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PubMed1985

Human lymphocyte-eosinophil interactions. I. Modulation of phytohemagglutinin-induced lymphocyte proliferation by eosinophils.

Do eosinophils modulate lymphocyte function? This question was studied by examining the effect of purified eosinophils (eos) on lectin-induced human lymphocyte proliferation. Intact resting or zymosan-stimulated eos or their extracts were cocultured with phytohemagglutinin-stimulated mononuclear cells in vitro and [3H]thymidine uptake was measured at 72 hr. Zymosan-stimulated eos consistently suppressed (up to 90%) the lectin-induced proliferative response by a noncytotoxic mechanism. Freeze-thaw extracts from zymosan-stimulated eos also significantly suppressed lymphocyte proliferation to a similar degree. The amount of suppression was directly proportional to the number of eos or the amount of extract added to the lymphocyte cultures. Intact resting eos and their extracts occasionally exhibited suppressive effects (up to 40%) on lymphocyte proliferation; this suppression, however, was always less than that of activated eos or their extracts. Eos pretreated with the protein synthesis inhibitor, pactamycin, exhibited significantly less suppressive activity, suggesting that a protein was responsible in part for the reduction in proliferation. The addition of superoxide dismutase or catalase to the eos-mononuclear cell cocultures did not reduce the amount of suppression observed, thus making it unlikely that active oxygen products were involved in the mechanism of suppression. Heating extracts from stimulated eos to 80 degrees C for 30 min resulted in partial loss of suppressive activity while extensive dialysis of the extracts had no effect. The studies reported here provide evidence that a nondialyzable and heat sensitive factor(s) produced by stimulated eos may exert feedback inhibition of lymphocyte function.

Catalase↗
PubMed1984

Translational initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells.

The association of mRNA and ribosomes with the cytoskeleton of eucaryotic cells may be important for protein synthesis and its regulation. HeLa cells were gently lysed with detergent, and soluble and cytoskeletal framework subfractions were prepared by centrifugation. We analyzed these fractions for ribosomes and confirmed earlier findings that polysomes are preferentially associated with the cytoskeletal fraction. The levels of initiation factors eIF-2, eIF-3, eIF-4A, and eIF-4B were quantitated by immunoblotting; all are enriched in the cytoskeletal fraction relative to the soluble fraction. Heat shock, fluoride, pactamycin , and cytochalasin caused the release of both ribosomes and initiation factors into the soluble fraction. However, treatment of the cytoskeletal fraction with EDTA or low levels of ribonuclease resulted in polysome degradation but no release. Therefore initiation factor association with the cytoskeletal framework correlates with the presence of ribosomes, whereas ribosome association does not require intact mRNA.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs.

We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, beta-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.

Animals↗
PubMedDate not supplied

A model system for T-lymphocyte differentiation: regulation of CD4 and CD8 gene expression in SL12.4 T-lymphoma cell clones.

The T-cell surface proteins CD4 (L3T4) and CD8 (Lyt2) are first expressed on thymocytes as they undergo maturation in the thymus. Two immature T-lymphoma cell clones SL12.4 and RS4.2 which constitutively express low or undetectable levels of CD4 and CD8 were used to investigate the activation of CD4 and CD8 gene expression. The protein synthesis inhibitors cycloheximide (CHX) and pactamycin rapidly and reversibly increased CD4 and CD8 mRNA in the cloned cell lines, suggesting that a labile inhibitor protein(s) may regulate the expression of these transcripts. Cell surface CD4 and CD8 proteins were transiently detectable following a pulse of CHX. Thymic epithelial cell lines also induced CD4 and CD8 mRNA and cell surface protein, as well as TCR-alpha mRNA when co-cultivated with SL12.4 T lymphoma cells. The increase in CD4 and CD8 was modest, but stable for at least 22 cell generations after the thymic epithelial inducer cells were removed. Epithelial cells of non-thymic origin did not cause induction of these T-cell differentiation markers in SL12.4 T-lymphoma cells. Since the induction elicited by thymic epithelial cells and protein synthesis inhibitors differed dramatically in kinetics and reversibility, it is likely that these inducers act, at least in part, via different mechanisms. This lymphoma model system may be useful for analysis of molecular events which occur in immature thymocytes undergoing differentiation.

