement for methionine observed in these transformed cells does not necessarily involve a deficiency in enzymes related to methionine synthesis.     

An in vitro model for induction of immunologic unresponsiveness to turkey gamma-globulin in primed mouse spleen cells, II. Antigen-specific suppressor cells.

Unresponsiveness induced to turkey γ-globulin (TGG) in cultures of TGG-primed spleen cells by incubation with high concentrations of soluble TGG (sTGG) was shown to involve a state of active suppression. Upon transfer to secondary cultures of primed spleen cells stimulated with an optimal dose of TGG-conjugated erythrocytes, such tolerant spleen cells were able to actively inhibit a secondary plaque-forming cell response to TGG in these cultures. Almost complete inhibition was observed with a tolerant cell to primed cell ratio of as low as 0.1. The suppression was antigen specific in that tolerant spleen cells which were suppressive for the secondary TGG response were unable to inhibit a primary response to sheep erythrocytes. T cells were shown to be required for the suppressor effect, in that (i) suppressor activity could be removed by complement-mediated lysis with an anti-Thy 1.2 antiserum and (ii) suppressor activity was retained in the effluent fraction after passage of suppressor spleen cells over a nylon wool column. Induction of the T-cell suppressor activity was found to be associated with a loss of T-cell helper activity within the TGG-pulsed cell population. The presence of adherent cells was not required for induction of suppressor activity. Furthermore, the suppressor effect was found to be resistant to 1000 R of γ irradiation.     

Brain cyclic AMP levels and the initiation of adult development in the Cecropia silkmoth.

A six-fold increase in the level of brain cyclic AMP is observed in chilled Cecropia pharate adults within 24 hr of transfer from the cold to room temperature. This increase is not observed in pupae chilled for a period insufficient to allow initiation of adult development, nor after injury to diapausing pupae. Other tissues show a variable and minor response during initiation. Injected dibutyryl cAMP will cause initiation in insufficiently chilled pupae, but not in dauer pupae. The possible relationship of this rise in cAMP to the process of initiation is discussed.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

Response of selected fetal organs to antineoplastic agents

This study was designed to quantitate the effects of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DIC) and 5-(3,3-bis(2-chlorethyl)-1-triazeno)-imidazole-4-carboxamide (BIC) on growth and selected components of rat fetal organs. Twelve-day pregnant rats were given single intraperitoneal injections of 600 mg/kg of DIC and 900 mg/kg of BIC and autopsied on day 21 of gestation. Fetal liver, brain, kidney, and placenta were removed, weighed, and assayed for total DNA, RNA, and protein. DIC significantly reduced weight, total DNA, RNA, and protein of all four fetal organs as compared to age-matched controls. The brain was most severely affected by this compound. BIC also significantly reduced weight, DNA, RNA, and protein of fetal brain, kidney, and placenta, but in fetal liver only weight and total protein were significantly depressed, while DNA and RNA remained essentially unchanged. The effect of BIC was maximal on the placenta.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

12-O-tetradecanoylphorbol-13-acetate stimulates release of arachidonic acid, prostaglandin E2 and prostaglandin F2 alpha from TPA nonproliferative variants of 3T3 cells

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, β-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.     

Glucocorticoid requirement for tyrosine aminotransferase induction by adenosine 3':5'-cyclic phosphate analogs in somatic cell hybrids.

The ability of adenosine 3′:5′-cyclic phosphate (cyclic AMP) analogs to induce l-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5; TAT) in a rat hepatoma (H35)-rat liver cell (BRL) hybrid (BF5) and a subclone which has lost 29 chromosomes (BF5-1-1) has been analyzed. Cyclic AMP analogs alone were unable to increase TAT activity in either hybrid cell line or in the “normal” liver cells despite three- to fivefold induction of this enzyme in the hepatoma parental cells. In contrast, dexamethasone by itself reproducibly increased TAT activity both in BF5-1-1 cells and in the parental H35 hepatoma cells. Pretreatment of the hybrid cells with dexamethasone revealed a synergistic increase in TAT activity when a cyclic AMP analog was added. From studies of the thermal stability and immunological inhibition of TAT activity, it is concluded that the low basal activity in BRL, BF5, and BF5-1-1 cells represents tyrosine transamination catalyzed by a different aminotransferase, whereas all the induced activity does represent bona fide TAT. The results suggest that functional TAT mRNA may not be present in significant quantities in the hybrid cells in the absence of adrenal steroids and that this could account for the inability of cyclic AMP analogs to exert their presumably translational effect on TAT synthesis.     

The regional metabolism of 5-hydroxytryptamine in mouse brain in vitro.

The relative rates of formation of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindoleacetic acid (5-HIAA) from exogenous 5-hydroxytryptamine, showed regional variations when examined in homogenates of seven separate areas of mouse brain. 5-HTOL production was highest in the cerebellum, and lowest in the corpus striatum, whereas the production of 5-HIAA was greatest in the hypothalamus. Addition of NADPH was shown to increase the formation of the alcohol catabolite in whole brain homogenates. The production of 5-HTOL decreased in the brain homogenates of mice which had previously been injected with phenytoin sodium or oxypertine, with the latter also causing a fall in overall 5-HT metabolism.     

Proline synthesis and redox regulation: Differential functions of pyrroline-5-carboxylate reductase in human lymphoblastoid cell lines

Pyrroline-5-carboxylate (P5C) reductase not only catalyzes the final step in proline biosynthesis but also mediates redox regulation. We examined these dual functions in human lymphoblastoid cell lines (REH, LHN₁₃) one of which (REH) is deficient in proline synthesis. In spite of the deficiency in proline synthesis in REH, the PC-mediated redox effect was relatively intact. The dissociation of these functions was due to qualitative as well as quantitative differences in the REH enzyme relative to its utilization of NADPH and NADH.     

An in vitro model for induction of immunological unresponsiveness to turkey gamma-globulin in primed mouse spleen cells. I. The secondary in vitro response and induction of unresponsiveness.

Spleen cells from mice previously immunized with turkey γ-globulin (TGG) were shown to give a vigorous secondary response in vitro when challenged in Mishell-Dutton cultures with TGG covalently coupled to pig erthrocytes (TGG-PRBC). However, 90–100% of the response could be abrogated by the incorporation of soluble TGG (sTGG) into the culture medium at concentrations greater than 1 mg/ml. Unresponsiveness, as measured by the absence of plaque-forming cells (PFC) in cultures receiving sTGG, was found to be antigen specific in that these cultures were still able to give normal PFC responses to sheep or burro erythrocytes. Spleen cells incubated with sTGG for short periods of time were shown to remain unresponsive after removal of sTGG from the culture and addition of TGG-PRBC. A 1-hr exposure period resulted in greater than 70% Unresponsiveness and a complete unresponsive state required only 8 hr of exposure. In contrast to the continued Unresponsiveness of sTGG-treated cells in vitro, spleen cells incubated with sTGG for 24 hr were fully responsive to an immunogenic challenge with alum-precipitated TGG when they were transferred into irradiated syngeneic mice. These data suggest that the readily induced unresponsive state in cultures of TGG primed cells may involve either a reversible antigen blockade of antigen-sensitive lymphocytes or a peripheral inhibition of reactive cells by suppressor lymphocytes.