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Total syntheses of SB-203207 (1) and sphingofungin E (2) were accomplished by utilizing an asymmetric desymmetrization strategy to introduce multiple stereogenic centers into meso starting compounds in a single step. Thus, rhodium carbenoid-mediated C H insertion into 3 provided the bicyclo[3.3.0]framework 4 for 1, while organocatalyst-mediated bromolactonization of 5 afforded 6 for the synthesis of 2. Similarly, we utilized desymmetric rhodium nitrenoid-mediated aziridination reaction of 5 to obtain a key intermediate required for a total synthesis of pactamycin (29), a complex aminocyclopentitol antibiotic featuring six contiguous stereogenic centers.

The crystal structures of the universal translation-initiation inhibitors edeine and pactamycin bound to ribosomal 30S subunit have revealed that edeine induces base pairing of G693:C795, residues that constitute the pactamycin binding site. Here, we show that base pair formation by addition of edeine inhibits tRNA binding to the P site by preventing codon-anticodon interaction and that addition of pactamycin, which rebreaks the base pair, can relieve this inhibition. In addition, edeine induces translational misreading in the A site, at levels comparable to those induced by the classic misreading antibiotic streptomycin. Binding of pactamycin between residues G693 and C795 strongly inhibits translocation with a surprising tRNA specificity but has no effect on translation initiation, suggesting that reclassification of this antibiotic is necessary. Collectively, these results suggest that the universally conserved G693:C795 residues regulate tRNA binding at the P site of the ribosome and influence translocation efficiency.

We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, interactions with the 30S subunit suggest a mechanism for its effects on ribosome function.

Overexpression of the translation initiation factor eIF4E leads to cell transformation and occurs in a number of human cancers [1]. mRNA translation and cell growth can be regulated through the availability of eIF4E to form initiation complexes by binding to eIF4G. The availability of eIF4E is blocked through the binding of members of a family of eIF4E-binding proteins (4E-BPs) [2] [3]. Indeed, cell transformation caused by the overexpression of eIF4E can be reversed by the overexpression of 4E-BPs [4] [5] [6] [7] [8]. To study the role of eIF4E in cell transformation, we developed a series of peptides based on the conserved eIF4E-binding motifs in 4E-BPs and eIF4G [9] linked to the penetratin peptide-carrier sequence, which mediates the rapid transport of peptides across cell membranes. Surprisingly, introduction of these eIF4E-binding peptides into MRC5 cells led to rapid, dose-dependent cell death, with characteristics of apoptosis. Single alanine substitutions at key positions in the peptides impair their binding to eIF4E and markedly reduce their ability to induce apoptosis. A triple alanine substitution, which abolishes binding to eIF4E, renders the peptide unable to induce apoptosis. Our data provide strong evidence that the peptides induce apoptosis through binding to eIF4E. They do not induce apoptosis through inhibition of protein synthesis, as chemical inhibitors of translation did not induce apoptosis or affect peptide-induced cell death. Thus these new data indicate that eIF4E has a direct role in controlling cell survival that is not linked to its known role in mRNA translation.

AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.

Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 90 degrees C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA.

The c-fms proto-oncogene encodes the receptor for a hematopoietic growth factor, CSF-1. Recently, the importance of c-fms and its ligand CSF-1 in malignancies of non-hematopoietic origin, such as breast, ovarian, endometrial, pulmonary, and trophoblastic cancers has been recognized. We have previously shown that glucocorticoids induce a large increase in c-fms mRNA and protein levels in breast carcinoma cell lines. In this report, we investigate the mechanism underlying such c-fms overexpression by dexamethasone. We show that dexamethasone treatment of two breast carcinoma cell lines (BT20-c-fms expressor, and SKBR3-co-expressor of both c-fms and CSF-1) does not increase the rate of c-fms gene transcription, suggesting a post-transcriptional mechanism of regulation of c-fms expression by dexamethasone. The effect of protein synthesis inhibition was studied to help determine whether there was a role for intermediary regulatory proteins in the regulation of c-fms expression. We find that several protein synthesis inhibitors interfere with dexamethasone induction of c-fms transcripts, suggesting the existence of regulatory proteins. These regulatory proteins do not appear to be constitutively expressed, as we show no effect of protein synthesis inhibition on c-fms transcript expression in resting BT20 cells. These findings suggest that the putative regulatory proteins are induced by dexamethasone. Furthermore, the addition of a protein synthesis inhibitor, pactamycin, to dexamethasone-treated BT20 cells results in a decrease in c-fms mRNA stability. There is no decrease of c-fms transcripts over the six hours of actinomycin-D chase in the dexamethasone-treated cells. However, when pactamycin is added to dexamethasone, the calculated c-fms mRNA half-life falls to seven hours, which represents a decrease of c-fms mRNA half-life to that measured in the resting cell, and in monocytes. Taken together, these results suggest the involvement of a dexamethasone-inducible c-fms mRNA stabilizing protein in the regulation of c-fms gene expression in breast carcinoma cell lines. Interestingly, dexamethasone has no effect on CSF-1 expression in SKBR3 cells. Protein synthesis inhibition however results in a reciprocal effect, with induction and stabilization of CSF-1 transcripts, which contrasts with the destabilization of c-fms mRNA. This reciprocal effect led us to postulate that CSF-1 may also contribute to the post-transcriptional regulation of c-fms mRNA expression in breast carcinoma cells. Therefore, the effect of addition of exogenous CSF-1 on c-fms mRNA expression in BT20 cells was studied. We demonstrate that exogenous CSF-1 is able to down-regulate c-fms mRNA expression by 3-fold in BT20 cells. This suggests that CSF-1, in addition to glucocorticoids, may play a role in the post-transcriptional regulation of c-fms expression in breast carcinoma cells. (Steroids 59:514–522, 1994)

The rate of tRNA synthesis in cultured Friend leukemia cells has been examined as a function of the variation in polyribosome structure produced by treatment with a variety of inhibitors of protein synthesis. The results indicate, in contrast to the conclusions of Bolcsfoldi (Bolcsfoldi, G. (1974) Exp. Cell Res., 88, 231–240), that no necessary relationship exists between the ribosome distribution and the rate of tRNA synthesis. Alternatively, it is observed that inhibitors of tRNA aminoacylation cause, in all cases, a decrease in the rate of tRNA synthesis whereas drugs which may stimulate the aminoacylation of tRNA cause, in all cases, an elevation of the rate of tRNA synthesis. It is concluded that tRNA synthesis in mammalian cells may be regulated by the relative levels of acylated and deacylated tRNA.