Animals↗
PubMed1986

Fast estimation of ATP/ADP ratio as a special step in pharmacological and toxicological studies using the cell-free translation systems.

We have developed a simple and effective reversed-phase HPLC procedure for rapid estimation of the ATP/ADP ratio in a cell-free translation system containing creatine kinase. Analysis of the acetone-extractable pool derived from a reticulocyte lysate cell-free system was carried out by automatic chromatography on S5CN-ODS stationary phase using a linear 10-65% pyridine elution gradient formed on the basis of methanol/water (9:1, v/v) mobile phase. This method was used to detect and characterize the inhibition of translation induced by considerable suppression of ATP resynthesis in vitro. It was shown that methyl mercury, unlike cycloheximide, pactamycin, CCl4 and barbituric acid, exerts inhibitory effect on the ATP regeneration in a cell-free translation system.

Adenosine Diphosphate↗
PubMed1994

Mitochondrial gene expression in Saccharomyces cerevisiae. IV. Effects of yeast cytosol on mitochondrial protein synthesis, degradation, and respiration.

It has been known for some time that the addition of a crude yeast cytosolic fraction to isolated mitochondria stimulates the rate of amino acid incorporation into protein in the isolated organelles. However, the mechanism and importance of this phenomenon relative to mitochondrial function has not been established. While it has been assumed that this effect is at the level of translation, the recognition that newly synthesized mitochondrial translation products are rapidly degraded in isolated yeast mitochondria raises the possibility that cytosol affects amino acid incorporation by inhibiting proteolysis. Using pulse-chase experiments we demonstrate that the rate constants of degradation of the nascent products are not affected by yeast cytosol. Further, not only is proteolysis not inhibited by cytosol, but the loss of label caused by proteolysis is actually increased. This increase is directly related to an increase in the size of the nascent product pool which increases simply as a consequence of increasing the rate of translation. By utilizing an approach in which the loss of label due to proteolysis is minimized, the true stimulatory activity of the cytosolic fraction on synthesis was determined (2.1-fold vs. 1.3-fold by the previous method). Pulse-chase experiments in the presence of pactamycin, an initiation inhibitor, demonstrate that yeast cytosol causes an initial increase in the rate of translational initiation without increasing the rate of elongation. However, at later intervals the yeast cytosol acts primarily to maintain the rate of elongation which falls steadily in the controls. Finally, the presence of yeast cytosol dramatically increases the length of incubation time in which the mitochondrial preparation consumes oxygen and maintains coupled respiration, parameters that fall rapidly in the controls. Thus, a yeast cytosolic fraction may function to promote the stability of the mitochondrial preparation, which in turn may account for the increase in rates of translation, particularly with regard to maintaining rates of elongation.

Cytosol↗
PubMed1989

Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells.

1. Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined. 2. Incorporation of [35S]methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide. 3. Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides.

Animals↗
PubMed2006

Molecular contacts between antibiotics and the 30S ribosomal particle.

