Extreme thermophiles produce two types of unusual polyamine: long linear polyamines such as caldopentamine and caldohexamine, and branched polyamines such as quaternary ammonium compounds [e.g. tetrakis(3-aminopropyl)ammonium]. To clarify the physiological roles of long linear and branched polyamines in thermophiles, we synthesized them chemically and tested their effects on the stability of ds (double-stranded) and ss (single-stranded) DNAs and tRNA in response to thermal denaturation, as measured by differential scanning calorimetry. Linear polyamines stabilized dsDNA in proportion to the number of amino nitrogen atoms within their molecular structure. We used the empirical results to derive formulae that estimate the melting temperature of dsDNA in the presence of polyamines of a particular molecular composition. ssDNA and tRNA were stabilized more effectively by tetrakis(3-aminopropyl)ammonium than any of the other polyamines tested. We propose that long linear polyamines are effective to stabilize DNA, and tetrakis(3-aminopropyl)ammonium plays important roles in stabilizing RNAs in thermophile cells.
The thymidine stereoisotopomer, (2′S)-[2′-2 H]thymidine, which incorporates deuterium in the S configuration at the furanosyl 2′ carbon, has been synthesized and its vibrational spectra have been recorded and compared with those of normal thymidine. Infrared and Raman spectra were collected from crystalline powders, the latter using 1063-and 514.5-nm excitations; ultraviolet resonance-Raman spectra were collected from aqueous solutions using 244-nm excitation. The results show, remarkably, that virtually all normal modes of thymidine involve some degree of vibrational coupling between the thymine base residue and the deoxyribose moiety. Nevertheless, systematic assignments and correlation of the spectral frequencies of thymidine and (2′S)-[2′-2 H]thymidine have been accomplished. A finding of importance for nucleic acid structure applications is that many prominent Raman marker bands of thymidine, assigned previously as thymine ring vibrations, in fact involve appreciable coupling with the C2′ methylene group of the attached sugar. Vibrational coupling between the base and sugar groups implies frequency dependence upon sugar conformation and allows the bands in question to be exploited as markers of deoxyribose ring pucker and glycosyl orientation in Raman spectra of DNA, antiviral drugs, and other thymine-containing nucleoside analogues. The present results also enable unambiguous and novel assignment of spectral bands to specific vibrational modes of the C2′ methylene group of thymidine as follows: C2′H 2 antisymmetric stretching (2995 cm -1 ), symmetric stretching (2956 cm -1 ), scissoring (1404 cm -1 ), and wagging (1174 cm -1 ). Additionally, probable assignments are deduced for the C2′H 2 twisting (1103 cm -1 ) and rocking modes (898 cm -1 ). Normal mode assignments are also proposed for many other vibrational bands of thymidine.
In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase ؍ agmatine ureohydrolase) accumulated N 1 -aminopropylagmatine (N 8 -amidino-1,8-diamino-4-azaoctane, N 8 -amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N 1 -aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N 1 -Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N 1 -Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N 1 -Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N 1 -aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N 1 -aminopropylagmatine to spermidine but does not act on agmatine.Polyamines play important roles in cell proliferation and cell differentiation. Common polyamines such as putrescine, spermidine, and spermine are distributed ubiquitously in cells and tissues at relatively high concentrations (1, 2).Thermus thermophilus, of which the genome project was completed using two strains, HB8 and HB27 (Structural-Biological Whole Cell Project at www.srg.harima.riken.go.jp/ thermus/j_index.htm and see Ref. 3, respectively), produces a variety of polyamines including unusually long polyamines and branched ones (4) (see Fig. 9C). These long and branched polyamines have a marked effect of protecting and stabilizing nucleic acids (5, 6) and of activating cell-free polypeptide synthesis at high temperature (7-9).In many organisms, such as bacteria, yeast, animals, and plants, the first step of polyamine biosynthesis is production of putrescine by decarboxylation of L-ornithine (see Fig. 9A). An additional or alternative pathway of putrescine biosynthesis that is often seen in plants and sometimes in bacteria is decarboxylation of L-arginine followed by hydrolysis of agmatine. Agmatine ureohydrolase or agmatinase, coded by the speB gene, catalyzes this second reaction. The next step is production of spermidine and spermine by the addition of an aminopropyl group to putrescine and spermidine, respectively. This reaction is catalyzed by spermidine or spermine synthase (putrescine/spermidine aminopropyltransferase) coded by the speE gene (1).To investigate the polyamine biosynthetic pathway in T. thermophilus, we constructed a disruption strain of the speB gene homolog of T. thermophilus. Disruption of the speB gene homolog resulted in drastic reduction of triamines, longer and branched polyamines without accumulation of agmatine, and in accumulation of an unknown compound. Double disruption of speB and speE gene homologs resulted in disappear...
Several 2-alkylthio- and 2-benzylthio derivatives of 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB) have been designed and synthesized from 5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole-2-thione. All compounds were evaluated for activity against human cytomegalovirus (HCMV) and/or herpes simplex virus type-1 (HSV-1). Three different cytotoxicity assays were used to determine if the compounds were toxic to uninfected cells. Most of the 2-alkylthio compounds were either inactive against HCMV and HSV-1 or were active only at concentrations at or near those which produced toxicity in uninfected cells. The best separation between activity against HCMV and cytotoxicity was observed with the 2-benzylthio analog 7. This prompted us to synthesize the substituted 2-benzylthio analogs 11-23 using a Topliss Tree approach. None of these compounds were more active than compound 7; most of the analogs were weakly active against both HCMV and HSV-1, but the activity was not separated from cytotoxicity. On the basis of both antiviral and cytotoxicity data, compound 7 was the best compound in the series. It was more active against HCMV than DRB (the 2-unsubstituted analog), acyclovir, and foscarnet, but it was less active than ganciclovir.
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