Trypanosoma cruzi: inhibition of protein synthesis by nitrofuran SQ 18,506

SQ 18,506 is a nitrofuran compound related to the trypanocide Lampit. In vitro, radiolabeled leucine, uridine, and thymidine were incorporated into macromolecular protein, RNA, and DNA in order to study growth inhibition of Trypanosoma cruzi. Our findings suggest that the primary effect of the drug is on protein synthesis and not mediated solely by inhibition of RNA synthesis as indicated by prior studies. The drug was also found to reduce markedly the uptake of uridine into the nucleotide precursor pool but to affect only slightly the formation of aminoacyl-tRNA.     

The effects of inhibitors of adenylate cyclase and phosphodiesterase on D. discoideum aggregation.

The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ22536-treated cells exhibited normal accumulation of their adenylate cyclase activity as measured in cell lysates but the amebae did not synthesize cAMP. The ability of this drug to compete for cAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of cAMP, suggest that SQ22536 functions as a cAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or cAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external cAMP.     

Trypanocidal activity of a nitrovinylfuran derivative (SQ 18,506) in mice

The curative action of trans-5-amino-3[2-(5-nitro-2-furyl)-vinyl]-1,2,4-oxadiazole (SQ 18,506), administered orally to mice infected with Trypanosoma (Trypanozoon) brucei rhodesiense (Wellcome CT strain), was dose- and time-related. The optimal regimen was 200 mg/kg at 12-hr intervals for 3 days, beginning shortly after the mice were inoculated. The earliest manifestation of the action of SQ 18,506 in vivo occurred 36 hr after treatment was begun, exemplified by suppression of trypanosomal reproduction. Trypanocidal effects became evident only after two full days of drug administration. The effectiveness of a relatively coarse formulation of SQ 18,506 (16 μm median particle size) was significantly lower than that of other preparations in which the median particle size was approximately 4 μm. A single intramuscular injection of SQ 18,506 (1 g/kg) given prior to a trypanosomal infection which, if untreated, would kill all the mice within 3.5 days, had a relatively slow onset of prophylactic action, reaching a peak when the interval between treatment and exposure was about 3 days. Prophylactic action essentially disappeared when the interval between treatment and exposure was longer than one week.Biochemical studies in vitro of trypanosomes previously exposed in vivo to SQ 18,506 for increasing periods of time indicated that this compound can inhibit various pathways of trypanosomal nuclec acid and carbohydrate metabolism, the former apparently more sensitive to the effects of the drug than the latter. These suggested modes of action can account for the profile of parasitemia in infected mice to which SQ 18,506 was repeatedly administered, which first showed evidence of suppression of trypanosomal reproduction followed by a precipitous drop in the population.     

Accumulation of guanosine tetraphosphate induced by polymixin and gramicidin in Escherichia coli

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA⁺) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.     

Inhibition of Protein Synthesis: A Mechanism of Amebicide Action of Emetine and Other Structurally Related Compounds*

New amebicides usually have been discovered by empirical screening procedures while other drugs, such as emetine, were adopted after their clinical efficacy was demonstrated. Emetine, puromycin, and cycloheximide are amebicides recently shown to act as inhibitors of protein synthesis in animal cells; the present study was designed to determine the general relationship of this mode of action to amebicidal activity. Our results indicate that amebicides structurally related to emetine inhibit protein synthesis in Entamoeba histolytica. It seems likely that the lethality of many amebicides may be attributed to specific effects on macromolecular synthesis in E. histolytica.     

Lack of influence of mitochondrial genetical information on the multiplication of Herpes simplex virus in HeLa cells

Mitochondrial DNA synthesis in HeLa cells is inhibited by 0.2 μg ethidium bromide/ml whereas nuclear DNA synthesis is essentially unimpaired under the same conditions. The action of ehtidium bromide on mitochondrial DNA appears to be completed within 18 hours of exposure to the drug. Total cellular macromolecular synthesis under ethidium bromide is initially decreased and at later times slightly stimulated. Ethidium bromide pretreatment of HeLa cells did not significantly affect the multiplication of Herpes simplex virus as compared with that in control cells.     