Crystal structures of complexes between ribosomal particles and antibiotics have pinned down very precisely the discrete binding sites of several classes of antibiotics inhibiting protein synthesis. The crystal structures of complexes between various antibiotics and ribosomal particles show definitively that ribosomal RNAs (rRNAs), rather than ribosomal proteins, are overwhelmingly targeted. The antibiotics are found at messenger RNA or transfer RNA binding sites and, most importantly, at pivot locations that are key for the structural rearrangements during the molecular mechanical steps in initiation, elongation, or termination of protein synthesis. We focus here on the 30S particle. Structurally, the antibiotics interact in many ways with RNA: (i) only with the phosphate groups (streptomycin); (ii) mainly with bases (hygromycin, spectinomycin); (iii) with a mixture of both (paromomycin, Geneticin); (iv) via magnesium ions (tetracycline) or a protein side chain (streptomycin). The antibiotics can mimic base stacking (pactamycin) or form pseudo-base pairing interactions with ribosomal bases (paromomycin and related aminoglycosides). Resistance strategies (mutations or methylations in rRNA or enzymatic modifications of the antibiotics) can generally be understood on the basis of the intermolecular contacts made between the antibiotics and rRNA residues in the crystal structures. In humans, toxicity of ribosomal antibiotics is most likely due, at least in part, to the sensitivity of mitochondrial ribosomes, since mitochondria evolved from a bacterial ancestor. Antibiotic families (e.g., aminoglycosides) form a set of invariant H-bonds to defined rRNA residues. When such residues are conserved in bacteria, but not in eukaryotes, resistance of eukaryotic ribosomes is observed. The structural knowledge, together with comparative genomic analysis, should allow for the development of new broad-spectrum antibiotics with higher selectivity toward bacterial ribosomes and less toxicity on eukaryotic cytoplasmic and mitochondrial ribosomes.

Aminoglycosides↗
PubMed1981

Early effects of dimethylnitrosamine on the initiation of protein synthesis in mouse liver.

1. Dimethylnitrosamine (37.5 mg/kg body wt.) was administered to mice by a single intraperitoneal injection, and the early effects on protein synthesis and related functions were studied in a liver S-30 system. 2. The incorporation of [14C]leucine into protein decreased rapidly after dimethylnitrosamine administration. The effect was associated with a decreased ability of the system to utilize methionyl-tRNAfMet and formyl-methionyl-tRNAfMet for 80 S ribosomal initiation-complex formation (primary initiation), and a loss of poly(A)-containing RNA from the postmicrosomal fraction. All the three effects developed simultaneously, and were clearly demonstrable within 15 min. 3. Initiation-complex formation in the polyribosomal fraction (re-initiation) was decreased to the same extent as the primary initiation, indicating that the initiation defect was not a result of the decrease in free mRNA. 4. The inhibition of initiation was only manifest at the joining of the 40 S pre-initiation complex to 60 S ribosomal subunits. It was not a result of methionyl-tRNAfMet deacylation. The functions between the formation of the methionyl-tRNAfMet-containing 80 S ribosomal complex and the first translocation on the ribosome were not involved, since the incorporation of formylmethionine into N-terminal polypeptides decreased to the same extent as the 80 S initiation-complex formation. 5. Inhibitors of protein synthesis (cycloheximide and pactamycin) decreased poly(A)-containing RNA in the postmicrosomal fraction in a similar way to dimethylnitrosamine.

Animals↗
PubMed1991

Abundance of hepatic metallothionein mRNA is increased by protein-synthesis inhibitors. Evidence for transcriptional activation and post-transcriptional regulation.

Ongoing protein synthesis is a prerequisite in the expression of some genes. We studied the effect of various protein synthesis inhibitors on the expression of the avian metallothionein (MT) gene. Chicken embryonic hepatocytes in culture were exposed to various concentrations of cycloheximide, puromycin and pactamycin. At concentrations which decreased total protein synthesis by about 90% each inhibitor increased MT mRNA accumulation approx. 5-fold at 9 h of incubation. Incubation with puromycin or zinc for 2 h markedly increased the rate of MT gene transcription. Estimates of the half-life of MT mRNA by using actinomycin D suggested for cycloheximide, but not puromycin, decreased the decay rate of MT mRNA. These data suggest the potential for post-transcriptional regulation of the avian MT gene. We conclude that different antibiotics increase the accumulation of hepatocyte MT mRNA by different mechanisms and that the possibility of multiple mechanisms should be considered in other studies of the role of protein synthesis in gene expression.

Animals↗
PubMed1971

Aurintricarboxylic acid: inhibitor of initiation of protein synthesis.

