Clustered genes for ribosomal ribonucleic acids in Escherichia coli.
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Centrifugation, Density Gradient↗
Biomedical subjects
S Spadari
Publications and source records attributed to S Spadari.
Isolation of the hybrid between ribosomal RNA and DNA of Bacillus subtilis.
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Bacillus subtilis↗
Some 6-aza-5-substituted-2'-deoxyuridines show potent and selective inhibition of herpes simplex virus type 1 thymidine kinase.
The synthesis and X-ray crystal structures of a series of 5-substituted-6-aza-2'-deoxyuridines is reported. These nucleoside analogues inhibit the phosphorylation of thymidine by HSV-1 TK but have no effect on the corresponding human enzyme. Detailed examination of one analogue proves it to be a competitive inhibitor of thymidine with a Ki of 0.34 microM and is a very poor substrate. The analogues are not substrates for the enzyme and also do not inhibit the degradation of thymidine by thymidine phosphorylase. Molecular modelling showed that the inhibitors fit well in the active site of HSV-1 TK, provided the conformation of the sugar moiety is the same for thymidine in the complex.
Antiviral Agents↗
Synthesis and molecular modeling of novel HSV1 uracil-DNA glycosylase inhibitors.
In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. Starting with the optimized model of binding between 6-(4'-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.
DNA Glycosylases↗
Structural determinants of HIV-1 reverse transcriptase stereoselectivity towards (beta)-L-deoxy- and dideoxy-pyrimidine nucleoside triphosphates: molecular basis for the combination of L-dideoxynucleoside analogs with non-nucleoside inhibitors in anti HIV chemotherapy.
We have compared the HIV-1 RT mutants containing the single substitutions L100I, K103N, V106A, V179D, Y181I and Y188L, known to confer NNI-resistance in treated patients, to HIV-1 RT wt for their sensitivity towards inhibition by D- and L-deoxy- and dideoxy-nucleoside tiphosphates. The results showed a differential effect of the substitutions on the affinity for both D- and L-enantiomers of deoxy- and dideoxy-nucleoside triphosphates and provide a rationale for the utilization of L-dideoxynucleoside analogs with NNI in combination chemotherapy.
Anti-HIV Agents↗
Interaction of beta-L-adenosine-5'-triphosphate (L-ATP) with human deoxycytidine kinase, human DNA primase and T4 DNA ligase: does the chance direct enzymatic enantioselectivity?
We demonstrate that L-ATP: 1) as well as its natural D-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase; 2) inhibits human DNA-primase and the ATP-dependent T4 DNA ligase. Thus, the lack of enantioselectivity of the enzymes is more frequent than it was believed a few years ago and we suggest that it would depend on chance more than on an evolutionary strategy.
Adenosine Triphosphate↗
Synthesis and in vitro activity of D- and L-enantiomers of 5-(trifluoromethyl)uracil nucleoside derivatives.
Recently, beta-L-nucleoside analogues have emerged as a new class of sugar modified nucleosides with potential antiviral and/or antitumoral activity. As a part of our ongoing research on this topic, we decided to synthesize 5-CF3-beta-L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic drug approved by FDA but only used in topical applications due to concomitant cytotoxicity. 5-CF3-beta-L-dUrd (7) as well as some other related L-nucleoside derivatives were stereospecifically prepared and tested in vitro against viral (HSV-1 and HSV-2) and human thymidine kinases (TK).
Antineoplastic Agents↗
DNA synthesis enzymes and proliferating cell nuclear antigen in normal and neoplastic nerve cells.
The activity of nuclear DNA polymerases alpha, beta and delta/epsilon, uracil-DNA glycosylase, thymidine kinase and the presence of Proliferating Cell Nuclear Antigen (PCNA) have been examined in developing rat glial cells, in rat and human glioma, in human neuroblastoma and in differentiated neuroblastoma cell lines in vitro. During glial development the activity of all enzymes tested, except DNA polymerase beta, markedly decreased, suggesting their coordinate regulation in respect to the proliferative state of the cells. Glioma and neuroblastoma cell lines restore the enzymatic activities that were no longer expressed in normal adult cells. Neuroblastoma cell lines induced to differentiate in vitro by retinoic acid showed a decline of the activities of DNA polymerase alpha, DNA polymerase delta/epsilon, uracil-DNA glycosylase and thymidine kinase similar to that observed during in vivo differentiation. We also demonstrate that PCNA is not detectable in glial and neuronal cells at all developmental stages, but can be found in tumor nerve cells. A possible use of enzymatic assays or anti-PCNA antibodies to detect brain tumors is discussed.
Animals↗
Induction of karyological damage by bleomycin in polyploidizating rabbit hepatocytes.
The effects of the antitumor drug bleomycin on polyploidizating hepatocytes of immature rabbits were investigated by combining cytochemical (static microfluorometry) and morphological (fluorescence and electron microscopy) approaches. In addition to a decelerating effect of the drug on the cell kinetics, there were several types of karyological damage (micronuclei, internuclear chromatin bridges, mitotic chromosome bridges), thus demonstrating a clastogenic activity of bleomycin on the DNA, Moreover, anomalous nuclear and cytoplasmic divisions suggest interference of the drug with other cellular structures (mainly the microtubule network) responsible for the cell kinetics.
