Exogenous Spermine Research Articles

Effects of exogenous polyamine and trypanocides on the DNA polymerase activities from Trypanosoma brucei brucei, mouse thymus and murine leukemia virus

The effects of exogenously added spermine on activated (gapped) DNA-directed and poly(dC) - (dG) 12–18-directed DNA synthesis were tested on the chromatographically separated DNA polymerase activities of Trypanosoma brucei brucei. Activated DNA-directed DNA synthesis by the Peak I (eluting from DNA-agarose at 0.15 M KCl) and Peak II (eluting at 0.3 M KCl) polymerase was consistently inhibited or stimulated, respectively, by exogenous spermine. Kinetic analysis revealed that inhibition of the Peak I enzyme with respect to template DNA occurred by a mixed mechanism, while a major factor in the stimulation of the Peak II enzyme by spermine appeared to be the polyamine-mediated reversal of ‘substrate inhibition’ by DNA at concentrations above 10 μg/ml. The apparent K m values of Peak I and Peak II DNA polymerases for activated DNA were determined to be 5 and 0.5 μg/ml, respectively. In contrast to the results observed with activated DNA, activation of Peak II-enzyme-catalyzed poly(dC)-directed DNA synthesis was similar at all template-primer concentrations. Peak I enzyme-catalyzed poly(dG) synthesis was either inhibited or slightly stimulated by spermine, depending upon the presence or absence of heteropolymeric DNA, respectively. Dose-dependent inhibition of DNA-directed DNA synthesis catalyzed by T. b. brucei DNA polymerases, murine thymus DNA polymerase α, and Rauscher murine leukemia virus reverse transcriptase by trypanocides was examined to determine a possible mechanism of selective toxicity by such agents. The drugs Antrycide (quinapyramine), pentamidine, imidocarb, Berenil (diminazene aceturate), WR-199-385 [2,5-bis(4-guanylphenyl)furan-2HCl] and isometamidium inhibited DNA polymerases of the eucaryotic cells at approximately the same degree, and at similar concentrations. The presence of spermine in reaction mixtures did not spare any drug inhibition. Stimulation of reverse transcriptase activity was observed in the presence of Antrycide and imidocarb, however, this could be negated by stimulatory amounts of spermine present in the reaction mixture. The results, obtained using an activated DNA-directed assay system, suggest that trypanosomal DNA polymerases are not the selective target of trypanocidal drugs currently available.

Spermine and aminoguanidine protect cells from chromosome aberrations induced by adenovirus during the G2 phase of the cell cycle.

Adenovirus uncouples DNA replication from polyamine biosynthesis and causes chromosome aberrations in rodent cells. Addition of polyamines protected infected cells from this chromosome damage. Spermine was the only individual polyamine which protected. The diamine oxidase inhibitor aminoguanidine also protected. Neither compound detectably reduced synthesis of viral early proteins. The protective effects of spermine and aminoguanidine were not additive. Maximal protection was obtained when the compounds were added 4.5 h before mitosis, but significant protection was observed up to 1.25 h before mitosis. This suggests that the compounds act in G2. In vitro, spermine bound strongly to DNA and protected it from mild endonuclease attack, but aminoguanidine did neither. We propose that viral infection causes a deficiency in spermine during a critical period G2, possibly accompanied by an increase in endonuclease activity. The resulting chromosome damage can be prevented by adding exogenous spermine, or by inhibiting the oxidative degradation of endogenous spermine.

Evidence for a sub-population of rat T-lymphocytes highly resistant to methylglyoxal bis[guanylhydrazone] activity

Methylglyoxal bis[guanylhydrazone] (methyl-GAG) inhibits the S-adenosylmethionine decarboxylase (SAMD) activity in cells [1]. SAMD is essential for polyamine biosynthesis and its inactivation by methyl-GAG resulted in G 1-phase-arrest of mitogen-induced lymphocyte blastic transformation (uptake of [ 3H]thymidine). The inhibitory-dose 50 of methyl-GAG for [ 3H]TdR uptake by thymic T-lymphocytes (August strain rats) stimulated by phytohaemagglutinin (PHA) was 100 μM; in contrast, the sensitivity of lymph node or splenic lymphocytes was considerably greater (ID 50 = 6.0–7.0 μM). Whereas only about 62–66% of the uptake of [ 3H]TdR by PHA-treated extrathymic T-lymphocytes was inhibited by low concentrations of the drug, there remained 34–38% of the uptake that was unaffected until cells had been exposed to much higher drug concentrations. In all cases, arrest of [ 3H]TdR uptake was prevented by exogenous spermine (20 μM). Results suggested that a sub-population of T-lymphocytes (thymus, 100%; lymph node and spleen, 34–38%) were highly resistant to the drug activity. Thymic lymphocytes of Sprague-Dawley strain rats were somewhat more resistant to the drug (ID 50 = 500 μM). Low concentrations of methyl-GAG enhanced [ 3H]TdR uptake by the lymphocytes.

Polyamine-polyamine oxidase interaction: part of maternal protective mechanism against fetal rejection.

