Alessandra Angeretti

Penetration ability of different irrigants into dentinal tubules

Dentinal tubules of human root canal walls were infected with a known bacterial isolate. The teeth were divided into two groups and the root canals instrumentated. Different types of canal irrigant were used for each group. In group A, 5% NaOCl was followed by a 10% EDTA rinse and neutralized with a final physiological solution rinse. In Group B, 10% EDTA, a tensioactive agent (TRITON), and 5% NaOCl were used in sequence, with a final physiological solution rinse to neutralize the action of the agents used. Histological examination of group A specimens showed a residual area of infection extending from the canal lumen to a mean depth of 300 microns. Histological examination of group B specimens showed an infection-free area of tubules to a mean depth of 130 microns. Below this was an infected area of variable extent. In some group B sections, no infection was found.

Mechanisms of viral inhibition by interferons.

Interferons (IFNs) are a family of related proteins grouped in four species (alpha, beta, gamma and omega) according to their cellular origin, inducing agents and antigenic and functional properties. Their binding to specific receptors leads to the activation of signal transduction pathways that stimulate a defined set of genes, whose products are eventually responsible for the IFN antiviral effects. Their action against viruses is a complex phenomenon. It has been reported that IFNs restrict virus growth at the levels of penetration, uncoating, synthesis of mRNA, protein synthesis and assembly. This review will attempt to evaluate evidence of the involvement of the IFN-inducible proteins in the expression of the antiviral state against RNA or DNA viruses.

MTA obturation of pulpless teeth with open apices: bacterial leakage as detected by polymerase chain reaction assay.

Polymerase chain reaction (PCR) followed by reverse dot blot was used to detect Enterococcus faecalis leakage through mineral trioxide aggregate (MTA) apical obturations of pulpless teeth with open apices. Prepared root canals of 34 extracted teeth were given a standard apical foramen opening and received orthograde apical obturation with MTA; three groups had 1-, 2-, or 3-mm thickness. Sterilized specimens were inoculated with E. faecalis and incubated in sterile medium. DNA extracted from the specimens was amplified by polymerase chain reaction, which yielded a specific segment of E. faecalis 16S rDNA. On day 10 of incubation, no specimens were contaminated. On day 50, almost 17% of specimens were contaminated, with no statistically significant difference between groups (Chi-square = 0.48; df = 2; p = 0.787). Therefore, MTA provides an adequate seal even in cases of orthograde apical obturation of pulpless teeth with open apices.

Expression of an Altered Ribonucleotide Reductase Activity Associated with the Replication of Murine Cytomegalovirus in Quiescent Fibroblasts

ABSTRACT Ribonucleotide reductase (RNR) is an essential enzyme for the de novo synthesis of both cellular and viral DNA and catalyzes the conversion of ribonucleoside diphosphates into the corresponding deoxyribonucleoside diphosphates. The enzyme consists of two nonidentical subunits, termed R1 and R2, whose expression is very low in resting cells and maximal in S-phase cells. Here we show that murine cytomegalovirus (MCMV) replication depends on ribonucleotide reduction since it is prevented by the RNR inhibitor hydroxyurea. MCMV infection of quiescent fibroblasts markedly induces both mRNA and protein corresponding to the cellular R2 subunit, whereas expression of the cellular R1 subunit does not appear to be up-regulated. The increase in R2 gene expression is due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse R2 promoter is induced following virus infection. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in R2 promoter activity. It was found that the viral gene M45, encoding a putative homologue of the R1 subunit, is expressed 24 and 48 h after infection. Meanwhile, we observed an expansion of the deoxyribonucleoside triphosphate pool between 24 and 48 h after infection; however, neither CDP reduction nor viral replication was inhibited by treatment with 10 mM thymidine. These findings indicate the induction of an RNR activity with an altered allosteric regulation compared to the mouse RNR following MCMV infection and suggest that the virus R1 homologue may complex with the induced cellular R2 protein to reconstitute a new RNR activity.

Human cytomegalovirus stimulates cellular dihydrofolate reductase activity in quiescent cells.

Human cytomegalovirus (HCMV) productively infects quiescent fibroblasts in which the levels of deoxynucleotide triphosphates (dNTPs) and cell functions involved in DNA metabolism are very low. Since sufficient dNTPs levels are essential for human HCMV replication, host cell enzymes involved in the biosynthesis of dNTPs might be expected to be stimulated by viral infection in quiescent cells. We report that HCMV infection of quiescent fibroblasts stimulates the activity of cellular dihydrofolate reductase (DHFR), a key enzyme in DNA precursor synthesis. We also demonstrate that suppression of DHFR activity by the specific inhibitor methotrexate prevents HCMV replication and DNA synthesis. These observations indicate that induction of DHFR activity by HCMV is required for efficient viral replication in quiescent fibroblasts.

