Effect of divalent and monovalent cations on calf thymus PCNA-independent DNA polymerase δ and its 3' → 5' exonuclease

AbstractRecent data suggest that DNA polymerases α and δ might have a coordinate functional role at the replication fork. In this communication we show that Mg2+ is likely the natural metal activator for both enzymes. Mn2+, a known mutagenic agent, is a competitive inhibitor of Mg2+ for DNA polymerase δ and acompetitive for DNA polymerase α. The 3'→ 5' exonuclease activity associated with DNA polymerase δ is not affected upon addition of Mn2+, Be2+, another mutagenic agent, on the other hand, has an inhibitory effect on the 3' → 5' exonuclease, but not on the DNA polymerase δ. The data presented might explain the mutagenic and carcinogenic potential of these two divalent cations

Design and synthesis of phosphonoacetic acid (PPA) ester and amide bioisosters of ribofuranosylnucleoside diphosphates as potential ribonucleotide reductase inhibitors and evaluation of their enzyme inhibitory, cytostatic and antiviral activity.

Continuing our investigations on inhibitors of ribonucleotide reductase (RNR), the crucial enzyme that catalyses the reduction of ribonu-cleotides to deoxyribonucleotides, we have now prepared and evaluated 5′-phosphonoacetic acid, amide and ester analogues of adenosine, uridine and cytidine with the aim to verify both substrate specificity and contribution to biological activity of diphosphate mimic moieties. A molecular modelling study has been conducted on the RNR R1 subunit, in order to verify the possible interaction of the proposed bioisosteric moieties. The study compounds were finally tested on the recombinant murine RNR showing a degree of inhibition that ranged from 350 μM for the UDP analogue 5′-deoxy-5′- N-(phosphon-acetyl)uridine sodium salt (amide) to 600 μM for the CDP analogue 5′- O-[(diethyl-phosphon)acetyl]cytidine (ester). None of the tested compounds displayed noteworthy cytostatic activity at 100–500 μM concentrations, whereas ADP analogue 5′- N-[(diethyl-phosphon) acetyl]adenosine (amide) and 5′-deoxy-5′- N-(phos-phon-acetyl)adenosine sodium salt (amide) showed a moderate inhibitory activity (EC50: 48 μM) against HSV-2 and a modest inhibitory activity (EC50: 110 μM) against HIV-1, respectively

Super-telomeres in transformed human fibroblasts

Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis

La logica del vivente in Maupertuis: dall’attrazione newtoniana al determinismo psicobiologico

After successfully relaunching the epigenetic theory of generation, reinterpreted in the light of Newtonian attraction and chemical affinities ("Vénus physique", 1745), Pierre-Louis Moreau de Maupertuis (1698-1759) deepened his theoretical investigations in the field of the genesis of organisms and species in the "Système de la Nature" (1754). In this work, in order to reconcile the scientific postulate of objectivity - which rejects the recourse to final causes - with the evident teleonomic properties of living beings, and at the same time recognizing inadequate for this purpose the mechanistic principles he advanced in the "Vénus physique", Maupertuis proposed a bold panpsychist theory of life, of Spinozian inspiration. Therefore, by postulating the matter alive, endowed with instinct and feeling, Maupertuis imagined that some form of intelligence or psychic memory, associated with matter, directed the development of living beings. In light of recent discoveries in biology, the original psychobiological determinism advanced by Maupertuis in the "Système de la Nature" appears to be a brilliant intuition of the logic of genetic and evolutionary processes.Dopo aver rilanciato con successo la teoria epigenetica della generazione, reinterpretata alla luce dell’attrazione newtoniana e delle affinità chimiche ("Vénus physique", 1745), Pierre-Louis Moreau de Maupertuis (1698-1759) approfondì le proprie indagini teoretiche nel campo della genesi degli organismi e delle specie nel "Système de la Nature" (1754). In quest’opera, al fine di conciliare il postulato di oggettività della scienza - che respinge il ricorso alle cause finali - con le evidenti proprietà teleonomiche degli esseri viventi, e riconoscendo nel contempo inadeguati a tale scopo i pur originali principii meccanicistici avanzati nella "Vénus physique", Maupertuis propose un’ardita teoria panpsichista del vivente, di ispirazione spinoziana. Postulando quindi la materia viva, dotata di istinto e di sentimento, Maupertuis immaginò che una qualche forma di intelligenza o di memoria psichica, associata alla materia, dirigesse lo sviluppo dei viventi. Alla luce delle recenti scoperte della biologia, l’originale determinismo psicobiologico avanzato da Maupertuis nel "Système de la Nature" appare una geniale intuizione della logica dei processi genetici ed evolutivi

Do DNA polymerases δ and α act coordinately as leading and lagging strand replicases?

AbstractThe activity ratio of DNA polymerases δ and α in calf thymus was found to be invariably 1:1, irrespective of extraction procedure (8 types) and subcellular localization (cytoplasm, nucleus and microsomes). This was established by separation of the two forms by hydroxyapatite chromatography and by their response to specific inhibitors and monoclonal antibodies. This finding supports the dimeric DNA polymerase model [(1980) J. Biol. Chem. 255, 4290–4303], which proposes that DNA polymerases δ and α act coordinately as leading and lagging strand enzymes, respectively, at the replication fork

Microwave-assisted synthesis and biological evaluation of novel uracil derivatives inhibiting human thymidine phosphorylase

New 5-chloro-6-substituted-uracil derivs. have been prepd. by microwave assisted-synthesis and tested in vitro as thymidine phosphorylase inhibitors. One of these compds. showed potent inhibitory activity, with an IC50 value in the submicromolar range. The biol. activity of the new compds. is discussed in terms of structure-activity relationship

Effect of combinations of antiviral drugs on herpes simplex encephalitis

Bryan M Gebhardt1, Federico Focher2, Richard Eberle3, Andrzej Manikowski4, George E Wright41LSU Eye Center, Department of Ophthalmology, Louisiana State University Health Sciences Center, New Orleans, LA, USA; 2Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy; 3Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, USA; 4GLSynthesis Inc., Worcester, MA, USAAbstract: 2-Phenylamino-6-oxo-9-(4-hydroxybutyl)purine (HBPG) is a thymidine kinase inhibitor that prevents encephalitic death in mice caused by herpes simplex virus (HSV) types 1 and 2, although its potency is somewhat less than that of acyclovir (ACV). The present study was undertaken to determine the effect of combinations of HBPG and either ACV, phosphonoformate (PFA), or cidofovir (CDF) against HSV encephalitis. BALB/c mice were given ocular infections with HSV-1 or HSV-2, and treated twice daily intraperitoneally for five days with HBPG, alone or in combination with ACV, PFA, or CDF. Animals were observed daily for up to 30 days, and the day of death of each was recorded. All of the combinations showed additivity, and the combination of HBPG + ACV appeared to be synergistic, ie, protected more mice against HSV-1 encephalitis compared with each drug given alone. Delay of treatment with HBPG for up to two days was still effective in preventing HSV-2 encephalitis. The combination of the thymidine kinase inhibitor HBPG and the antiherpes drug ACV may have synergistic activity against HSV encephalitis. The development of a potent and safe combination therapy for the prevention and/or treatment of HSV infection of the central nervous system can improve the outcome of this infection in humans.Keywords: antivirals, herpetic encephaliti