Mechanism of Action of Showdomycin

The effect of showdomycin on the syntheses of deoxyribonucleotides from various pyrimidine and purine derivatives was studied in cell-free systems from E. coli.

The formations of deoxycytidine phosphates, deoxyuridine phosphates, deoxyguanosine phosphates and deoxyadenosine phosphates from the corresponding ribonucleoside diphosphates were all inhibited by low concentrations of showdomycin. The formation of deoxythymidine phosphates from dUMP was also very susceptible to the antibiotic. These inhibitory actions of showdomycin could be reversed by a sulfhydryl compound (mercaptoethanol) but not by nucleosides, in contrast to a previous finding that the inhibitory action of this antibiotic on the cell growth was reversed by compounds belonging to both of these groups.

N-Ethylmaleimide (NEM), a thiol reagent which has a structure related to the aglycone moiety of showdomycin, was also found to be a potent inhibitor of both the reduction of CDP and the methylation of dUMP as showdomycin. A mercurial thiol reagent, p-chloromercuribenzoic acid (PCMB), however, was found to be inactive against the methylation of dUMP although the salvage synthesis of dUMP was inhibited by low concentrations of this reagent.

The formations of deoxythymidine phosphates and of deoxyuridine phosphates from their respective pyrimidine bases and a deoxyribosyl donor were quite resistant to showdomycin.     

Mechanism of Action of Showdomycin

Among the syntheses of DNA, RNA and protein in Escherichia coli cells, the DNA synthesis was found to be preferentially inhibited at lower concentrations of showdomycin. At such lower concentrations of this antibiotic, serious decreases in the synthesis of deoxycytidine phosphates and in de novo synthesis of deoxythymidine phosphates were found in parallel with the decrease in the synthesis of DNA, although the syntheses of other pyrimidine nucleotides were not significantly diminished. The salvage synthesis of deoxythymidine phosphates was very resistant to this antibiotic. The inhibitory action of this antibiotic on DNA synthesis could be reversed by the concomitant addition of a thiol compound or a nucleoside. When a nucleoside was added after the completion of the inhibition by showdomycin, the recovery of the DNA synthesis from the inhibition was detected only after the recovery of the syntheses of pyrimidine ribotides, pyrimidine deoxyribotides and RNA have become distinct.     

Mechanism of Action of Showdomycin

¹⁴C-Labelled showdomycin was rapidly taken up by Escherichia coli K-12 cells. The showdomycin uptake was highly temperature dependent, sensitive to azide and N-ethyl-maleimide, but was only partially inhibited by treatment with high concentration of iodoacetic acid.

The uptake of showdomycin was inhibited by a wide variety of nucleosides but not by purine and pyrimidine bases, nucleotides, ribose or ribose-5-phosphate. The inhibition of showdomycin uptake by adenosine was of a competitive type.

Since nucleosides inhibited the uptake of showdomycin but did not facilitate its efflux, they must play a role of inhibitors to the entry of the antibiotic into cells.

Removal of extracellular showdomycin by washing, or inhibition of its subsequent entry into cells by the addition of nucleosides or sulfhydryl compounds resulted in a rapid decrease in the intracellular level of the antibiotic during subsequent incubation.     

Synthesis and Studies of 3′-C-Trifluoromethyl-β-D-ribonucleosides Bearing the Five Naturally Occurring Nucleic Acid Bases

Abstract

3′-C-Trifluoromethyl-β-D-ribonucleoside derivatives bearing the five naturally occurring nucleic acid bases have been synthesized. All these derivatives were prepared by glycosylation reactions of purine and pyrimidine bases with a suitable peracylated 3-C-trifluoromethyl ribofuranose precursor. After deprotection, the resulting title nucleoside analogues were tested for their inhibitory properties against the replication of HIV, HBV and several RNA viruses. However, none of these compounds showed significant antiviral activity.     

Formation of l-Butyl-5-hydroxy-methylpyrrole-2-carboxaldehyde in the Maillard Reaction between Lactose and n-Butylamine

A new mutant Shm^r-001-1 has been isolated by treating showdomycin-resistant mutant Shm^r-001 cells with N-methyl-N′-nitro-N-nitrosoguanidine. This mutant was resistant to high level of showdomycin, and took up practically no showdomycin and little pyrimidine nucleosides, and it showed different ability to take up purine nucleosides. Strains Shm^r-001–1, Shm^r-001, and K–12 (wild type) were compared: in susceptibility to showdomycin, in ability to take up the antibiotic and various nucleosides, on effects of other nucleosides on entry of particular nucleosides, and on kinetics of the entry of nucleosides and showdomycin. From these experiments, at least three different nucleoside transport systems were observed in Escherichia coli K–12 cells: the first system was common to adenine nucleosides, pyrimidine nucleosides, and showdomycin; the second system was common to adenine nucleosides, guanine nucleosides, inosine, pyrimidine nucleosides, and showdomycin; and the third system was common to adenine nucleosides, guanine nucleosides, and inosine. The first system was not observable in Shm^r-001 cells. In Shm^r-001–1 cells both the first and the second systems were no longer detectable but the third system was found to be active.     

