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Pactamycin and sparsomycin, antitumor drugs which act synergistically with endotoxins, also potentiate the lethal toxicity of mice by Mycoplasma fermentans , strain K10. Sparsomycin (50 μg/20 g mouse, injected intraperitoneally 1 h after various doses of M. fermentans ) exerted a minimal degree of synergism with few extra deaths, but with prolonged appearance of nonlethal disease. Pactamycin (75 μg in the same protocol) increased susceptibility to Mycoplasma -induced lethal toxicity approximately 1,000-fold. The optimal response was noted when pactamycin was administered in the period between 6 h before and 3 h after the administration of 10 8 colony-forming units of viable mycoplasmas. Doses of 12.5, 25, or 50 μg increased severity of signs, but only 75 μg/mouse produced synergism of the lethal effect. Methylprednisolone partially negated the synergism when injected simultaneously with the pactamycin 1 h after the cells. The lethal synergistic effect occurred with four species of Mycoplasma in addition to M. fermentans .

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.

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 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.

The reversible inhibition of protein synthesis at the 75 –95% level in the early zygote of Fundulus results in a specific series of developmental failures dependent upon the times of inhibitor pulse initiation. The severity of the morphogenetic failure is inversely related to the time of initiation and directly to the length of the pulse. The defects reflect the time dependent serial order of events in morphogenesis. The defects range from failure of cleavage through disorders of blastulation, failure of axiation, anencephaly to microcephaly and are entirely predictable. With the exception of cleavage failure the pattern is identical with that found using pulses of actinomycin D in a similar manner. The agent used was pactamycin, an antibiotic which reversibly inhibits amino acid incorporation into protein by disturbing the assembly of the functional ribosomal complex. The significance of time dependent protein synthesis as an active expression in morphogenesis of similarly time dependent information flow via RNA synthesis is discussed.

Pactamycin does not inhibit the overall initiation factor- and GTP-dependent binding of [ 35 S]Met-tRNA f to rabbit reticulocyte ribosomes but does prevent the formation of Met-puromycin, provided that the antibiotic is present during the course of the binding reaction. These data indicate that pactamycin blocks the synthesis of a functional peptide-chain initiation complex. Sucrose density gradient centrifugation analysis of binding reactions shows that pactamycin causes the accumulation of an initiation complex on the smaller ribosomal subunit (smaller initiation complex), to which the 60S ribosomal subunit either cannot join or with which it forms a larger inactive 80S initiation complex that falls apart under the conditions used for isolation. The smaller initiation complex formed in the presence of pactamycin differs from the normal intermediate in peptide-chain initiation in being much more resistant to degradation by pancreatic RNase. In the presence of pactamycin, the inactive smaller complex can also form on mRNA to which an unaffected ribosomal couple is already attached, forming an oligoribosome lacking a larger ribosomal subunit or a “1.5 mer.” These effects of pactamycin can be overcome to a considerable degree by elevation of the Mg 2+ concentration.

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.

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.

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.

Antibiotics that interfere with protein synthesis have been distinguished by their effect on procaryotic or euraryotic cells and extracts, or both; by their binding or action on the smaller or larger ribosome subunit; and by their action on the Por A-site of the ribosomal-mRNA complex. Sparsomycin, chioramphenicol. gougerotin and amicetin all inhibit peptide bond formation and interact with the larger ribosomal subunit. Pactamycin, streptomycin and the tetracyclines bind to the 30S ribosomal subunit from bacteria; however pactamycin also interacts with the smaller (40S) ribosomal subunit from mammalian cells. At low concentrations (10-6 M) pactamycin inhibits primarily initiation of protein synthesis. On the other hand fusidic acid and thiostrepton inhibit translocation and interfere with the function of the larger ribosomal subunit. Also G factor has recently been implicated in fusidic acid action. Antibiotics interfere with nucleic acid synthesis and function either by complexing with DNA or by inactivating or competing with the other components involved in nucleic acid replication reactions. Antibiotics which complex with DNA include actinomycin, chromomycin, mithramycin. olivomycin, the anthracyclines and mitomycin. Antibiotics which directly inactivate the RNA polymerase include rifamycin, streptovaricin and streptolydigin. Another inhibitor, kanchanomycin, appears to combine features of the antibiotics which activate the DNA template and those which inactivate the RNA polymerase. In the antibiotics, nature has provided man with exquisitely potent and specific agents that have proved useful as drugs in the treatment of disease and as tools in the study of biological processes. Some antibiotics have been employed clinically for their antitumour and immunosuppressive properties, others for their effectiveness in infections of bacterial or viral origin. Although certain antibiotics work only on procaryotes (cells lacking a nucleus, such as bacteria), others are toxic only to eucaryotes (cells with a nucleus) or to both types of organisms. This discussion will be limited to a consideration of the mechanisms of action of only a few of the antibiotics that Present address: National Institute of Mental Health. Bethesda, Md, USA.

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.

