Effects of differentiated membranes on the developmental program of the cellular slime mold.

In the preceding paper isolated aggregation phase membranes (prepared from Dictyostelium discoideum cells which had proceeded through 12–14 hr of the developmental cycle) were found to be capable of preventing the aggregation and subsequent morphological development of vegetative cells when mixed with these and plated under normal conditions for slime mold development. In this paper we have extended the investigations on the nature of this interaction by monitoring the display of several developmentally controlled enzymes. It appears that exogenously applied aggregation phase membrane preparations are capable of influencing biochemical events inside D. discoideum cells through their interaction with the cell surface. This interaction leads to the induction or accumulation of some developmentally controlled enzymes, as well as the repression or excretion of others. The results suggest that the formation and maintenance of correct cell-cell contacts during normal development may be of crucial importance. They also show that changes in the specific activity of some developmentally controlled enzymes may in certain conditions be wholly divorced from both morphogenesis and the normal sequence of induction.     

Inhibition of the development of Dictyostelium discoideum by isolated plasma membranes

The life cycle of Dictyostelium discoideum can be divided into two mutually exclusive phases: growth and development. A distinguishing characteristic of the two phases is the absence of intercellular communication during vegative growth, and the many forms of such interaction during development. We have investigated the role of the cell surface membrane during the aggregation and development of this organism. We have asked the question: Are there molecules on the cell surface which are necessary for aggregation, and if so, can they be isolated in a biologically active membrane preparation? Further, when do these molecules appear during normal development, and does the interaction between two neighboring cell surfaces signal the cell or affect their subsequent development in any way? We have been able to isolate a partially purified plasma membrane fraction which is capable of specifically blocking the aggregation of other cells. Additional characterization of this preparation suggests that isolated aggregation phase membranes display a new, or newly exposed, heat-stable component which is capable of interacting with vegetative cells in such a way as to halt development.     

The ability of nonspecific T-cell stimulators to induce helper-cell-dependent increases in either polyclonal or isotype-restricted Ig production in vivo

In order to delineate the various roles of T cells in B-cell activation, mice were exposed to a variety of specific or nonspecific T-cell stimuli including mitogens, e.g., concanavalin A, adjuvants, e.g., complete Freund's adjuvant, and colchicine plus nonimmunogenic doses of antigen, anti-lymphocyte serum, and pathogens and their spleens analyzed for total class-specific immunoglobulin-secreting cells as indicators of helper cell generation. The results demonstrate that, depending on the mode of stimulation, markedly different Ig-secreting cell response patterns were induced, differing with respect to their kinetics and the isotype induced. In contrast to polyclonal T-cell stimuli such as concanavalin A and 17X lethal malaria which induced increases in all classes of Ig-recruiting cells, injection of many T-cell-activating agents resulted in the selective production of IgG clones in particular IgG 1. Such findings are discussed in terms of the different mechanisms of T-cell help and provide further evidence for functional heterogeneity in the T-helper-cell pool.     

Proteins in cerebrospinal fluid and plasma of fetal rats during development

Rat mammary epithelial tumor cells from the line Rama 25 can grow into three-dimensional, multicellular structures when cultured on floating collagen gels. These structures include branching tubules reminiscent of ducts in glands, and the production of the tubules is studied here as a model of glandular morphogenesis. The cell line contains cells of two types which can be cloned and grown separately. Tubules are formed by neither cell type alone, but by combinations. The behavior of the two cell types suggests a mechanism for the growth of glands.     

Analysis of 2',5'-oligoadenylates in cells and tissues

Complex mixtures of 2',5'-oligoadenylates are formed in cells and tissues under several different circumstances, and methods for analyzing such mixtures are reviewed. Separation is achieved by high-performance liquid chromatography and quantitation by competition-binding assays, using three different types of antibodies or a specific binding protein, or by functional assay, using preparations of an endonuclease specifically activated by some of the 2',5'-oligoadenylates. Representative results from three different biological systems are presented. The function of 2',5'-oligoadenylates as activators of intracellular RNA degradation is discussed, along with the possibility that these compounds may serve as signals for other intracellular regulatory processes.     

Butyrate-induced glycolipid biosynthesis in HeLa cells: properties of the induced sialyltransferase.