Aurintricarboxylic acid prevents the attachment of bacteriophage messenger RNA to ribosomes. As a consequence, initiation of protein synthesis in cell-free extracts prepared from Escherichia coli or rabbit reticulocytes is inhibited at concentrations of dye that do not prevent chain extension. Its properties can be distinguished from other agents that inhibit protein synthesis, including sodium fluoride, cycloheximide, and pactamycin.

Animals↗
PubMed1972

Mechanism of hormonal induction of tyrosine aminotransferase studied by measurement of the concentration of growing enzyme molecules.

Proteins labeled in cultured hepatoma cells grown in the presence of radioactive amino acids and pactamycin consist primarily of growing polypeptide chains initiated before addition of the antibiotic. Nascent tyrosine aminotransferase (EC 2.6.1.5) molecules were labeled in this way, and their radioactivity after completion was measured immunologically. Cells exposed to the synthetic adrenal steroid, dexamethasone, contain 10- to 18- times as much nascent enzyme as uninduced cells. These results indicate that induction control is not at the level of polypeptide-chain elongation or release, but probably operates on either the amount of messenger RNA or the rate of specific polypeptide-chain initiation.

Amino Acids↗
PubMed1973

Protein initiation in eukaryotes: formation and function of a ternary complex composed of a partially purified ribosomal factor, methionyl transfer RNA, and guanosine triphosphate.

A protein factor contained in a 1 M KCl extract of L-cell ribosomes and partially purified by chromatography on DEAE-cellulose forms a specific ternary complex with rat-liver Met-tRNA(f) and GTP. The complex is measured by its quantitative retention on nitrocellulose membranes. Complex assembly is optimal at 100 mM KCl and 0.2 mM MgCl(2), and is independent of mRNA and of ribosomes. The GTP requirement can be replaced over 65% by its methylene analogue GDPCH(2)P, indicating that GTP hydrolysis is not involved. Complex formation is inhibited by 10 muM aurintricarboxylic acid, but is unaffected by 100 muM pactamycin, 100 muM fusidic acid, or by excess uncharged methionine tRNA(f). The ternary complex is relatively stable and appears at the void volume during filtration on Sephadex G-100. At 1-3 mM MgCl(2) and in the presence of other factors, the ternary complex is implicated in protein initiation by (i) its capacity to bind to the 40S ribosomal subunit to form a 48S complex; and (ii) the subsequent association of the 48S complex with a 60S subunit to form a functional "80S complex."

Adenosine Triphosphate↗
PubMed1976

mRNA-dependent synthesis of authentic precursor to human placental lactogen: conversion to its mature hormone form in ascites cell-free extracts.

Messenger RNA derived from term placenta directs the synthesis of human placental lactogen (hPL, molecular weight 22,200) in an ascites 30,000 X g post-mitochondrial supernate (S-30). When the S-30 is fractionated into ribosome and cell-sap (S-100) fractions, and these are recombined for incubation, term placental mRNA directs the synthesis of a protein with a molecular weight of 25,000. This protein contains authentic hPL tryptic peptides. This suggested that during the separation of ribosomes and S-100 a component responsible for cleavage was lost. A 1.0 M sucrose cushion was used for the preparation of ribosomes and S-100 and membranous material accumulated at the sucrose interphase. When this membrane fraction was added back to the ribosome-S-100 system only hPl was formed. Cleavage was greatest when membranes were added within the first few minutes of incubation. In a run-off system composed of term polysomes, ascites S-100, and the inhibitor of initiation, pactamycin, the 25,000 molecular weight material, referred to as pre-hPL, was also synthesized. These data strongly suggest that (i) pre-hPL is an authentic percursor to hPL, (ii) cleavage of the precursor primarily occurs on nascent, ribosome-bound peptide chains, and (iii) pre-hPL is the primary gene product.

Cell-Free System↗
PubMed1995

A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro.