The effect of 7-methylguanosine 5′-monophosphate (m7G5′ p) on translation of partially purified globin mRNA and of polysome-associated endogenous globin mRNA has been studied. Under identical experimental conditions, with 0.4 mM m7G5′ p, translation with partially purified globin mRNA is inhibited 50%; translation with endogenous globin mRNA is inhibited 10%. The inhibition of protein synthesis by m7G5′ p occurs at a step before the first peptide bond formation as evidenced by studies with pactamycin; 0.4 mM m7G5′ p inhibited the first dipeptide synthesis 43% when the partially purified globin mRNA was used whereas 15% inhibition was observed with the endogenous mRNA. The inhibition of m7G5′ p appears to be related to the structural integrity of globin mRNA.

Cell-survival (of DON and L1210 cells) after treatment with cytotoxic compounds was assessed by measuring cloning efficiency, exclusion of trypan blue and erythrosin B, [ 51 Cr] release, and attachment of DON cells to glass. Cell survival as measured by cloning efficiency did not correlate with survival measured by any of the other methods. We found that the stainability of cells after drug exposure depended on the cell line used. For example, after 3 h exposure to tubercidin although 100% of both DON and L1210 cells were killed (on basis of cloning efficiency), only 11% of DON cells and 68% of L1210 cells were dead as indicated by staining with erythrosin B. The stainability of cells also depended on the particular drug used. For example, after 24 h exposure of L1210 cells to adriamycin and tubercidin (both killed >99% of cells on basis of cloning efficiency) 21% of cells exposed to adriamycin and 99% of cells exposed to tubercidin were stained. The results obtained with several other cytotoxic compounds are discussed.

The addition of 0.167 to 4.0 mM cAMP to gel-filtered rabbit reticulocyte lysates stimulates the initial rate and the extent of polypeptide synthesis. The stimulation is at the initiation step of polypeptide synthesis as measured by the (i) increased dipeptide, methionyl-valine, accumulation in the presence of the specific initiation inhibitor, pactamycin, and (ii) increased formation of the 40 S and 80 S initiation complex when gel-filtered lysates are incubated with [35S]Met-tRNAFMet. Furthermore, a synergistic stimulation of protein synthesis is observed when cAMP and hexose phosphates (which alone elicit a 1.8-fold stimulation of protein synthesis) are added simultaneously to gel-filtered rabbit reticulocyte lysates. These results indicate that cAMP and hexose phosphates are both essential to maintain the high rate of initiation.

Migration of 40 S ribosomal subunits on messenger RNA, detected previously in experiments using the antibiotic edeine (Kozak, M., and Shatkin, A.J. (1978) J. Biol. Chem. 253, 6568-6577) has now been observed in the presence of other inhibitors of initiation. 40 S subunit migration has been detected in both wheat germ and reticulocyte lysates treated with edeine, pactamycin, or sodium fluoride. The variety of structurally unrelated inhibitors that mediate this effect argues against the interpretation that migration is a drug-induced artifact. Indeed, limited migration of 40 S ribosomes occurs upon simply lowering the magnesium concentration, in the absence of inhibitors. Thus, migration seems to be an inherent property of 40 S ribosomal subunits and might be involved in the mechanism by which eukaryotic ribosomes select initiation sites in messenger RNA.

Methyl mercury inhibits in vitro protein synthesis in the rabbit reticulocyte lysate cell-free translation system and simultaneously leads to reduction of the ATP/ADP index. It has been established that there is a close relationship (r = 0.86) between the rates of ATP resynthesis and protein synthesis in vitro within a wide range of the methyl mercury concentrations tested (0.0001-1.0 mumol/ml). Ammonia, CCl4, cycloheximide and pactamycin inhibit translation in vitro without affecting ATP resynthesis. Methanol does not cause substantial alterations of the parameters of the cell-free translation system. Thus, there are at least two essentially different causes of poison-induced in vitro translation blocking. Suppression of ATP resynthesis (methyl mercury) and direct effect on protein synthesis (cycloheximide, CCl4, etc.)

Earlier I found that a variety of stimuli to proliferation of cultured human fibroblasts caused an increase in the rate of putrescine transport into the cells. This paper reports the effects of cycloheximide on putrescine transport in stationary and growing cultures. Cycloheximide in concentrations that inhibited protein synthesis caused increased putrescine transport in serumstarved and density-inhibited cultures. Similar effects were found with pactamycin, also an inhibitor of protein synthesis. Actinomycin D in concentrations that suppressed messenger RNA (mRNA) synthesis, did not cause increased putrescine transport. When both serum and cycloheximide were added to serum-starved cultures, the increase in putrescine transport was greater than when serum alone was added. However, cycloheximide had an inhibitory effect when added 1–2 h after addition of serum. These results suggest that one or more rapidly metabolizing proteins may be important in the regulation of putrescine transport and initiation of cell growth.

