Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

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.     

Change in kinetic property of M1-type pyruvate kinase from hyperbolic type to sigmoidal type by limited proteolysis with an acid fraction from rabbit liver

An acid extract of rabbit liver contained M1-type pyruvate kinase inactivating activity, and was separated to three fractions. The optimal inactivation of the enzyme with Fraction II (Mr 42,000) was observed at pH 5.5, and this inactivation was completely prevented by leupeptin and antipain, but not by pepstatin. With Fraction III (Mr 22,000), on the other hand, optimal inactivation of the enzyme was observed at pH 8-9, and was not prevented by these inhibitors. The kinetic properties, with phosphoenolpyruvate, of the enzyme were changed from hyperbolic type to sigmoidal type by the limited proteolysis with Fractions II and III. The subunit molecular weight of the enzyme (57,300) was decreased to 55,800 via 56,400 in the former case and to 56,400 in the latter case.     

Activation/suppression of the immune response in vitro by antigen and dextran sulfate: 2. The role of T-cell subsets determined by cell separation and partition analysis

Culture of spleen cells with dextran sulfate (DxS) and antigen at various different cell densities revealed a T-cell-dependent regulatory pathway not observed in conventional culture. This finding can be explained by the frequent presence in the cultures of a helper cell and the less frequent presence of a suppressor cell, both activated by antigen and DxS. The classic, radioresistant, antigen-specific, helper T cell was not regulated by this newly revealed pathway. The highly frequent, DxS-dependent helper T cell is Lyt-1⁺2^?. The suppressive effect is mediated by a Lyt-1⁺2⁺ population consisting of helpers and latent suppressors that can be made active by DxS or Lyt-1⁺ cells. The specificity of the Lyt-1⁺ helper cells was not established, but the high frequency observed implies a nonspecific mechanism. The specificity of the suppressor effect was not determined by these experiments. This regulatory mechanism is similar to the phenomena exhibited by polyclonally activated T-cell populations.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.     

Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli.

The tyrT gene codes for one of the tyrosirie tRNA species. Using the Casadabatn (1976a) technique, strains of Escherichia coli were isolated in which the lac structural genes are fused to the promoter of the tyrT gene. This procedure involved obtaining a number of insertions of phage Mu DNA in the tyrT gene, lysogenizing the Mu insertion strains with a λplac-Mu hybrid phage, and selecting Lac⁺ derivatives of such lysogens. In a number of Lac⁺ strains thus obtained, the synthesis of β-galactosidase, the product of the lacZ gene, is regulated in a similar fashion to the synthesis of stable RNA. The fusion strains were shown directly to be tyrT-lac fusions by demonstrating that a Mu insertion in the tyrT gene when genetically recombined into the presumed fusion, inactivates the expression of the lac genes. This result shows that tyrT gene sequences are fused to and control the expression of the lac genes in these strains. This is the first report in which genes which code for proteins have been fused to a stable RNA gene in vivo.     

Activation of macrophage adenylate cyclase by stimulants of the oxidative burst and by arachidonic acid--two distinct mechanisms

The effect of agents stimulating the oxidative burst (OB) in oil-elicited guinea pig peritoneal macrophages (MPs) on cyclic adenosine 3′,5′-monophosphate (cAMP) levels was examined. We found that: (i) Phorbol myristate acetate (PMA), the Ca²⁺ ionophore A23187, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and opsonized zymosan, elevated cAMP levels two- to fivefold; (ii) the biologically inactive PMA analog, 4-O-methyl-PMA, was proportionally less effective than PMA in stimulating cAMP accumulation; (iii) increased levels of cAMP were evident after 10 min of incubation with the stimulants, in the presence of the phosphodiesterase inhibitor 3-isobutyl methylxanthine (IBX); (iv) basal cAMP levels in MPs increased proportionally with the extracellular Ca²⁺ concentration; (v) the cAMP-elevating effect of all stimulants (with the exception of A23187) was more pronounced in low Ca²⁺ media, associated with lower basal cAMP levels. A23187 did not elevate cAMP levels in the absence of extracellular Ca²⁺; (vi) short-term incubation of MPs with arachidonic acid and with the arachidonic acid precursor, linoleic acid, induced an increase in the level of cAMP; (vii) the elevations in cAMP levels induced by OB stimulants were enhanced, not blocked, by mepacrine, 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin or aspirin, demonstrating that prostaglandin (PG) synthesis was not involved; (viii) the cAMP-elevating effect of arachidonic and linoleic acids was blocked by ETYA and indomethacin, indicating that it was mediated by PGs. The mechanism by which OB stimulants elevate cAMP levels could not be determined but changes in the cellular level of Ca²⁺ seem to play a pivotal role.     