In Vitro Characterization of the Anti-Bacterial Activity of SQ109 against Helicobacter pylori

The most evident challenge to treatment of Helicobacter pylori, a bacterium responsible for gastritis, peptic ulcers and gastric cancer, is the increasing rate of resistance to all currently used therapeutic antibiotics. Thus, the development of novel therapies is urgently required. N-geranyl-N''-(2-adamantyl) ethane-1, 2-diamine (SQ109) is an ethylene diamine-based antitubercular drug that is currently in clinical trials for the treatment of tuberculosis (TB). Previous pharmacokinetic studies of SQ109 revealed that persistently high concentrations of SQ109 remain in the stomach 4 hours post oral administration in rats. This finding, combined with the need for new anti- Helicobacter therapies, prompted us to define the in vitro efficacy of SQ109 against H. pylori. Liquid broth micro-dilution was used for susceptibility studies to determine the antimicrobial activity of SQ109 against a total of 6 laboratory strains and 20 clinical isolates of H. pylori; the clinical isolates included a multi-drug resistant strain. All strains tested were susceptible to SQ109 with MIC and MBC ranges of 6-10 µM and 50-60 µM, respectively. SQ109 killing kinetics were concentration- and time-dependent. SQ109 killed H. pylori in 8-10 h at 140 µM (2MBCs) or 4-6 h at 200 µM (~3MBCs). Importantly, though the kinetics of killing were altered, SQ109 retained potent bactericidal activity against H. pylori at low pH. Additionally, SQ109 demonstrated robust thermal stability and was effective at killing slow growing or static bacteria. In fact, pretreatment of cultures with a bacteriostatic concentration of chloramphenicol (Cm) synergized the effects of typically bacteriostatic concentrations of SQ109 to the level of five-logs of bacterial killing. A molar-to-molar comparison of the efficacy of SQ109 as compared to metronidazole (MTZ), amoxicillin (AMX), rifampicin (RIF) and clarithromycin (CLR), revealed that SQ109 was superior to MTZ, AMX and RIF but not to CLR. Finally, the frequency of resistance to SQ109 was low and electron microscopy studies revealed that SQ109 interacted with bacterial inner membrane and cytoplasmic content(s). Collectively, our in vitro data demonstrate that SQ109 is an effective monotherapy against susceptible and multi-drug resistant strains of H. pylori and may be useful alone or in combination with other antibiotics for development as a new class of anti- Helicobacter drugs.     

The Action of Mercaptoethanol on Cell Division in Synchronized Tetrahymena1

SYNOPSIS. Action of mercaptoethanol (ME) on cell division and macromolecular synthesis was examined in Tetrahymena synchronized for division. Cells continuously exposed to increasingly higher concentrations of ME divided with progressively longer division delays showing a dosage-dependent response to the agent. Division was blocked in 2 × 10^?2 M ME. Many cells cytolyzed in high concentrations of ME (4 × 10^?2 M); others became spherical and motility decreased. Non-delaying concentrations of ME (2 × 10^?2 M) had little or no effect on protein synthesis but decreased DNA and RNA synthesis 10 and 35%, respectively. Blocking concentrations inhibited incorporation of phenylalanine, thymidine and uridine 35, 60, and 85%, respectively. It is suggested that the mode of action of ME is mediated thru inhibition of macromolecular synthesis essential for cell division and thru inhibition of formation of disulfide bridges between protein subunits.     