The structural proteins of Semliki Forest virus are translated as a 130K polyprotein from which the amino terminal capsid protein is cleaved immediately after being completed. We have followed the intracellular pathway of capsid protein from polysomes to the nucleo-capsids by pulse-chase experiments. After a 1-min pulse given in the middle of the infection cycle the newly formed capsid protein was quantitatively associated with the large ribosomal subunits in the polysomes. After a 2-min chase 40 to 60% of the capsid proteins disappeared from the polysomes, appearing simultaneously in the nucleocapsid pool. Since the released capsid protein could never be found free in the cytoplasm, we conclude that it was transferred directly from the polysomal ribosomes to the nucleocapsid pool. Those capsid proteins which were not transferred from the polysomes were released into the monosome pool after a 5-min chase. During longer chase periods the label in monosomes decreased slowly in parallel to a corresponding increase in the amount of labeled nucleocapsids, suggesting that monosome-associated capsid protein was also transferred to the nucleocapsid pool, albeit at a greatly reduced rate. The rapid “polysomal transfer” of capsid protein was more dominant late than early in the infection. Puromycin, cycloheximide, and sparsomycin, added immediately after the pulse, inhibited to a large extent the transfer of capsid protein to the nucleocapsid, showing that the “polysomal transfer” plays an important role in the assembly of the nucleocapsid. Inhibition of initiation of protein synthesis with pactamycin had no effect on the pathway of the pulse-labeled capsid protein.

An in vitro system using vesicular stomatitis virus (VSV) infected HeLa cell S10 extracts, optimized for transcription and translation, replicated and assembled VSV genome-size RNA into nucleocapsids containing at least N protein. The in vitro synthesized nucleocapsids (RNPs) were RNase resistant and had the same buoyant density in CsCl as virion nucleocapsids. Full-length (+) and (−) RNA were synthesized in about the same ratio as in vivo . In vitro replication was dependent on protein synthesis or at least a pool of VSV RNP proteins. This system will be useful for identification of the host cell factors necessary for replication and the relationship of L and NS proteins in transcription and replication.

The induction of thymidylate (TMP) synthetase in Tetrahymena pyriformis occurred following a shift from complex medium to defined medium, or following addition of cyclic guanosine-3′,5′-monophosphate (cGMP) to complex medium. Inhibitors of macromolecular synthesis were used to probe the role of culture conditions in the enzyme induction. In defined medium actinomycin D minimally decreased the induction of enzyme activity, implying that control of enzyme synthesis is not exerted at the transcriptional level. Cycloheximide markedly inhibited the induction of TMP synthetase by defined medium, suggesting the requirement for protein synthesis in the induction process. In contrast, in complex medium where the activity of the synthetase was low (spec. act.

Nylon wool-purified splenic T cells from herpes simplex virus (HSV) immunized mice could be induced in vitro to yield large quantities of secondary cytotoxic T lymphocytes (CTLs) with specificities for HSV and histocompatibility (H-2) antigens. The in vitro approaches used for generating secondary CTLs consisted of culturing memory T cells either a) in medium containing supernatant from concanavalin A (Con A) stimulated spleen cells (CM), b) coculturing them with mitomycin C treated L cells coated with ultraviolet (UV) inactivated HSV, or c) stimulating them in vitro with purified UV-inactivated HSV alone. The primary aim of the present study was to characterize the nature of virus-cell interaction(s) that renders target cells susceptible to the lytic activity of anti-HSV CTLs. The results revealed that addition of irreversible protein synthesis inhibitor pactamycin to the target cells, prior to infection , could block substantially (ca. 50%) the lysis of targets by anti- HSV CTLs. The magnitude of CTL-mediated lysis against targets incubated with the drug canavanine which apparently blocked the synthesis of late virus-encoded polypeptides was of relatively low level when compared to untreated targets (i.e. 23 ± 2.5% vs. 46 ± 3.2% for HSV-1 CTLs and 26 ± 2.2% vs. 39 ± 4.2 for HSV-2 CTLs) which implies that virus specific late polypeptide(s) are involved in the recognition phenomenon. However, anti-HSV CTLs could also mediate an adequate level of specific lytic activity towards sygeneic enucleated cells (cytoplasts) which were exposed to HSV and would not allow the synthesis of new virus encoded antigen(s). Taken together, the data suggest that very early after HSV infection, the putative virus specific antigen(s) involved in CTL recognition event is derived directly from input virions i.e. externally, whereas late in the infectious cycle, it is the newly synthesized (from within) viral antigen(s) which is of relevance in the context of this phenomenon.

Sindbis virus 26S RNA is the principal species of virus-specific RNA found in the infected cell; it is derived from a one third segment of virion 42S RNA. When translated in cell-free extracts from mouse ascites cells or rabbit reticulocytes, 26S RNA directed the synthesis primarily of the 33,000 dalton virus capsid protein, and the protein products were in the form of free peptides rather than peptidyl-tRNA. In contrast, the polypeptides synthesized in either extract in response to Sindbis virus 42S RNA were heterogeneous, ranging in molecular weight from 33,000 to 190,000, and were largely in the form of peptidyl-tRNA. The number of independent initiation sites on the 26S and 42S RNAs was determined by analyzing a tryptic digest of reaction products labeled with yeast N-formyl- 35 S-methionyl-tRNA F met . The 26S RNA appeared to contain a single initiation site, and this site could also be found in varying amounts in different preparations of 42S RNA. However, a second initiation site, distinct from that of 26S RNA, was the major site in 42S virion RNA. These results suggest that 42S virion RNA contains two potential sites for initiation of protein synthesis. Only one of these may be active, however, and it is postulated that the second site functions primarily, if not exclusively, in the subgenomic 26S RNA species. In this regard, Sindbis virus 42S RNA may represent a novel form of a eucaryotic messenger RNA.