Animals↗
Inhibition of human DNA ligase by anthracyclines and distamycins.
Antitumor anthracyclines and distamycins behave as strong inhibitors of human replicative DNA ligase. The enzyme appears sensitive to a specific occupancy of the minor groove of DNA. Inhibition by anthracyclines takes place after enzyme adenylation, does not correlate with DNA unwinding potency but strictly correlates with the presence of an amino group on the sugar. In contrast to the non-toxic distamycin A, its antitumor derivative FCE24517 inhibits DNA joining, enzyme adenylation and AMP-driven DNA topoisomerisation. Our data favour a model in which multiple enzymic targets contribute to the activity of these antitumor drugs.
Antibiotics, Antineoplastic↗
Arabinose furanosyl thymidine: uptake, phosphorylation and incorporation into DNA of mammalian cells.
The naturally occurring nucleoside analogue arabinosyl thymidine is known as an anti-herpes and anti-cancer agent. The biologically active form is arabinosyl thymidine triphosphate (Ara-TTP), which inhibits cellular and viral DNA-polymerases and thus interferes with DNA replication. Using two murine erythroleukemia cell lines, Friend cell clone F4-6 and F4-12N, the latter being thymidine kinase deficient (TK-) cells transformed to a TK+ phenotype with the HSV TK gene, we have determined 1) the role of cellular and herpes simplex virus thymidine kinase (HSV TK) in the uptake of Ara-T into the cells; 2) the subsequent phosphorylation of intracellular Ara-T to Ara-TMP, Ara-TDP and Ara-TTP; 3) the incorporation of Ara-TTP into the DNA. Incorporation into DNA was studied under different conditions, including selective inhibition of the different cellular DNA polymerases by aphidicolin (that inhibits polymerases alpha and delta) and dideoxythymidine (that preferentially inhibits polymerases beta and gamma). The uptake of Ara-T into the methanol soluble pool of the cells depends upon its phosphorylation to Ara-TMP, which is more efficiently performed by the HSV TK than by the cellular TK, thus explaining the sensitivity of HSV infected cells to Ara-T. However, using increasing concentrations of Ara-T, we have shown that phosphorylation also occurs in normal control cells due to the cellular thymidine kinase. More than 90% of Ara-T is phosphorylated in the cell, and more than 60% of total Ara-T(MP, DP, TP) exists in the triphosphate form.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Developmental activity profile of DNA polymerases delta and alpha in rat neurons suggests a coordinated in vivo function.
DNA polymerase delta, a fourth cellular DNA polymerase, might play an important role in cellular events. The properties of the enzyme suggested possible roles in nuclear DNA replication and DNA repair. By using specific assays that allow the determination of DNA polymerases delta and alpha in crude enzyme fractions (1), we show here that in developing rat neurons, which stop dividing before birth, both DNA polymerases delta and alpha drop sharply in an identical pattern from a high level with the approach of term and disappear at approximately three weeks of postnatal age. The results suggest that DNA polymerases delta and alpha might have a coordinated function during DNA replication in eukaryotic cells.
Aging↗
DNA polymerases and DNA topoisomerases as targets for the development of anticancer drugs.
Studies of a variety of compounds designed as derivatives of prototype active molecules aphidicolin and doxorubicin are reported. So far none of the aphidicolin simpler analogues is as active as the parental molecule. Ten anthracycline analogues, characterized for their cytotoxicity, antitumor activity and inhibition of the relaxing activity of purified human DNA topoisomerase II can be divided into five groups. The majority of the tested compounds shows properties very similar to those of doxorubicin. Epirubicin shows extremely high inhibitory activity toward the relaxing property of topoisomerase II but its antitumor activity and cytotoxicity are similar to those of the former group. The third group includes a compound with extremely high cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity typical of anthracyclines and good antitumor activity but which has no specific inhibitory activity on topoisomerase II. A fifth group includes a totally inactive compound. Our results suggest that the inhibition of human DNA topoisomerase II is only partially correlated with antitumor activity.
Animals↗
Aphidicolin inhibits in vitro the activity of pseudorabies virus (PRV) DNA polymerase and in vivo the viral proliferation.
We have purified and characterized Pseudorabies virus (PRV) DNA polymerase from infected TK- mouse cells. PRV DNA polymerase has a 3'- > 5' exonuclease activity; it is stimulated by ionic strength, requires magnesium for optimal activity and it is more sensitive to aphidicolin than eukaryotic and HSV-1 replicative DNA polymerases. Aphidicolin inhibits in vitro PRV DNA polymerase competitively with respect to dCTP with a Ki of 0.06 microM and completely blocks viral growth in vivo at 4.4 microM. The high sensitivity to aphidicolin of animal herpesvirus DNA polymerases might allow a topical use of this drug in the treatment of animal herpesvirus keratitis and stomatitis.
Animals↗