Mode of Action of Pesticin

The mode of action of pesticin, a bacteriocin produced by many strains of Pasturella pestis, was studied. Pesticin action on macromolecular synthesis of a sensitive strain of Escherichia coli, strain , was found to have features similar to those of colicin E2-317 acting on the same strain. After exposure to pesticin, deoxyribonucleic acid synthesis was arrested and ribonucleic acid was degraded, but little effect was observed on protein synthesis. Pesticin, like colicin E2-317, induced lysogenic E. coli (P1), but, unlike the colicin, was active in the presence of dinitrophenol. Trypsin was found to reverse pesticin action up to 15 min after its addition at 40 C to E. coli . Pesticin action was studied on three sensitive bacterial strains, P. pestis 2C, P. pseudotuberculosis, and E. coli strain , which vary widely in their optimal growth temperature. P. pestis grows best at 29 C, P. pseudotuberculosis at 37 C, and E. coli at 40 C. It was found that pesticin action on all three strains was optimal at 40 C. Whereas the titer of pesticin was the same on all three strains when determined on agar, E. coli was the most sensitive to pesticin action in broth. No action of pesticin in broth on P. pseudotuberculosis was observed unless Ca ions were added. The effect was not immediate; that is, the cells had to be grown in a medium containing Ca⁺⁺ before they displayed sensitivity to pesticin.     

The application of pH-sensitive fluorescent dyes in lactic acid bacteria reveals distinct extrusion systems for unmodified and conjugated dyes

Membrane translocation is a crucial issue when addressing the activity of both cell-penetrating and antimicrobial peptides. Translocation is responsible for the therapeutic potential of cell-penetrating peptides as drug carriers and can dictate the killing mechanisms, selectivity and efficiency of antimicrobial peptides. It is essential to evaluate if the internalization of cell-penetrating peptides is mediated by endocytosis and if it is able to internalize attached cargoes. The mode of action of an antimicrobial peptide cannot be fully understood if it is not known whether the peptide acts exclusively at the membrane level or also at the cytoplasm. Therefore, experimental methods to evaluate and quantify translocation processes are of first importance. In this work, over 20 methods described in the literature for the assessment of peptide translocation in vivo and in vitro, with and without attached macromolecular cargoes, are discussed and their applicability, advantages and disadvantages reviewed. In addition, a classification of these methods is proposed, based on common approaches to detect translocation.     

Arylmethanol and thiosemicarbazone influence of plasmodial macromolecular synthesis and cell stability

The influence of mefloquine and additional antimalarial drugs on (3H)adenosine uptake, macromolecular synthesis, and cell stability was determined in rodent red cells parasitized with Plasmodium berghei. High arylmethanol to cell ratios induced lysis, whereas inhibition of macromolecular synthesis occurred at lower drug/cell ratios. A 2-acetylpyridine thiosemicarbazone rapidly suppressed (3H)adenosine uptake, and also blocked macromolecular synthesis by parasitized cells.     

Actions of vasopressin and isoprenaline on the ionic transport across the isolated frog skin in the presence and the absence of adenyl cyclase inhibitors MDL12330A and SQ22536.

1. The effects of both adenyl cyclase inhibitors (MDL12330A and SQ22536) have been studied on the ionic transport induced by vasopressin and isoprenaline across the frog skin. 2. MDL12330A inhibits the vasopressin action on the short-circuit current (SCC), confirming that this effect is cAMP-mediated. 3. On the other hand, isoprenaline action on the SCC is unaffected by MDL12330A. However, this lack of effect is not a sufficient argument against the role of cAMP in this action; in fact, as MDL12330A is also an inhibitor of cAMP phosphodiesterase, this action could mask the inhibitory effect of the drug on adenyl cyclase. 4. By using the other adenyl cyclase inhibitor (SQ22536), probably deprived of effect on the cAMP phosphodiesterase, we obtained a strong inhibition of isoprenaline action on the SCC. Thus we conclude that the actions of isoprenaline on the ionic transport across the frog skin are also cAMP-mediated.     

STUDIES ON THE MECHANISM OF ACTION OF PEDERINE

Pederine, a drug extracted from the coleopter Paederus fuscipes, inhibits the growth of in vitro cultured cell lines at concentrations of the order of 1.5 nanogram/ml. Cytological examination shows a generalized cytotoxic effect. Analysis of macromolecular syntheses by the use of radioactive precursors shows that pederine causes an almost immediate block of protein and DNA synthesis, without affecting RNA synthesis. The effects on the synthesis of the two types of macromolecules remain nearly simultaneous even at the lowest active concentrations of pederine. Studies with cell-free systems show that the drug inhibits protein synthesis, whereas it is ineffective on the DNA-polymerizing activity. It seems, therefore, that the drug acts primarily on the amino acid-polymerizing system, and that the effect on DNA is secondary. This idea is strengthened by the observation that puromycin, a specific inhibitor of protein synthesis, also affects promptly DNA synthesis of in vitro cultured cells. Other authors have shown the same phenomenon with a number of inhibitors of protein synthesis; the properties of pederine support, therefore, the view that continuous protein synthesis is necessary for the maintenance of DNA replication in higher organisms.     