Egg laying of the two-spotted spider mite, Tetranychus urticae (Koch), was effectively inhibited by the antibiotic actinomycin D, especially in the first 3 days after treatment, when there was no significant effect on mortality of the adults. After 7 days, the material was significantly acaricidal. Other antibiotics such as pactamycin, novobiocin, ampicillin, anthelmycin D. sarkomycin, humulon, puromycin, Carzinophilin® (from Streptomyces sahachiroi ), and mitomycin C were significantly toxic to adult females (mortalities of 76–100% in 7 days). Most of these antibiotics evidently caused some suppression of egg laying at lower dilutions that were sublethal to some adults. Some purine and pyrimidine derivatives and some dye compounds had no marked effects on the mites.

In tests of several antibiotics on the green peach aphid, Myzus persicae (Sulzer), reproduction (viviparous) was significantly inhibited in various degrees by cytovirin, cycloheximide, antimycin A, actinomycin D, pactamycin, hygromycin B, ampicillin trihydrate, vancomycin, fungichromin, and some other chemicals. In tests of a group of synthetic materials, 2-thiouracil and apholate caused some inhibition. Of these materials, the autibiotics novobiocin and anthelmycin werc notably toxic to the aphids.

The optimal fermentation conditions for the production of pactamycin, a new antitumor antibiotic, by Streptomyces pactum var. pactum were investigated. The optimal pH range for growth was 6.5 to 7.0. The optimal temperature for the growth of the culture and the production of the antibiotic was investigated in a medium containing Cerelose, blackstrap molasses, Pabst yeast, Kay Soy, CaCO 3 , and KCl. Since maximal growth and maximal production efficiency was obtained at 32 C, all subsequent fermentations were conducted at this temperature. Pactamycin was bound to the mycelium in different amounts, depending on the fermentation conditions, and could be extracted with acetone. Good yields (216 μg/ml) of pactamycin could be obtained in a medium containing Cerelose, soy-peptone, calcium carbonate, and potassium chloride. Analysis of the biochemical changes during fermentation indicated that pactamycin was produced during the later autolytic phase.

A crude mixture of polypeptide chain initiation factors (0.5 M KCl ribosomal wash) from reticulocyte ribosomes was fractionated by DEAE-cellulose column chromatography. Among several initiation factors obtained from the column, one factor eluting at 0.22-0.25 M KCl showed a remarkable ability to overcome the inhibition of Met-puromycin and 80S initiation complex formation caused by the antibiotic, pactamycin. Earlier experiments had shown that pactamycin does not prevent the binding of Met-tRNA f to the small ribosomal subunit but does interfere with the joining of the 60S ribosomal subunit to form the 80S initiation complex. A Lineweaver-Burk plot of initial rates of Met-puromycin formation showed that the interaction of the factor and pactamycin was of a competitive type. In the absence of the factor, [ 35 S]Met-puromycin was not synthesized and [ 35 S]Met-tRNA f bound only to the small ribosomal subunit. The amount of [ 35 S]Met-tRNA f bound to 80S ribosomes bearing endogenous mRNA and the amount of [ 35 S]Met-puromycin formed were directly related to the amount of factor added. Thus, this factor can be termed a “joining factor,” and a simple assay of its activity can be devised based on its ability to overcome the pactamycin inhibition of the puromycin reaction.

Pactamycin primarily inhibited protein synthesis in KB cells and Bacillus subtilis with much less effect on RNA or DNA synthesis. Pactamycin injected into rats at a level of 0.82 mg/kg caused about 50 per cent inhibition of both hydrocortisone- and tryptophan-induced increase in tryptophan pyrrolase in rat liver. In the cell-free system isolated from Escherichia coli , polyproline synthesis was inhibited 50 per cent at 0.75 μg/ml, although polyphenylalanine synthesis was not inhibited even at 100 μg/ml. Pactamycin did not affect the formation of prolyl-sRNA. Also, in agreement with the above observations, pactamycin inhibited the incorporation into protein of prolyl-sRNA and not of phenylalanyl-sRNA. These results indicate that pactamycin inhibited some step in the transfer of amino acyl-sRNA to ribosomes. Ribosomes isolated from the liver of pactamycin-treated rats had 30 per cent of the activity of control ribosomes, whereas the activity of the 105,000 g supernatant was not affected. Pactamycin, unlike chloramphenicol, inhibited protein synthesis by the rabbit reticulocyte cell-free system at high poly U level.

Puromycin induced the release of nascent peptides from ribosomes. We have studied this effect in vivo using protoplasts of Bacillus megaterium. Chloramphenicol, erythromycin and sparsomycin inhibited the reaction, but chlortetracycline, bottromycin and pactamycin did not. In the presence of chlortetracycline, both chloramphenicol and sparsomycin completely inhibited the puromycin reaction, whereas erythromycin allowed release of much of the nascent peptide. We suggest that chloramphenicol and sparsomycin inhibit the peptidyl transferase reaction, and that erythromycin may inhibit the translocation reaction(s).