Gene rearrangement during the ontogeny of T- and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contain premature terminations codons (ptcs). We performed sequence analysis of reverse transcriptase-polymerase chain reaction products from fetal and adult thymus and found that newly transcribed TCR-beta pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-beta transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-beta exons triggered a decrease in mRNA levels. Ptc-bearing TCR-beta transcripts were selectively depressed in levels in a cell clone that contained both an in-frame and an out-of-frame gene, thus demonstrating the allelic specificity of this down-regulatory response. Protein synthesis inhibitors with different mechanism of action (anisomysin, cycloheximide, emetine, pactamycin, puromycin, and polio virus) all reversed the down-regulatory response. Ptc-bearing transcripts were induced within 0.5 h after cycloheximide treatment. The reversal by protein synthesis inhibitors was not restricted to lymphoid cells, as shown with TCR-beta and beta-globin constructs transfected in HeLa cells. Collectively, the data suggest that the ptc-mediated mRNA decay pathway requires an unstable protein, a ribosome, or a ribosome-like entity. Protein synthesis inhibitors may be useful tools toward elucidating the molecular mechanism of ptc-mediated mRNA decay, an enigmatic response that can occur in the nuclear fraction of mammalian cells.

Animals↗
PubMed2000

The fate of membrane-bound ribosomes following the termination of protein synthesis.

Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.

Endoplasmic Reticulum↗
PubMed2007

Binding mode of the first aminoacyl-tRNA in translation initiation mediated by Plautia stali intestine virus internal ribosome entry site.

Eukaryotic ribosomes directly bind to the intergenic region-internal ribosome entry site (IGR-IRES) of Plautia stali intestine virus (PSIV) and initiate translation without either initiation factors or initiator Met-tRNA. We have investigated the mode of binding of the first aminoacyl-tRNA in translation initiation mediated by the IGR-IRES. Binding ability of aminoacyl-tRNA to the first codon within the IGR-IRES/80 S ribosome complex was very low in the presence of eukaryotic elongation factor 1A (eEF1A) alone but markedly enhanced by the translocase eEF2. Moreover, eEF2-dependent GTPase activity of the IRES/80 S ribosome complex was 3-fold higher than that of the free 80 S ribosome. This activation was suppressed by addition of the antibiotics pactamycin and hygromycin B, which are inhibitors of translocation. The results suggest that translocation by the action of eEF2 is essential for stable tRNA binding to the first codon of the PSIV-IRES in the ribosome. Chemical probing analysis showed that IRES binding causes a conformational change in helix 18 of 18 S rRNA at the A site such that IRES destabilizes the conserved pseudoknot within the helix. This conformational change caused by the PSIV-IRES may be responsible for the activation of eEF2 action and stimulation of the first tRNA binding to the P site without initiation factors.

Animals↗
PubMed1974

On the relation of products of activated lymphocytes to cell-mediated cytolysis.

Experiments have been designed to test the hypothesis that soluble mediator production and T-cell-mediated cytotoxicity are necessarily related phenomena, and that soluble mediators may be involved in the mechanism of cytolysis. To this end, agents known to inhibit T-cell-mediated lysis in vitro have been studied for their effects on the production of two lymphocyte-derived mediators, lymphotoxin (LT) and migration inhibitory factor (MIF). A clear dissociation between mediator production and cell-mediated cytolysis was found using inhibitors of protein synthesis. Pactamycin and emetine, in doses of 10(-7) M to 10(-6) M, suppressed production of MIF and LT with only slight effect on killing of mastocytoma cells by immune T cells. On the other hand colchicine and vinblastine inhibited T-cell-mediated cytolysis in a dose-related manner but had no significant effect on either MIF or LT production, A striking dichotomy was also observed after augmentation of intracellular cyclic 3'5' adenosine monophosphate (cAMP) levels with cholera enterotoxin. Increased cAMP levels were associated with abrogation of direct lytic activity, but were without significant effect on MIF or LT production in guinea pigs or mice. These findings indicate that mediator production and direct lymphocyte-mediated cytolysis can be experimentally dissociated and represent independent cell-mediated immune functions.

Animals↗