Mode of Action of Novobiocin in Escherichia coli

The mechanism of action of novobiocin was studied in various strains of Escherichia coli. In all strains tested except mutants of strain ML, the drug immediately and reversibly inhibited cell division, and later slowed cell growth. The previously described impairment of membrane integrity, degradation of ribonucleic acid (RNA), and associated bactericidal effect were found to be peculiar to ML strains. The earliest and greatest effect in all strains was an inhibition of deoxyribonucleic acid (DNA) synthesis; RNA synthesis was inhibited to a lesser extent, and cell wall and protein synthesis were affected later. The inhibition of nucleic acid synthesis was accompanied by an approximately threefold accumulation of all eight nucleoside triphosphates. Since novobiocin does not inhibit nucleoside triphosphate synthesis, degrade DNA, or immediately affect energy metabolism, it must inhibit the synthesis of DNA and RNA by direct action on template-polymerase complexes.     

Effect of salvin on the synthesis of macromolecules in Staphylococcus aureus 209P

The effect of salvin and its active component, carnazolic acid, on the synthesis of macromolecular compounds in the cells of S. aureus 209P was studied. It was shown that the inhibitory action of salvin on the synthesis of peptidoglycane in the culture was defined by the presence of carnazolic acid in its composition. In the bactericidal concentration, carnazolic acid was twice as less active as salvin and inhibited incorporation of labeled precursors into RNA and protein. The findings grounded the conclusion that some nonidentified components of salvin with low antimicrobial activity contained in it in insignificant quantities had an additional inhibitory effect on the process. Comparative study of salvin and antibiotics with the known mechanisms of action such as benzyl penicillin or chloramphenicol revealed a certain similarity in the action of salvin and benzyl penicillin on incorporation of labeled precursors into the macromolecular compounds of S. aureus 209P.     

Macromolecular synthesis in E. coli, isolated mitochondria and L5178Y cells in the presence of hydroxyurea or formamidoxime

The effects of formamidoxime and hydroxyurea over a 10⁵ concentration range were studied on macromolecular synthesis in E. coli, L5178Y mouse leukemic cells, isolated rat liver mitochondria and isolated rat cerebral cortex mitochondria. In E. coli 2 mg per ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 20% and 17%, DNA synthesis by 91% and 96%, protein synthesis by 54% and 60% and lipopolysaccharide synthesis by 65% and 48%. In L5178Y mouse leukemic cells 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 41% and 24%, DNA synthesis by 90% and 97%, protein synthesis by 59% and 44% and glycoprotein synthesis by 83% and 50%. In isolated rat liver mitochondria 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 43% and 52%, DNA synthesis by 42% and 56% and protein synthesis by 18% and 30%. Glycoprotein synthesis was not affected. In isolated rat cerebral cortex mitochondria 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 50% and 44%, DNA synthesis by 59% and 66% and protein synthesis by 48% and 40%. Glycoprotein synthesis again was not affected. Lower concentrations of the drugs produced less inhibition of macromolecular synthesis in each of the systems.     

Effects of hormones and inhibitors of macromolecular synthesis on the follicle cells of Rhodnius

The response of the follicle cells of Rhodnius prolixus to JH in vitro was found to be independent of de novo macromolecular synthesis as exemplified by the failure of Actinomycin D, and puromycin to inhibit the response at any but the highest concentrations employed. C₁₈ JH was found to be a more effective gonadotropin than C₁₆ JH in this in vitro study. Neither methyl palmitate nor ecdysone mimiced or antagonised the action of JH on the follicle cells in vitro. Follicle cells of ovaries removed from allatectomised mated females failed to respond to C₁₆ JH and ecdysone in vitro.