It has been established previously that the maintenance of expression of prespore specific genes of Dictyostelium discoideum is prevented by the translational inhibitor cycloheximide. The drug had no effect upon the level of transcripts of the other genes examined, prestalk-specific or cell type non-specific (Mehdy, M., Ratner, D. and Firtel, R., (1983) Cell 32, 763–771). We have now characterized the cellular specificity and temporal profiles of mRNA accumulation of additional Dictyostelium cDNA clones. Other inhibitors of in vivo protein synthesis have been examined, with emetine shown to be a particularly effective but reversible agent. Four structurally and mechanistically distinct translational inhibitors each prevented the reaccumulation of prespore transcripts in cyclic AMP-primed disaggregated amoebae. These results establish a role for protein synthesis in the transcription or transcript stability of prespore genes.

Fourteen temperature-sensitive ( ts ), transformation-defective mutants have been isolated from mutagenized Schmidt-Ruppin Rous sarcoma virus. We report that, while in cells infected with most of the mutants all parameters of cell transformation were coordinately suppressed, certain ts mutants induced the ability of infected cells to multiply in soft agarose and to express the tumor-specific surface antigen (TSSA), in the absence of morphological conversion. Studies with inhibitors of protein synthesis have shown that cycloheximide (Ch) and pactamycin (Pac) act differently on focus formation from Pu and that the heat-labile src gene product of these mutants may be spontaneously reactivated following a downshift to permissive temperature. Our data also indicate the existence of two classes of mutants regarding the response of infected cells to Pu. In most cases, focus formation is inhibited by Pu when infected cells are shifted to 37°. However, cells infected with three ts mutants resume morphological transformation at permissive temperature in the presence of this inhibitor. In addition, the fact that the same mutants induce high levels of TSSA expression at restrictive temperatures suggests that the two properties may be linked.

Aurintricarboxylic acid and pactamycin inhibited initiation factor catalyzed reassociation of ribosomal subunits to form 80S couples and subsequent polyphenylalanine synthesis although their effects were qualitatively different. The two inhibitors prevented the formation of 80S monomers if they were present with 40S subunits in the reassociation mixture before addition of large subunits; they did not inhibit protein synthesis nor reassociation if they were added with the 60S subunits after formation of a small subunit initiation complex. Thus creation of a 40S initiation complex precedes addition of the large subunit and formation of an 80S monomer. An additional finding was that aurintricarboxylic acid preferentially inhibited the formation of inactive 40S–60S couples.

The precursor to the major core polypeptide (pVII) of adenovirus type 5 has five methionine-containing tryptic peptides, while the mature core polypeptide (VII) after processing has only four. A technique utilizing the drug pactamycin has been used to determine the relative order (N terminal to C terminal) of the tryptic peptides within polypeptide pVII. Taking the order of these peptides into account and correlating it with the peptide missing from polypeptide VII, we are able to conclude that a region near the amino terminus of polypeptide pVII is removed during proteolytic processing.

The effect of inhibitors of protein synthesis on macrophage mediated cytotoxicity was determined. Cytotoxicity was inhibited by concentrations of pactamycin and actinomycin D that almost completely inhibited protein synthesis by human macrophages derived from peripheral blood monocytes. In addition, cytotoxicity was also inhibited when 10 −5 M of pactamycin were added to the macrophage monolayers at the time of addition of the tumor cells or 30–60 minutes after addition of the tumor cells whereas no inhibition of cytotoxicity occurred when this drug was added more than one hour after the initiation of the cytotoxic reaction.

The antitumoral and antibacterial drug pactamycin can be radioactively labeled by iodination without loss of biological activity. Using the labeled pactamycin, the ribosomal binding site of the drug on rat liver ribosomes has been studied by affinity labeling techniques taking advantage of the photoreactive acetophenone group present in the molecule. When 40 S ribosomal subunits are labeled, one major spot of radioactivity is found associated to protein S25. In addition, weaker spots related to proteins S14/15, S10, S17 and S7 can also be detected in the autoradiogram of the two-dimensional gel slab. Since pactamycin inhibits protein synthesis initiation, the proteins forming its binding site must be related to some step of this process. By comparison with results from pactamycin affinity labeling of Escherichia coli ribosomes (Tejedor, F., Amils, R. and Ballesta, J.P.G. (1985) Biochemistry 24, 3667–3672) these proteins could lie in the mRNA and initiation factors binding region of the rat liver ribosome.

In an earlier study, we established long-term bulk (uncloned) human, T-cell (BTC) cultures which displayed EBV-specific and HLA-restricted lytic activities. The present study demonstrates the feasibility of isolating and propagating clones of CTLs which express EBV specificity and HLA restriction. A total of 7 independent cloned cultures derived from 2 of the initially established BTC cultures were found to exert reproducible, preferential lytic activity towards EBV-infected autologous or HLA-A/B-matched allogenic B blasts. The conclusion that the cloned effectors were indeed exerting EBV-specific and HLA-restricted lysis was supported by the results of cold-target inhibition experiments. In addition, it was found that a monoclonal anti-HLA antibody (W3/62) could effectively block the observed lytic effect. Data have been presented suggesting that very early after exposure of B blasts to high multiplicity of EBV, the putative virus-specific component(s) involved in the CTL recognition event is derived directly from the envelope of input EBV, i.e. «from without» . Such susceptible EBV-infected blasts could be converted to the insusceptible state following brief exposure to papain, which presumably removes membrane-integrated EBV-envelope material from infected cell surfaces. Using this approach, the time required for cell-surface expression of newly synthesized EBV component(s), conferring susceptibility to virus-specific CTLs, was investigated. The results indicate that EBV-infected and papain-treated B blasts acquire optimal susceptibility to the lytic effect of effector T cells 3-4 days following infection. This conversion of infected blasts from the insusceptible to the susceptible state was blocked by the protein-synthesis inhibitor pactamycin only when added immediately and not at 6 h after papain treatment. On the other hand, papain-treated infected blasts incubated in presence of phosphonoacetic acid, a known inhibitor of EBV-DNA synthesis, were lysable by the effectors. Taken together, the results imply that EBV-specific envelope protein(s) inserted in targetcell surfaces either «from without» or de novo synthesized (from within) presumably represent the putative target(s) for virus-specific CTLs in the context of HLA-A/B antigen(s).