Human retroplacental blood serum significantly (p less than 0.01) suppresses the in-vitro uptake of 3H-thymidine--that is, synthesis of deoxyribonucleic acid--by spontaneously growing human lymphocytes in the presence of exogenous spermine, but only in concentrations with a higher polyamine oxidase activity than that found in maternal peripheral blood serum during pregnancy. These findings together with observations that the placenta is rich in spermine and that interaction of polyamine oxidase and substrate arrests cell proliferation suggest that such interaction might represent a localised immunoregulatory mechanism in the placental bed, which might contribute to the protection of the fetoplacental unit from possible maternal immune rejection.

Effects of spermine on the detection of induced forward mutation at the can1 locus in yeast: Evidence for selection against canavanine-resistant mutants

The effect of exogenous spermine tetrahydrochloride (0.5 mg/ml) on hydrazine- and nitrous acid-induced forward mutation to canavanine resistance ( CAN1 → cna1, normal to defective arginine permease) was examined in stationary-phase haploid Saccharomyces cerevisiae. Post-treatment cell division (specifically DNA replication) is required for hydrazine mutagenesis at this locus, whereas nitrous acid mutagenesis exhibits, in addition, asignificant post-treatment-independent component. Spermine addition only during mutagenic treatments in buffer did not affect mutagen cytotoxicity, but did not result in a slight yet consistent decrease in induced mutation frequencies. Addition of spermine to the yeast extract-peptone-dextrose (YEPD) post-treatment growth medium resulted in dramatic reductions of induced mutation frequencies, which could be alleviated by pregrowth in spermine-containing YEPD. Such a medium was found to cause an apparent temporary growth inhibition for almost 40 h, after which the growth rate of the culture increased rapidly. Cultures “recovering” from spermine inhibition were no longer inhibitable by spermine in fresh medium, suggesting an outgrowth of spontaneous and/or induced spermine-resistant derivatives. Genetic analysis of one isolated revealed a single dominant nuclear gene conferring resistance by some means other than defective spermine uptake. Growth of this mutant was only slightly inhibited by spermine (20% increase in doubling time), while mutation expression remained high. Results of competitive growth experiments indicated that spermine-containing YEPD exerted a selection pressure against canavanine-resistant cells, while YEPD by itself did not. The mechanism for this selection is not presently understood. With respect to replication-dependent induced mutation at CAN1, our initial observation of a strong apparent antimutagenic action of spermine was found to be best explained by this specific selection against can1 mutants. This underscores the need for caution in the interpretation of experiments designed to study physiological modification of mutagenic potential.

Studies on vaginal malodor. II Laboratory model.

Various factors contribute to the formation of malodor in intravaginal collagen sponges (CS) worn for several days by sexually active persons. To study this problem we developed an in vitro model: CS was infiltrated with semen and incubated at various pH's or in the presence of various drugs, at 37° and 95% O2-5% CO2 in humid atmosphere. The odor was measured by subjective olfactometry as well as quantitated and characterized by gas liquid chromatography. Based on previous findings concerning the disappearance of polyamines from the semen in sponges with malodor and present detection of maximal malodor formation at pH 7.4 and absence of malodor with heat-inactivated semen we propose that malodor forms through the enzymatic oxidation of polyamines or diamines possibly by diamine oxidase (DAO) of the semen. Direct assay of DAO showed that all factors inhibiting or stimulating the malodor affected the activity of DAO in the same direction. Thus, while copper ions were stimulatory to odor and DAO activity, zinc ions, acid environment (3.4 pH), exogenous spermine administration, isoproniazid and pargyline, both known inhibitors of MAO's and DAO's, reduced malodor and DAO activity. We found that more intensive malodor developed in sponges made of polyurethane, cellulose or spongin (sea sponge) than of collagen. Whole semen caused greater malodor formation than prostatic fluid and seminal plasma. Both washed spermatozoa and vaginal secretion did not induce any subjectively detectable offensive odor in sponges. Addition of a mixture of nonpathogenic microorganisms to the semen did not enhance the formation of subjectively detectable malodor. GLC showed characteristic differences between volatile compounds formed by ejaculate and bacteria. While most of the malodor formed by the semen-CS system was enzymatically related, some evidence indicates a portion of malodor to be nonenzymatic process. It is concluded that the most feasible method preventing the semen related vaginal malodor is to keep the CS acidic (pH 5) and to impregnate sponges with low concentrations of zinc sulfate (40 mg/sponge), unless the sponge is removed 24 h after the intercourse, washed and reinserted.

Spermidine biosynthesis during germination and subsequent vegetative growth of Bacillus megaterium spores.

Spermidine biosynthesis was extremely low early in germination of Bacillus megaterium spores and the spermidine level remained constant. Rapid synthesis began after 130 min and thereafter accounted for the increase in spermidine level which began at this time. Biosynthesis was greatly (>84%) diminished by exogenous spermine or spermidine. Arginine and ornithine were both converted efficiently into spermidine, but arginine was the more immediate precursor as shown by isotope competition studies and by the absence of ornithine decarboxylase and the presence of arginine decarboxylase. Exogenous putrescine was not incorporated into spermidine, although it was taken up rapidly and degraded.