The effects of oestradiol on nucleoside transport in rat uterus

1. By using the non-metabolized cytidine analogue, cytosine arabinoside, it was possible to examine the mechanism of nucleoside transport in the immature rat uterus in the absence of intracellular utilization of the permeant. It was demonstrated that the uptake of cytosine arabinoside is not accumulative and that it can be competitively inhibited by the addition of a second nucleoside, uridine. Introduction of a concentration gradient of uridine from the medium towards the intracellular water promotes the counterflow of cytosine arabinoside out of the cells against its concentration gradient. These properties indicate that a facilitated-diffusion system is involved in nucleoside transport in the uterus. Further counterflow studies have shown that the transport system has a broad specificity for purine and pyrimidine nucleosides and that it is distinct from the processes that mediate the uptake of sugars, amino acids and purine and pyrimidine bases. 2. Oestradiol injection has no effect on the initial rate of cytosine arabinoside uptake in vitro. The increased amount of the analogue taken up per uterus is simply due to the expansion of the uterine volume that accompanies oestrogen action. 3. It is concluded that the striking increase in uridine uptake, observed in vivo in uteri from oestrogen-treated rats, does not result from an increase in the initial rate of nucleoside transport into the intracellular space of the tissue.     

Enzymatic Synthesis of 2′,5′-Dideoxv Purine Nucleosides and Related Compounds

Abstract

A wide range of 2′,5′-dideoxy-nucleosides, including 6- substituted purine, pyrazolo[3,4-d]pyrimidine and 1-deazapurine derivatives, has been enzymatically prepared using purine nucleoside phosphorylase. Specificity towards cleavage by bacterial versus mammalian purine nucleoside phosphorylase was evaluated.     

LACK of DNA SYNTHESIS INHIBITORY ACTIVITY IN an IMMUNOSUPPRESSOR OBTAINED FROM SPLEEN

A tissue extract derived from bovine spleen which is an immunosuppressor in vivo inhibits the incorporation of the two DNA pyrimidine nucleosides but does not inhibit the incorporation of purine nucleosides. the results indicate that the immunosuppressive action of the spleen extract is not mediated via inhibition of cell division.     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K(m) = 13.2 microM) and D-inosine (K(m) = 253 microM). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K(m) > 500 microM). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation.     

Factors Which Increase Acid Production in Milk by Lactobacilli

The stimulation by yeast extract of acid production in milk by various lactobacilli was studied. It was found that supplementing milk with purine and pyrimidine bases and amino acids allowed nearly maximal acid production by Lactobacillus bulgaricus strain 7994, L. acidophilus 4796, 4356, and 4357, and L. leichmannii 326 and 327. Further supplementation with deoxyribotides allowed maximal acid production by L. acidophilus 204, but L. acidophilus 207 required adenosine or adenylic acid. L. casei strain 7469 showed no appreciable response to the amino acids or purine and pyrimidine bases, and is presumed to require an unidentified factor in corn steep liquor.     

Configurational effects on conformational properties of cyclic nucleotides. I. Theoretical calculations of conformer preferences in α-nucleoside 3′,5′ cyclic monophosphates

A comparative study has been made of the configurational effects on the conformational properties of α- and β-anomers of purine and pyrimidine nucleoside 3′,5′,-cyclic monophosphates and their 2′-arabino epimers. Correlation between orientation of the base and the 2′-hydroxyl group have been studied theoretically using the PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The effect of change in ribose puckering on the base-hydroxyl interaction has also been studied. The result show that steric repulsions and stabilizing effects of intramolecular hydrogen bonding between the base and the 2′-hydroxyl (OH) group are of major importance in determining configurations of α-anomers and 2′-arabino-β-epimers. For example, hydrogen bonding between the 2′-hydroxyl group and polar centers on the base ring is clearly implicated as a determinant of syn-anti preferences of the purine (adenine) or pyrimidine (uracil) bases in α-nucleoside 3′,5′-cyclic monophosphates. Moreover, barrier heights for interconversion between conformers are sensitive to ribose pucker and 2′-OH orientations. The result clearly show that a change in ribose-ring pucker plays an essential role in relieving repulsive interaction between the base and the 2′-hydroxyl group. Thus a C2′-exo-C3′-endo (₂T³) pucker is favored for α-anomers in contrast with the C4′-exo-C3′-endo (₄T³) from found in β-compounds.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