We have described ongoing studies designed to elucidate the mechanisms of action of the cytotoxic antibiotics PM and NCS. PM selectively blocks polypeptide chain initiation in eukaryotes by two effects, which may be related: (1) the joining of the 60S ribosomal subunit to the smaller initiation complex is interfered with. This action can be overcome by adding excess of a factor, obtained by DEAE-cellulose chromatography of a crude initiation factor preparation, that is normally required for joining activity; (2) under conditions where joining is not prevented, there is still a block in the formation of peptides beyond the initial dipeptide. These data can be explained by a model of PM action in which PM does not interfere with the ribosomal joining reaction per se , but prevents the release and reuse of the joining factor, and in so doing, blocks a step in elongation after formation of the initial dipeptide and its translocation to the P-site on the ribosome. It is possible that the block in tripeptide formation is at the level of the use of the ribosomal A-site. The selectivity of PM for polypeptide initiation may be determined by the availability of its binding site only at any early stage in initiation. NCS has been found to inhibit thymidine incorporation into DNA in vivo and to place single stranded nicks in DNA both in vivo and in vitro . While it is premature to relate the cutting of DNA by NCS in vitro to these and other effects of the drug in vivo , the test tube reaction is interesting in its own right and has revealed information on the molecular requirements for the cutting reaction. Mercaptoethanol or some other sulfhydryl compound and a pH of 7.5 are required for maximal cutting. DNA strand scission by NCS produces 5′-phosphoryl end groups on all four deoxyribonucleotides, although in preliminary experiments dGMP and TMP appear to be favored. From protection experiments, it appears that thymidylic acid in DNA is required for interaction between DNA and NCS. How these effects of NCS are related, if at all, to the block at G2 of the cell cycle (45) remains to be elucidated.

The effect of cytochalasin A and B, colchicine and vinblastine on tumor cell killing by macrophages activated in vitro with lymphocyte mediators was examined. Both cytochalasins reversibly inhibited the killing of tumor cells by activated macrophages. Kinetic studies with cytochalasin B suggested that this drug exerts its effect on an early step of the cytotoxic process. Additional studies revealed that the drug inhibited the binding of tumor cells by activated macrophages. Colchicine inhibited both the binding and the killing of tumor cells by activated macrophages, whereas its structural analogue, lumicolchicine, had no effect on either macrophage function. Vinblastine also inhibited the binding and killing of tumor cells. However, this drug no longer inhibited tumor cell binding at low concentrations ( −6 M ) that still inhibited tumor cell killing. Further, vinblastine inhibited tumor cell killing when added late to an ongoing cytolytic reaction. These results suggest that the cytochalasins, colchicine and vinblastine inhibit macrophage mediated cytotoxicity by preventing intimate contact between the effector macrophages and their targets. In addition, vinblastine also appears to inhibit a later step of the cytolytic process, possibly the secretion of a cytotoxic macrophage product.

The translation of poly(A)-rich and poly(A)-poor populations of Encephalomyocarditis viral RNA was studied in Ehrlich ascites cell-free extracts. The poly(A)-rich viral RNA was translated 2–3 times more efficiently than the poly(A)-poor RNA. Both viral RNA populations were found to be similar with respect to susceptibility to nuclease attack as well as their ability to initiate and carry out protein synthesis early in the translation reaction. Later in the reaction, however, translation of the poly(A)-poor RNA was markedly reduced whereas translation of the poly(A)-rich RNA continued. These results are consistent with the hypothesis that poly(A) plays a role in the re-utilization and longevity of mRNA.

The in vivo injection of 400 μg of pactamycin into normal and partially hepatectomized mice resulted in the rapid deaggregation of their liver polyribosomes and accumulation of ribosomal monomers. When nascent polypeptide chains were labelled in vivo, the accumulated monomers were free of protein radioactivity. The previous injection of cycloheximide prevented the deaggregation of polyribosomes. The proportion of polyribosomes relative to the total ribosome population was measured in normal and regenerating livers from 10–60 s after the injection of pactamycin. Their rate of deaggregation was the same in both situations and it was such as could be expected from the in vivo rate of translation measured by other means elsewhere. These results are taken as further evidence that the efficiency with which polyribosomes are utilized for protein synthesis is the same in normal and regenerating livers.

Combinations of pactamycin and Escherichia coli 026: B6 Boivin lipopolysaccharide (LPS) or Salmonella minnesota R595 lipid A, as a bovine serum albumin-lipid A complex, administered simultaneously, killed BALB c mice synergistically. The enhanced lethality of the combination was not the result of a reduced rate of elimination of LPS or pactamycin from the circulation. Pactamycin administered iv, but not ip, accumulated in the lungs immediately after injection; thereafter the drug progressively disappeared from lung tissue. Concurrent administration of LPS did not alter the accumulation of pactamycin in the lungs nor its rate of elimination from blood and lung. The amount of pactamycin recoverable after incubation with liver or lung homogenates was markedly decreased at both 4 and 37°C. No products of the antibiotic were detected chromatographically after incubation with homogenates. Pactamycin and LPS in combination increased the in vitro fragility of hepatic and renal lysosomes, as measured by the release of cathepsin and β-glucuronidase; the effect of the combination was approximately equivalent to the sum of effects of LPS and pactamycin alone. Concanavalin A and cyclic adenosine 3′,5′-monophosphate did not modify the lethality of pactamycin and LPS, singly or in combination, whereas caffeine, ethylenediamine tetraacetate, or 6α-methylprednisolone protected mice from the lethal action of LPS and the synergistic combination but not that of pactamycin. Lethal doses of LPS and the synergistic combination, but not pactamycin, provoked marked hypothermia and elevated concentrations of blood urea nitrogen. These results indicate that pactamycin rendered the mice more susceptible to LPS and that LPS was the causal lethal agent.