CMP-sialic acid:lactosylceramide sialyltransferase is induced in HeLa cells by butyrate which also causes the cells to undergo morphological changes including the extension of neurite-like processes. The activity of this enzyme is more than 20-fold higher in butyrate-treated cells than in cells grown without this short chain fatty acid. In vitro synthesis of hematoside from endogenous acceptors is also elevated in cells grown in the presence of butyrate. The levels of induced enzyme activity are influenced by the pH of the culture medium, being higher in more acidic cultures, but are not affected markedly by varying the cell density over a wide range. Detergent is required for in vitro sialyltransferase activity, and this activity is stimulated almost fivefold by cardiolipid. The optimum pH for in vitro activity is 6.0 and the apparent K_m value for lactosylceramide is 3.5 × 10^?5m. Although there are several sialyltransferase activities in HeLa cells, the induced enzyme is specific for lactosylceramide.     

An altered response of virally transformed 3T3 cells to ouabain

The effect of ouabain on K⁺ transport was examined in 3T3 and virally transformed 3T3 cells. A 10 min exposure to ouabain (10⁻³ M) produced approximately 40% inhibition of the unidirectional K⁺ influx in all cell lines. In 3T3 cells the response was not significantly altered by up to 70 min exposure to the drug. In contrast, the continued exposure of transformed cells to ouabain produced a time-dependent increase in the K⁺ influx. This increased influx was shown to be accompanied by an increase in the K⁺ efflux. The results suggest that, in transformed cells, ouabain produces both an inhibition of Na⁺-K⁺ exchange and a stimulation of K⁺-K⁺ exchange. The latter was shown to be more readily reversible than the former.     

Clustering of the DNA sequences complementary to repetitive nuclear RNA of HeLa cells.

We have studied the hybridization profile of heterogeneous nuclear RNA from HeLa cells across DNA density gradients, and found that components in the high molecular weight fraction of heterogeneous nuclear RNA of HeLa cells hybridize to discrete density fractions on the light and heavy sides of the DNA. The conditions used for hybridization in this work allowed the detection of only those components in the RNA complementary to reiterated sequences in the DNA. These sequences in HnRNA are known to include double-stranded regions, which can be isolated readily. The double-stranded RNA shows a pattern of hybridization across a DNA density gradient which is similar to that of total HnRNA. It is concluded that the repeated sequences in HnRNA are complementary to clusters of repeated sequences in the DNA.     

Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells.

Ribonucleoprotein particles containing either heterogeneous nuclear RNA or polyribosomal messenger RNA were isolated from growing HeLa cells in order to compare their respective protein components. The major obstacle to analysing the proteins bound to HeLa cell mRNA proved to be the cosedimentation of a large fraction of the mRNP² particles with ribosomal subunits following puromycin or EDTA disassembly of polyribosomes. This was circumvented by oligo(dT)-cellulose chromatography, in which essentially all of the ribosomal subunits passed through the column without retention, while approximately 80% of the pulse-labeled, poly(A)-containing mRNP became bound and could be eluted with formamide. Polyacrylamide gel electrophoresis of the non-bound fraction (ribosomal subunits) revealed polypeptides between 15,000 and 55,000 molecular weight, with no detectable components greater than 55,000. The oligo-(dT)-bound mRNP contained a much simpler protein complement, consisting of three major components having molecular weights of 120,000, 76,000 and 52,000.In the case of the nuclear ribonucleoprotein particles that contain heterogeneous nuclear RNA, oligo(dT)-cellulose chromatography revealed two classes of particles. The first contained 10 to 20% of the hnRNA, did not bind to oligo(dT)-cellulose in 0.25 m-NaCl, 10 mm-sodium phosphate buffer, pH 7.0 (4 °C), and contained primarily a single polypeptide component having an estimated molecular weight of 40,000 (“informofers”). A second population of hnRNP particles comprised approximately 80% of the hnRNA, displayed strong binding to oligo(dT)-cellulose at 0.25 m-NaCl, and contained a very complex population of proteins, having molecular weights between 40,000 and 180,000, the same as unfractionated hnRNP. The results indicate that, at the resolution of gel electrophoresis and at the sensitivity of Coomassie blue dye, the proteins bound to HeLa cell hnRNA are qualitatively distinct from those bound to polyribosomal mRNA and, in addition, that the hnRNP proteins are the more complex of the two. These results are discussed in relation to the possible nucleotide sequence elements in hnRNA and mRNA to which these specific proteins are bound.     