New perspectives on the roles of pyrimidines in the central nervous system

Since 1956, when exogenous uridine and cytidine were found to be necessary for the maintenance of perfused rat brain function, the co-existence of de novo synthesis, salvage pathways and removal of pyrimidine bases in the CNS has been a controversial subject. Here, we review studies on metabolites and enzymes of pyrimidine metabolism through more than 60 years. In view of known and newly-described inherited pyrimidine and purine disorders - some with complex clinical profiles of neurological impairments - we underline the necessity to investigate how the different pathways work together in the developing brain and then sustain plasticity, regeneration and neuro-transmission in the adult CNS. Experimentally, early incorporation studies in animal brain slices and homogenates with radio-labelled nucleosides or precursors demonstrated salvage activity or de novo synthesis. Later, the nucleoside transporters and organic anionic transporters underlying uptake of metabolites and anti-pyrimidine drugs in the CNS were identified. Recently, the expression of de novo enzymes in glial cells and neurons was verified using (immuno) histochemical and in-situ-hybridization techniques. Adult brain was shown to take up or produce all pyrimidine (deoxy) ribonucleosides or, after uptake and phosphorolysis of nucleosides, to make use of ribose for different purposes, including energy. More recently, non-canonical pyrimidine bases (5mC, 5hmC) have been found most notably in brain, pointing to considerable postreplicative DNA metabolism, with the need for pyrimidine-specific enzymes. Even more perspectives are emerging, with advances in genome analysis and in the manipulation of expression from the gene.     

Showdomycin,a nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase

Showdomycin [2-(β-d-ribofuranosyl)maleimide] is a nucleoside antibiotic containing a maleimide ring and which is structurally related to uridine. Showdomycin inhibited rat brain (Na⁺ + K⁺)-ATPase irreversibly by an apparently bimolecular reaction with a rate constant of about 11.01·mol^?1·min^?1. Micromolar concentrations of ATP protected against this inhibition but uridine triphosphate or uridine were much less effective. In the presence of K⁺, 100 μM ATP was unable to protect against inhibition by showdomycin. These observations show that showdomycin inhibits (Na⁺ + K⁺)-ATPase by reacting with a specific chemical group or groups at the nucleotide-binding site on this enzyme. Inhibition by showdomycin appears to be more selective for this site than that due to tetrathionate or N-ethylmaleimide. Since tetrathionate is a specific reactant for sulfhydryl groups it appears likely that the reactive groups are sulfhydryl groups. The data thus show that showdomycin is a relatively selective nucleotide-site-directed inhibitor of (Na⁺ + K⁺)-ATPase and inhibition is likely due to the reaction of showdomycin with sulfhydryl group(s) at the nucleotide-binding site on this enzyme.     

Regulation of pyrimidine biosynthesis by purine and pyrimidine nucleosides in slices of rat tissue

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [¹⁴C]-NaHCO₃ into orotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia-stimulated incorporation of [¹⁴C]NaHCO₃ into orotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, but not uridine, was found to have the additional effects of inhibiting the conversion of [¹⁴C]orotic acid to UMP and depleting the tissue slices of PRPP. Since PRPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [¹⁴C]NaHCO₃ into orotic acid and the incorporation of [¹⁴C]orotic acid into UMP by depriving CPSase II and OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de novo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of orotic acid to UMP in tissue slices.     