The effect of drugs such as puromycin and cycloheximide, which inhibit protein synthesis, on the accumulation of La Crosse virus S genome RNAs in vivo has been examined. We have found that if these drugs are added to the cultures before infection, minuscule amounts of S-mRNA can be detected late in infection. Genome replication, on the other hand, cannot be detected at any time. When these drugs are added later in infection when RNA synthesis is well established, S-mRNA accumulation decreases in a dose-dependent manner proportional to the effect of these drugs on protein synthesis. This decrease cannot be accounted for by increased turnover of the mRNA in the presence of the drug. S genome replication, curiously, was found to be hypersensitive to the effects of these drugs. Our results confirm those of Abraham and Pattnaik (1983) that ongoing protein synthesis is required for the accumulation of complete bunyavirus S-mRNA.

At 1 h after intraperitoneal administration of D -amphetamine sulphate (15 mg/kg), rat brain polyribosomes show disaggregation accompanied by reduced capacity for in vitro peptide chain elongation. The direct action of amphetamine on cell-free protein-synthesizing systems was therefore explored. When brain or liver polyribosomes from untreated rats were incubated with pH 5 enzyme peptide chain elongation was not inhibited by the addition of 4 mM amphetamine to the medium. On the other hand, an initiation-dependent system consisting of rat liver or brain mRNA and wheat germ S-30 fraction showed inhibition of [3H]leucine incorporation by 50% when 4 mM amphetamine were added. The metabolites of amphetamine, p-hydroxyamphetamine and p-hydroxynorephedrine, had no inhibitory action in either system, but the potent neurotoxin p-chloroamphetamine was a more powerful inhibitor of initiation than amphetamine. By using [3H]amphetamine, it was shown that amphetamine binds to the 80-S ribosomes of the wheat germ system. This binding depended on the presence in the system of natural liver or brain mRNA or several synthetic mRNAs, but was not promoted by polyuridylic acid as the messenger. Significantly, polyuridylic acid-dependent polyphenylalanine synthesis by the wheat germ system was not inhibited by amphetamine or p-chloroamphetamine. Therefore, it was concluded that amphetamine inhibits protein synthesis by interfering with initiation through a step related to formation of the mRNA ribosome complex.

Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, unlike most retroviruses, preassembles core structures in the cytoplasm of infected cells during morphogenesis. We have shown previously ( J. Bradac and E. Hunter, 1984 , Virology 138, 260–275) that M-PMV virions contain 5 gag-encoded polypeptides. In this report, the translational order of the proteins encoded on the gag and env genes of M-PMV has been determined by pactamycin mapping. The proposed order of the viral proteins on their respective precursors is env: NH2-gp85-gp20-COOH; gag: NH2-p10-pp24/pp16-p12-p27-p14000H. Myristic acid-labeled virions contained a single radioactive protein, p10, supporting the mapping of this molecule to the amino terminus. From these studies it is clear that M-PMV contains an unusual additional gag polypeptide, p12, for which no function has been assigned to date. A remarkable similarity exists in the size and organization of M-PMV and mouse mammary tumor virus (MMTV) gag polypeptides; suggesting a common ancestor to these two viruses.

We have developed a simple and effective reversed-phase HPLC procedure for rapid estimation of the ATP/ADP ration 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, CCl 4 and barbituric acid, exerts inhibitory effect on the ATP regeneration in a cell-free translation system.

Viral ribonucleoprotein (RNP) particles isolated from vesicular stomatitis virus (VSV)-infected cells synthesized genome-length, complementary viral RNA, in addition to viral messenger RNA, in the presence of uninfected HeLa S10 extracts. The newly synthesized viral RNA was assembled into an RNP-like structure. RNA replication in vitro ceased when protein synthesis was blocked with pactamycin. Antibody raised against VSV NS protein inhibited in vitro RNA replication as well as mRNA synthesis. Anti-N protein also inhibited RNA replication, although it has no effect on the synthesis of mRNAs. Anti-G and anti-M IgG had no effect on either reaction. Anti-L IgG stimulated RNA replication 1.5- to 2-fold, lthough the synthesis of mRNA was inhibited.

The major methylated bases of tRNA of Bacillus subtilis are 5-methyluracil (m5U), 7-methylguanine (m7G) and 1-methyladenine (m1A). In addition, tRNA from stationary phase cultures always contains considerable amounts of an unidentified methylated pyrimidine derivative, “P.” Trimethoprim, which inhibits tetrahydrofolate (FH4)-dependent transmethylation reactions, was used to accumulate m5U-deficient tRNA in B. subtilis. With Escherichia coli enzymes the methyl moiety of S-adenosylmethionine (SAM) can be incorporated into the tRNA, and 0.7 m5U residue is formed per one tRNA molecule. The FH4-dependent formation of m5U in tRNA of B. subtilis as well as SAM-dependent methylations are affected by antibiotics which interfere with ribosomal functions. tRNA from pactamycin- or chloramphenicol-treated cells serves as substrate for SAM-dependent m5U- and m7G-specific tRNA methyltransferases from E. coli. With homologous SAM-dependent tRNA methyltransferases considerably fewer, but specific, transmethylations were found. tRNA obtained from a methionine-requiring stringent mutant of B. subtilis starved for methionine in the presence of chloramphenicol accepts the methyl moiety from SAM with homologous enzymes. The pattern of the methylated bases upon in vitro methylation of this tRNA agrees well with the pattern of methioninederived methylated bases in total tRNA of B. subtilis. Significant alterations were found in the relative distribution of tRNA isoacceptors from pactamycin-treated B. subtilis, which may reflect an accumulation of premature tRNAs.