Activation of adenylate cyclase by beta-adrenergic receptors: Investigation of rate limiting steps by simultaneous assay of high affinity agonist binding and GDP release

We report the development and application of a novel assay for high affinity binding of the agonist [³H]hydrozybenzyl-isoproterenol simultaneously with the agonist-promoted release of membrane bound [³²P] GDP in the frog erythrocyte beta-adrenergic receptor system. We find that under various assay conditions both events occur with the same rate, ranging from 0.05 to 0.5 min^?1. Addition of the non-hydrolyzable guanine nucleotide, guanylyl-imidodiphosphate simultaneously increases the rate of high affinity agonist binding and agonist promoted GDP release. In addition, the guanine nucleotide analog decreases the steady state level of high affinity agonist binding and increases the steady state level of agonist promoted GDP release with comparable potencies of 0.5 μM and 0.1 μM, respectively. The decrement in the steady state level of high affinity agonist binding (180 fmol/mg protein) due to the guanine nucleotide analog is in the same range as the reciprocal increment in the extent of agonist-induced [³²P] GDP release (180 fmol/mg protein). The concommittant activation of adenylate cyclase, by submaximal concentrations of the agonist [³H]hydroxybenzylisoproterenol and guanylylimido-diphosphate under similar assay conditions proceeds with the same rate as for the two other measured functions of the system, i.e. ³H-agonist binding and agonist-promoted [³²P] GDP release. This represents the first attempt at comparing the time course of adenylate cyclase activation with that of agonist binding and GDP release under similar assay conditions. The results indicate that GDP is not released prior to but rather coincident with formation of the complex of the hormone receptor with the regulatory protein and that enzyme activation proceeds with the same time course as agonist binds to the receptor. It is concluded that both high affinity agonist binding and GDP release represent integral aspects of the rate limiting step in the enzyme activation mechanism.     

Purification of amyloglucosidase

Amyloglucosidase (glucoamylase; EC 3.2.1.3) has been purified from the culture filtrates of Aspergillus candidus Link var. aureus using hydrophobic interaction chromatography and DEAE-cellulose treatment. The enzyme thus obtained has a specific activity of 329 units/mg protein with the overall recoveries between 70 and 75%. The process appears to be of industrial promise.     

Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy

The effect of specific DNA binding of the cAMP . cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP . cAMP receptor protein complex induces a kink of 30 to 45 degrees in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.     

Absence of hydrocortisone from cytoplasmic hormone-protein complexes formed in vivo after administration of biologically active doses of [3H]hydrocortisone

After administration of [³H]hydrocortisone to adrenalectomized rats, hormone-protein complexes were isolated from liver cytosol by DEAE-cellulose chromatography. After application of biologically active and inactive doses of hydrocortisone five binding components were detected eluting at the same salt concentrations as the hormone-protein complexes observed after incubation of cytosol with [³H]hydrocortisone in vitro. The isolated hormone-protein fractions were acidified and extracted with ethylacetate and the steroids were analyzed by thin-layer chromatography. No significant amount of hydrocortisone could be detected in any of the complexes formed in vivo 5–60 min after administration of biologically active doses of hydrocortisone. 3ξ,11β,17α,20ξ, 21-Pentahydroxypregnane, steroidal carboxy acids, glucuronides and a very polar conjugate of hydrocortisone were found in the different fractions. After an in vivo dose of hydrocortisone of about 1/5000th of the minimal dose required for enzyme induction, hydrocortisone could be found in all cytoplasmic hormone-protein complexes formed. In contrast to the cytoplasmic hormone-protein complexes, hydrocortisone could be readily demonstrated in nuclei isolated after the administration of biologically active doses of hormone, although acid metabolites were found to represent the main part of the radioactive compounds present in the nuclei. These acid metabolites were located in ronide on the basis of its chromatographic behavior. The biological significance of this conjugate of hydrocortisone as well as that of the extremely polar conjugate found in fraction DE-3 cannot be understood on the basis of the published data pertaining to biological functions and metabolism of glucocorticosteroids.Our finding that no ‘classical’ glucocorticosteroid receptor can be detected in rat liver cytosol raises again the question of the way in which hydrocortisone and its active metabolites enter the nucleus. On the basis of the published data, the possibility cannot be ruled out that glucocorticosteroids are transported via the endoplasmic reticulum. A transport by this way has been inferred for the uptake of sodium and inulin by liver nuclei [40–42].     

Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes,using a rapid immunological procedure for the isolation of plasma membrane

The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 μM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a ‘high K_m’ form, whereas the particulate activity had both ‘high K_m’ and ‘low K_m’ components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 μM substrate concentration, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both ‘high K_m’ and ‘low K_m’ phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at μM levels, in the liver. The ‘low K_m’ enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellylar membranes in hepatocytes.The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisiting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.     

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF₁ are attached by stalks to an aggregate of CF₀.The CF₁-CF₀ complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF₀, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF₀ and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.