Kinetics of aggregation of αs1 -casein/ca2+ mixtures: charge and temperature effects

Time-dependent light-scattering studies have been made on mixtures of α_s1 -casein and Ca²⁺ at fixed temperature over a range of [Ca²⁺] and [α_s1 -casein], and also as functions of temperature- Measurements were also made of the extent of precipitate formation in the casein/Ca²⁺ mixtures, using centrifugation. The results are analysed in terms of a monomeroctamer equilibrium between calcium caseinate particles followed by a Smoluchowski aggregation in which only the octamers can participate. The equilibrium constant is dependent upon the charge on the protein/Ca²⁺ particles, and hence can be related to the extent of binding of Ca²⁺ to the α_s1 -casein. The Smoluchowski constant is likewise shown to be charge-dependent. The variation of the reaction rate with temperature can be ascribed solely to the changing charge of the α_s1 -casein/Ca²⁺ complex caused by changed binding of Ca²⁺ at different temperatures.     

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells. II. Effect of a soluble "costimulator" factor(s) in primary and secondary mixed leukocyte culture

The ability of a concanavalin A-stimulated spleen cell supernatant (“costimulator”) to overcome the effects of impaired CD and LD antigen presentation by metabolically inactivated stimulator spleen cells was examined in the primary and secondary cytolytic T lymphocyte (T_c) response. (i) Cells inactivated by ultraviolet irradiation or mild glutaraldehyde treatment, which were unable to stimulate primary cytolytic activity on their own, generated near maximal responses in the presence of costimulator. The 30-fold lower efficiency of splenic membrane fragments as antigen in primary MLC with the supernatant indicated that the damage to immunogenicity caused by membrane isolation was not equivalent to that caused by uv light and glutaraldehyde, as has previously been assumed. (ii) Comparison of the relative effects of antigen and costimulator demonstrated that costimulator played the dominant regulatory role in primary MLC, increasing sensitivity to suboptimal antigen doses 10- to 30-fold; neither antigen nor the supernatant appeared preferentially to control the strength of the secondary response. (iii) Metabolically inactivated adult and untreated neonatal spleen cells failed to release costimulator activity in response to concanavalin A. However, the ability of the neonatal cells to induce a primary cytolytic response suggested that costimulator production by the stimulator cells themselves is not essential for primary T_c activation, and supports the hypothesis that the lack of primary immunogenicity of inactivated spleen cells reflects their failure to induce costimulator production by the responder population.     

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA- protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.     

Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells.

Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.     

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of oligo(U)-containing heterogeneous nuclear RNA from control and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-treated HeLa cells

Most (54–79%) of the heterogeneous nuclear RNA (hnRNA) which contains oligo(U) sequences was specifically retained on columns of poly(A) Sepharose and separated from hnRNA which lacked oligo(U) sequences. The isolation of oligo(U)-containing hnRNA was maximized by treating the hnRNA with HCHO prior to chromatography. This permitted an initial characterization of the oligo(U)-containing hnRNA. Experiments suggest that HCHO denatured the hnRNA and rendered the oligo(U) sequences accessible to poly(A) Sepharose. In denatured hnRNA, the percentage of molecules which contained an oligo(U) sequence increased with the size of the hnRNA; 32–57% of the large hnRNA [8–13 kilobases (kb) long] contained an oligo(U) sequence while only 11–14% of the 2-kb-long hnRNA contained an oligo(U) sequence. The number of oligo(U) sequences per molecule was also measured in denatured hnRNA of varying length. While the largest hnRNA class analyzed (13 kb) was found to contain the highest percentage of oligo(U)-containing molecules (57%), the 8- and 2-kb-long hnRNA fractions contained a greater total number of oligo(U)-containing molecules. The percentage of hnRNA molecules which contained an oligo(U) sequence, the number of oligo(U) sequences per molecule, and the size of the oligo(U) sequence were similar in both control hnRNA and the fraction of hnRNA (~30%) which is resistant to inhibition by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole.     

Analysis of mixed leucocyte culture (MLC) reactive cells after in vitro priming. Changes in avidity of T cell receptors.

Mixed leucocyte cultures were examined for populations of T cells responding to secondary stimulation with the priming antigen. Two such populations are described, one of which is stimulated optimally by low, the other by high doses of antigen. Both cell populations are sensitive to anti brain θ serum and complement, but are physically separable by size and by adherence on macrophage monolayers. The anti-brain θ-sensitive population stimulated by low antigen doses consists of larger cells with immunoglobulin-moieties on their surfaces.