Murine cytomegalovirus induces expression and enzyme activity of cellular dihydrofolate reductase in quiescent cells

Murine cytomegalovirus (MCMV) productively infects quiescent fibroblasts in which the levels of nucleoside triphosphate precursors and cell functions involved in DNA metabolism are minimal. It appears that MCMV has evolved molecular pathways in order to ensure the presence of nucleoside triphosphate precursors for the viral DNA polymerase. Here, we report that MCMV infection of quiescent NIH 3T3 cells markedly stimulates transcription, expression and activity of the cellular dihydrofolate reductase (DHFR), a key enzyme in the synthesis of DNA precursors. DHFR stimulation by MCMV is sensitive to UV irradiation and seems to depend on expression of the viral immediate-early protein pp89. Finally, it has been demonstrated that suppression of virus-induced DHFR activity by the specific inhibitor methotrexate prevents MCMV DNA replication. These observations indicate that induction of host cell DHFR activity by MCMV is required for viral DNA synthesis in quiescent fibroblasts.

Trans-activation of the mouse cytomegalovirus immediate early gene enhancer by ras oncogenes

The ras gene family encodes 21K proteins that reside on the inner face of the plasma membrane and bind GTP and GDP with an equally high affinity. Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a viral Harvey-ras (v-Ha-ras) cDNA, together with a plasmid (pCMVCAT) carrying the immediate early (IE) enhancer of the murine cytomegalovirus (MCMV) linked to the chloramphenicol acetyltransferase (CAT) reporter gene strongly stimulated CAT activity. Basal levels of pCMVCAT expression as well as trans-activation by v-ras plasmid were both inhibited by cotransfection of an expression vector containing the dominant inhibitory mutant gene Ha-ras Asn-17. This indicates that the p21ras protein is responsible for these activities. High pCMVCAT activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. To define the cis-acting DNA elements in the MCMV IE enhancer responsible for this trans-activation by p21ras protein, we constructed several plasmids containing the CAT gene under control of MCMV IE enhancers that were deleted in different regions. The CAT assays demonstrated that several sequences were responsive to p21ras protein. These sequences are scattered throughout the IE enhancer, upstream of the transcription start site, and contain responsive elements that are homologous to the binding sites for cellular transcription factors such as NF kappa B, AP1, ATF and SP1. Activation of the p21ras protein may thus be one of the signals that regulate IE genes transcription during MCMV infection.

The thymidylate synthase inhibitor ZD1694 potently inhibits murine and human cytomegalovirus replication in quiescent fibroblasts.

Tomudex (ZD1694) is a quinazoline-based folate analog and a powerful inhibitor of cellular thymidylate synthase and is approved in Europe for use in oncology. Here the first evidence of its activity against murine and human cytomegalovirus (MCMV and HCMV) is reported. ZD1694 irreversibly inhibited the replication and DNA synthesis of both viruses in quiescent fibroblasts. The corresponding 50% effective concentrations were 0.006 and 0.002 microM respectively, whereas the 50% cytotoxic concentration was >10 microM for both murine and human quiescent fibroblasts. A similar antiviral effect was observed against two ganciclovir-resistant HCMV strains isolated from AIDS patients. Taken as a whole these results demonstrate that cellular thymidylate synthase plays an essential role in viral replication and that ZD1694 merits further investigation as anticytomegaloviral agent.

Regulation of Gene Expression by Interferons

IFNs are a family of proteins produced by various cells following stimulation by biological or synthetic inducers. The interaction with the membrane receptor is followed by the activation of the expression of particular genes that are responsible for their antiviral, antiproliferative and immunomodulatory properties. Initiation of transcription is an early and critical event in the control of eukaryotic gene expression. In this review we will briefly discuss the mechanisms exploited by IFNs to control at transcriptional level the expression of inducible genes. In particular we will focus on some characteristics if cis-acting DNA elements that are located upstream from the initiation site for RNA transcription and of nuclear trans-acting factors that are required for modulation of gene expression by IFNs.

Overexpression of cellular dihydrofolate reductase abolishes the anticytomegaloviral activity of methotrexate

SummaryCytomegalovirus (CMV) stimulates numerous cellular pathways upon infection. One of these pathways involves activation of dihydrofolate reductase (DHFR), an essential enzyme in the biosynthesis of purines and thymidylate. Here we report that methotrexate (MTX), an inhibitor of DHFR, suppresses murine CMV replication at the level of DNA synthesis in quiescent NIH 3T3 cells. However, MTX has no antiviral activity in NIH 3T3 sublines resistant to MTX due to DHFR overexpression. These results directly link MTX antiviral activity to DHFR and demonstrate that DHFR plays an essential role for CMV replication in quiescent cells.