We have studied the binding of [3H]pactamycin to Escherichia coli ribosomes as influenced by the formation of the f2 RNA protein synthesizing complex. Pactamycin binds rapidly at 0 °C to free 30-S ribosomal subunits and to 70-S ribosomes not attached to viral mRNA, but does not bind to 50-S ribosomal subunits. Binding to the 30-S ribosomal subunits has the highest specific activity and reaches a maximum of one molecule of antibiotic per particle as analyzed by sucrose gradients. Single ribosomes and polyribosomes bearing f2 RNA bind pactamycin very poorly, if at all, at 0 °C and overall binding is inversely related to the number of ribosomes attached to the viral mRNA. On the other hand, 30-S ribosomal subunits continue to bind pactamycin in the presence of f2 RNA. As shown with the use of polypeptide chain elongation inhibitors, active protein synthesis is not necessary for the inhibition of pactamycin binding to ribosomes and restoration of binding is produced by puromycin which causes polyribosome degradation. If pactamycin is present at 35 °C during mRNA attachment and polyribosome formation, the antibiotic will bind to mono- and polyribosomes but this binding is slower and partially sensitive to chain elongation inhibitors. Possible explanations for the two types of pactamycin binding to ribosomes are discussed.

The inhibitors of initiation pactamycin, cycloheximide, NaF, aurintricarboxylic acid and pederine have been found to inhibit the reaction of initiator tRNA with puromycin, as measured by the formation of N- formyl [ 35 S]methionylpuromycin . None of these inhibitors, with the exception of aurintricarboxylic acid, inhibits, however, the binding of the initiator tRNA to ribosomes, as measured by the binding of [35S]Met-tRNAf to unwashed or washed ribosomes. This binding is stimulated by the codon AUG; the stimulatory activity of this trinucleotide decreases with time even when ribosomes are kept at 0°C. The ribosomal components that bind initiator tRNA have been analyzed by sucrose gradient centrifugation; Met-tRNAf is bound by polyribosomes, 80-S ribosomes and 40-S ribosomal subunit. Those compounds that inhibit elongation as well as initiation, like cycloheximide, pactamycin and pederine, interfere presumably with a biochemical step common to both processes.

1. 1. The content of free nucleotides and nucleosides, determined by u.v. measurement, did not change significantly during 24 hours. 2. 2. The antibiotics cycloheximide, pactamycin, puromycin and fusidic acid inhibited [3H]leucine incorporation into rat liver proteins by about 90%: in the case of cycloheximide and pactamycin the effect continued at least over 3 or 4 hours, whereas even high doses of puromycin and fusidic acid were effective not longer than 90–150 minutes. 3. 3. When protein synthesis was inhibited by these antibiotics, [3H]uridine incorporation into three RNA fractions at 0700 and 1100 hours was altered: at 0700 hours, the labeling of the 65° C fraction was increased in all cases. At 1100 hours a relative decrease in the labeling of the 65° C fraction and a relative increase in the 4° C fraction could be observed. Puromycin had no effect at that time of day. 4. 4. Two hours after an injection of cycloheximide at 0700 hours the content of RNA in the nucleus increased, but remained at the normal level at 1500 hours. In both cases a pronounced increase followed by a decrease of the nuclear RNA content occurred within the first 60 minutes. 5. 5. The results support the assumption that protein-associated transport processes or permeabilities are involved in the circadian pattern of RNA labeling.

Three inhibitors of protein synthesis, cycloheximide, sparsomycin, and pactamycin, depressed to similar extents the formation of protein and DNA in the livers of partially hepatectomized rats at all the levels of the antibiotics tested. Inhibition of DNA replication was not due to reductions in the activities of the kinases that convert thymidine to TTP or of ribonucleotide reductase.

Pactamycin at low concentrations ( ⩽ 10 −6 M ) selectively inhibits protein synthesis by rabbit reticulocyte ribosomes which is dependent on the addition of initiation factors (KCl wash). The completion of existing nascent peptide chains is unaffected by these levels of antibiotic but higher concentrations also block chain elongation. These results contrast with those obtained with inhibitors such as chlortetracycline and sparsomycin which block the KCl wash-independent incorporation of amino acids into polypeptide as effectively as the KCl wash-dependent incorporation.

The mechanism of action of pederine, an inhibitor of protein synthesis extracted from the coleopter Paederus fuscipes , has been investigated. Polysome disaggregation takes place in intact cells incubated with low concentrations (10–20 ng/ml) of pederine. At higher concentrations protein synthesis is inhibited without concomitant run off of polysomes. Polysomes incubated in the presence of pederine are inactivated irreversibly. This has suggested that pederine binds to ribosomes. Puromycin releases only 50 % of the growing polypeptide chains in the presence of pederine. Moreover, pederine inhibits the formation of N -formyl-methionylpuromycin, which occurs when the reticulocyte cell free system is incubated with N -formyl-methionyl-tRNA in the presence of puromycin. These results have suggested that pederine may interfere with the translocation of both the initiator tRNA and peptidyl-tRNA from the aminoacyl to the peptidyl site. In addition, other ribosomal functions are probably affected by this drug. Polysome disaggregation caused by pederine results most likely from an imbalance between the rate of initiation and that of elongation. At low pederine concentrations initiation seems to be preferentially inhibited. After incubation with high levels of pederine polysomes are remarkably homogeneous in sedimentation behaviour.