The Assimilation of Nitrate-nitrogen by Nitrogen-starved Cells of Platymonas (Tetraselmis) striata (Prasinophyceae)

Nitrate refeeding of nitrogen-starved cells of Platymonas striataresulted in approximately a doubling of average cellular nitrogenwithin 24 h. All the nitrate-nitrogen removed from the culturemedium could be accounted for as non-nitrate nitrogen withincells. Thus no significantly sized nitrate pool existed in Platymonasstriata and no assimilated nitrogen was lost from the cellsto the medium over the 48 h period studied. The slight fallin average cellular nitrogen which occurred from 24 to 36 hcould be attributed to cell division. The majority (70–80per cent) of the assimilated nitrate was recovered in the trichloroaceticacid (TCA)-insoluble fraction. There was some increase in thepercentage of nitrogen found in the TCA-soluble fraction duringthe period of most rapid nitrate assimilation (0–24 h).This presumably reflects an inability of the cells to assimilatelow-molecular-weight metabolic intermediates into macromoleculesat the same rate at which they were being formed. The majorityof the TCA-soluble fraction could be accounted for in termsof amino acids, purine and pyrimidine bases and ammonia. Cellswith adequate nitrogen nutrition seemed to maintain amino acidand purine + pyrimidine base nitrogen pools of about 0.8–0.9and 0.3–0.4 pg per average cell respectively. Algal amino acids, algal purine and pyrimidine bases, algal ammonia     

Production of Ribotides of 4-Hydroxy- and 4-Aminopyrazolo[3, 4-d]pyrimidine by Brevibacterium ammoniagenes

Brevibacterium ammoniagenes ATCC 6872 was previously reported to accumulate large amounts of IMP, AMP, ADP, ATP, GMP, GDP and GTP from the corresponding purine bases. The organism was also reported to convert various derivatives of purine and 8-azapurine to the corresponding ribotides.

Using the similar process, ribotidation of pyrazolo[3, 4-d]pyrimidines was attempted, and it was found that the same organism was able to produce remarkable amounts of 4-hydroxy-1-β-d-ribofuranosylpyrazolo[3, 4-d]pyrimidine 5′-monophosphate (HPP-RP) from 4-hydroxypyrazolo[3, 4-d]pyrimidine (HPP, allopurinol) and 4-amino-1-β-d-ribofuranosylpyrazolo[3, 4-d]pyrimidine 5′-monophosphate and 5′-diphosphate from 4-amino-pyrazolo[3, 4-d]pyrimidine.

The crystals of HPP-RP (Na-salt) were isolated from the cultured broth of Br. ammoniagenes incubated with HPP, and characterized based on UV-spectra, IR-spectrum, NMR and others.

It was also found that HPP-RP was converted to the corresponding riboside by hydrolysis in aqueous solution (pH 4.0 ~ 9.0) for 6 hr at 140°C. The hydrolysis of HPP-RP was also accomplished with various organisms.     

A New Synthesis of Hepialone [(R)-2-Ethyl-6-methyl-2,3-dihydro-4H-pyran-4-one], the Principal Component from Male Sex Scales of the Ghost Moth,Hepialus californicus B

Microorganisms that produce 5-methyluridine (ribothymidine) directly from purine nucleosides and thymine were screened from our stock cultures. Of the 400 strains tested, Erwinia carotovora AJ- 2992 was found to possess the most potent ability as to production of 5-methyluridine from guanosine and thymine. In the presence of intact cells of Er. carotovora AJ-2992 as the enzyme source, 222 mm 5-methyluridine was produced from 300 mm guanosine and 300 mm thymine at 60°C on 48 hr incubation. The enzymatic production of 5-methyluridine by Er. carotovora AJ-2992 was found to involve the following two successive reactions via ribose-1-phosphate as an intermediate: phosphorolysis of purine nucleosides to ribose-1-phosphate and purine bases by purine nucleoside phosphorylase, followed by condensation of ribose-1-phosphate and thymine into 5-methyluridine by pyrimidine nucleoside phosphorylase.     

Use of Tetrahymena pyriformis to Evaluate the Effects of Purine and Pyrimidine Analogs1

SYNOPSIS Eighty-four purine and pyrimidine analogs were evaluated for growth inhibition of Tetrahymena pyriformis. The most toxic were 2-fluoroadenine, 2-fluoroadenosine, 6-methylpurine, a series of 5-fluoropyrimidines, and a series of adenine derivatives substituted in the 9-position. 2-Fluoroadenine was metabolized to the ribonucleoside triphosphate and was incorporated into nucleic acids; its inhibition of growth was reversed by high levels of adenine. 6-Methylthiopurine ribonucleoside was phosphorylated, but only to the monophosphate derivative. Contrasting T. pyriformis with mammalian cells gave clues to the mechanism of action of some of the agents. 6-Mercaptopurine, 6-methylthiopurine ribonucleoside, and 6-thioguanine, all potent pseudofeedback inhibitors of de novo purine biosynthesis in mammalian cells, are not toxic to T. pyriformis, which lacks the de novo purine pathway; this implies that inhibition of de novo purine biosynthesis by them underlies their growth inhibition of mammalian cells.