Previous work has shown that translation of the encephalomyocarditis (EMC) viral ribonucleic acid (RNA) generates at least three primary products, polypeptides A, F, and C. The A and C polypeptides then undergo post-translational cleavages to complete the production of the stable viral polypeptides (delta, beta, gamma, alpha, G, I, F, H, and E). In this communication we show that A, F, and C are produced in equimolar amounts giving further support to the theory that the RNA of picornaviruses has only a single site for the initiation of protein synthesis. The biosynthesis of viral proteins in EMC virus-infected HeLa cells was studied in the presence of pactamycin at concentrations which preferentially inhibit the initiation of protein synthesis. The amount of each polypeptide formed during the residual period of protein synthesis observed after the addition of pactamycin was used as a criterion for ordering the genes on the viral RNA. The results obtained indicate that the primary gene products are ordered on the EMC viral RNA 5' --3' A-F-C and that the stable products are ordered delta-beta-gamma-alpha-G-I-F-H-E. Moreover, the intermediate chains B and epsilon map in the capsid region, whereas the intermediate chain D maps in the E region. This order is largely consistent with previously established relationships of the viral polypeptides and thus indicates that pactamycin is a valid tool for "genetic" mapping of polycistronic RNA molecules with single initiation sites.

Methionyl tRNA binding to wheat embryo ribosomes is catalyzed either by two initiation factors, or by one initiation factor together with a transfer factor. The bound Met-tRNA is reactive with puromycin, however, only when the binding reaction is catalyzed by the two initiation factors. If pactamycin is added to the binding reaction, the bound Met-tRNA is again unreactive with puromycin. These results suggest a two-step sequence for Met-tRNA binding, with puromycin reactivity occurring only as a consequence of the second step. Pactamycin appears to be a specific inhibitor of the reaction converting bound Met-tRNA to puromycin reactivity.

Pactamycin inhibits protein synthesis in B. amyloliquefaciens as expected but the cells quickly recover from this inhibition. This recovery is not due to acquisition of resistance; the results are compatible with the metabolic removal of the antibiotic.

THE marked sensitivity in vitro1 of various metabolic pathways in T. equiperdum to actinomycin D, mitomycin C, porfiromycin, phleomycin, and pactamycin indicated that these tumour-inhibitory antibiotics may be trypanocidal in vivo. Our results show this to be so with all except pactamycin, and also that phleomycin and porfiromycin in particular cured a high percentage of infected mice at certain dosages.

In a globin synthesizing system containing reticulocyte lysate and [ 35 S] met-tRNA f , low levels of pactamycin cause an accumulation of radioactivity on the monosomes and small oligosomes concomitant with the breakdown of polysomes. About 50% of the ribosome-bound radioactivity corresponds to methionyl-valine, the initial dipeptide of globin chains, with negligible amounts of tri- or other oligopeptides. This suggests that the site of its action is after the formation of the first peptide bond. The ribosome-bound radioactivity in the presence of sparsomycin, an inhibitor of chain elongation, is in di-, tri- and oligopeptides. Sparsomycin levels (10 −5 M) that cause almost complete inhibition of polypeptide synthesis have only a small inhibitory effect on the pactamycin-induced accumulation of methionyl-valine. Fusidic acid and chlortetracycline do not cause accumulation of any significant amounts of methionyl-valine.

By examination of the virus-specific polypeptides formed after the addition of pactamycin, an inhibitor of protein chain initiation, to infected cells, it has been possible to tentatively locate the virus coat proteins at the amino terminus of the large, virus-specific protein precursor, and, therefore, to assign the coat protein cistron to the 5′ end of the RNA.

Combinations of pactamycin and Salmonella typhosa 0901 endotoxin, administered simultaneously, killed more BALB/c mice than comparable doses of either agent alone The slopes of the dose-response curves for combinations of endotoxin and pactamycin were parallel to both that for endotoxin alone and the antitumor drug alone; therefore, no new mechanism of toxicity has been evoked by the combination. The synergistic toxicity of endotoxin and pactamycin was due to an in vivo interaction rather than a direct reaction between the two agents. Phenobarbital pretreatment protected the mice from toxicity of the antitumor agent alone but not from the lethal action of the combination. Pretreatment with endotoxin increased the resistance of the mice to both endotoxin alone and the combination. Third agents unable to protect mice from the synergistic toxicity of endotoxin and pactamycin were α[ p -(fluoren-9-ylidenemethyl)-phenyl]-2-piperidine-ethanol, neomycin, phenylbutazone, polymyxin B, and tybamate. Prednisolone pretreatment alleviated the toxicity of the combination. For the restricted series of killed bacteria and bacterial products tested for capability to enhance the toxicity of pactamycin, only gram-negative bacterial cells were potent. These results indicated that pactamycin rendered the mice more susceptible to endotoxin and that endotoxin was the causal lethal agent.

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.

In confluent cultures normal chick embryo fibroblasts become highty agglutinable by concanavalin A within 6 hours after their synthesis of protein is inhibited by cycloheximide, pactamycin, or emetine. When growing cells are similarly treated, they fail to become more agglutinable. Apparently, molecules which maintain the normal structure of the cell surface must be replaced continually when cell growth is contact inhibited.