Pactamycin levels in serum or urine could be determined either by a tissue culture assay or the disc-plate assay using Bacillus subtilis. After intravenous administration, the antibiotic level in the sera of various animal species decreased rapidly, with complete disappearance of the activity within 30 min. This decrease was not attributable to inactivation by blood or irreversible binding by tissues. Excretion in urine accounted for less than 1 per cent of the total antibiotic injected. In vitro, pactamycin was degraded by liver slices of dog at the rate of 487 μg/hr per g liver slice. If the counterpart of the in vitro-system operates in vitro, then the rapid inactivation of pactamycin and low recoveries in vitro can be explained. Cancer patients treated with the drug also showed low blood levels and poor recovery from urine; however, patients suffering from extensive liver metastasis had higher blood levels and excreted more pactamycin than those with no liver involvement. This could be attributed to the inability of the metastatically involved liver to degrade the antibiotic and could account for the somewhat more violent reactions of these patients to the drug.

Pactamycin and dextran sulphate were tested for their ability of inhibit, specifically, peptide chain initiation in a liver cell-free system. The incorporating ability of ribosomes from normal, hypophysectomized and growth-hormone-treated rats was then assayed in the absence of peptide chain initiation. 1. 1. Pactamycin inhibited protein synthesis but not, specifically, the initiation step. 2. 2. Dextran sulphate inhibited the poly U-stimulated incorporation of phenylalanine when added to ribosomes before the polymer. It did not inhibit incorporation in the absence of poly U if added after incubation had commenced. It was concluded that dextran sulphate specifically inhibited chain initiation. 3. 3. Dextran sulphate inhibited the basal level of incorporation when added to ribosomes before cell sap and other factors. It was concluded that some of the incorporation observed in the liver cell-free system results from initiation of peptide chain synthesis, but that this occurs only in the first moments of incubation. 4. 4. The differences in incorporating ability of liver ribosomes of normal, hypophysectomized and growth-hormone-treated rats persisted even when the initiation of peptide chain synthesis had been inhibited by dextran sulphate. It is suggested that growth hormone may act to increase the ratio of active to inactive ribosomes.

The effects of pactamycin on peptide chain initiation were studied using a reconstituted system comprised of reticulocyte ribosomes, poly r(A-U-G) messenger and peptide chain initiation factors, and the transfer of methionine from precharged Met-tRNA f Met into di-, oligo- and polymethionine products was measured. In the presence of low concentrations of pactamycin (10 −6 M), polymethionine synthesis in the above system was markedly reduced, and a concomitant increase in the synthesis of met-met and met-val dipeptides was observed. The latter dipeptide was presumably synthesized in response to endongenous hemoglobin messenger. Aurintricarboxylic acid and sparsomycin also inhibited polymethionine synthesis in response to poly r(A-U-G) messenger. Whereas aurintricarboxylic acid inhibited pactamycin induced dipeptide synthesis, sparsomycin had no significant effect on dipeptide synthesis in the presence of pactamycin.

In recent years several antibiotic inhibitors of aminoacyl-sRNA binding to bacterial ribosomes have been described (Suarez and Nathans, 1965; Hierowski, 1965; Chang, Sih and Weisblum, 1966; Pestka & al. , 1965; Kaji and Kaji, 1965; Ennis, 1966; Yamaguchi and Tanaka, 1967; Vazquez and Monro, 1967). Of these, only chlortetracycline (CTC) has been tested for its effect on the specific binding of peptidyl-sRNA to ribosomes (Rychlik, 1966; Goldberg and Mitsugi, 1967; Gottesman, personal communication, 1967). When ribosomes are in excess, poly A-dependent binding of polylysyl-sRNA to ribosomes is not affected by levels of CTC (lo* M) that maximally inhibit both binding of lysyl-sRNA to ribosomes and polypeptide formation. Under these conditions polylysyl-sRNA is presumed to bind directly to the donor site on the ribosome and can interact with puromycin in the absence of added soluble factors (Rychlik, 1966; Goldberg and Mitsugi, 1967; Gottesman, personal communication, 1967). It would be of considerable interest to have an agent that inhibits the binding of peptidyl-sRNA to the ribosome and thus interferes with initiation of polypeptide synthesis. We now report on the antibiotic pactamycin, an inhibitor of protein synthesis (Colono Bhuyan, 1967), which interferes with the specific binding of (14C) polylysylsRNA to E. coli ribosomes at concentrations that inhibit polylysine synthesis.

Evidence is presented to show that rabbit reticulocyte ribosomes contain a significant component of completed α-globin which is still attached to tRNA (α-globyl-tRNA). Additional data are presented to show that contamination by labeled supernatant hemoglobin or labeled α-globin from the free α-globin pool present in reticulocytes is not a significant factor in these results. Some 4.6% of the nascent α-globin chains are present as α-globin-tRNA, instead of 0.71% as predicted on the basis of the assumption that the size distribution of nascent globin chains is uniform. On the other hand β-globyl-tRNA comprises 0.69% of the nascent β-globin chains. This value coincides closely with the predicted value for nascent β-globin chains uniformly distributed in size along the polysome. Further evidence is presented to show that both α-globyl-tRNA and β-globyl-tRNA exhibit the kinetic properties expected for normal intermediates of soluble hemoglobin biosynthesis following inhibition of the initiation of protein synthesis by pactamycin.

Preferential labeling of COOH-terminal sequences in newly synthesized fibronectin was achieved by short term incorporation of radiolabeled amino acids in the presence of pactamycin, an inhibitor of polypeptide chain initiation. The labeled fibronectin was then cleaved with cathepsin D under conditions that yield a large (137,000-dalton) fragment that lacks collagen-binding properties, and a smaller (72,000-dalton) fragment that retains the ability of fibronectin to bind to collagen. Determination of the relative specific radioactivities of the two fragments leads us to conclude that the collagen-binding domain in fibronectin is located in the NH2-terminal third of the polypeptide chain and not in a COOH-terminal region as previously indicated by other structural studies.