Biomedical subjects

A Angeretti

Publications and source records attributed to A Angeretti.

At least 19 recordsLinked to original sources

PubMed2000

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.

Animals↗

PubMed2000

Expression of an altered ribonucleotide reductase activity associated with the replication of murine cytomegalovirus in quiescent fibroblasts.

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.

Animals↗

PubMed1999

Overexpression of cellular dihydrofolate reductase abolishes the anticytomegaloviral activity of methotrexate.

Cytomegalovirus (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.

3T3 Cells↗

PubMed1999

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.

Cells, Cultured↗

PubMed1998

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.

3T3 Cells↗

PubMed1997

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.

Anti-Bacterial Agents↗

PubMed1995

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.

Animals↗

PubMed1995

cAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by ras oncogene products.

Products of ras oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfected with prasZip6 plasmid, a mammalian expression vector containing a v-Ha-ras cDNA, together with p(delta)ACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of p(delta)ACMVCAT were also observed in cell lines carrying stably transfected ras oncogenes. Further support for the role of the CRE sequence in MCMV enhancer activation comes from the finding that v-Ha-ras expression increases the activity of a reporter gene, beta-galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha-ras (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha-ras (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21ras. Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by ras oncogenes.

3T3 Cells↗

PubMed1995

Modulation of HIV-LTR activity by ras oncogenes.

Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a v-Ha-ras gene, together with a plasmid carrying the human immunodeficiency virus (HIV) long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, significantly stimulated CAT activity. High HIV LTR activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. By contrast an inactivated form of ras (Ha-ras Asn-17) did not stimulate the HIV-LTR but strongly inhibited its basal activity. Activation of the p21ras protein may thus be one of the signals that regulate LTR driven transcription during HIV infection.

3T3 Cells↗

PubMed1994

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.

3T3 Cells↗

PubMed1994

Characterization of nuclear factors involved in 202 gene induction by IFN-alpha, viruses or dsRNA in murine leukemia cells.

When treated with IFN-alpha, L1210 leukemia cells express high levels of the mouse 202 gene mRNA after a few hours. Three tandem copies of a 43 bp fragment (GAbox) homologous to the IFN-stimulatable response element (ISRE), located in the 5'-flanking region of the 202 gene, were linked to the reporter CAT gene and transiently transfected into L1210 cells. The data suggest that the GA box is sufficient to confer transcriptional inducibility upon IFN stimulation. Binding assays, using the labeled GA box as a probe, demonstrated the presence of a retarded complex, designated GAbfl, in the nuclear extracts of L1210 cells treated with IFN-alpha. This complex is absent in the extracts of L1210 cells treated with ssRNA viruses or synthetic dsRNA. Moreover, photoaffinity cross-linking experiments revealed that GAbfl contains a protein of about 50 kDa. Altogether these results demonstrate that antiviral state induction by IFN-alpha in L1210 cells is preceded by GAbfl binding to the ISRE of the IFN-inducible genes.

Animals↗

PubMed1993

Effect of interferon-alpha on immediate early gene expression of murine cytomegalovirus.

Interferon-alpha (IFN-alpha) significantly reduced the replication of murine cytomegalovirus (MCMV) in mouse embryo fibroblasts derived from the susceptible mouse strain C3H/HeJ. When infectious virus production was measured, a strong decrease in virus titer was observed in IFN-treated cells at a multiplicity of infection (moi) of 1 and 0.5 pfu/cell. Analysis of virus-specified mRNAs by Northern blot assay revealed that IFN-alpha had a significant effect on the expression of viral mRNAs at 48h. In particular, the mRNAs of the major immediate early (IE) transcription units, IE1, IE2, and IE3, were impaired by IFN-alpha. In addition, decrease of IE1 mRNA synthesis was accompanied by a reduction of the major IE product (pp89), as revealed by Western blot assay. These results suggest that IFN-alpha may inhibit MCMV replication by directly impairing IE gene transcription.

Animals↗

PubMed1992

Effects of interferon alpha on murine cytomegalovirus replication.

The present study examined the protective effect of IFN-alpha against mouse cytomegalovirus (MCMV) infection in embryo fibroblasts (MEF) of genetically resistant (C3H/HeJ) and susceptible (C57BL/6) mouse strains. At a M.O.I. of 1 IFN-alpha was protective in C3H/HeJ-MEF but not in C57BL/6-MEF. Dot-blot analysis during MCMV replication in C3H/HeJ-MEF showed that IFN-alpha pretreatment reduced the steady state level of immediate early and late mRNAs but partially reduced early gene expression.

Animals↗

PubMed1988

Use of a crude extract or a purified antigen of Trichomonas vaginalis for the detection of secretory antibodies by ELISA.

A crude aqueous extract (T2) or a gel-filtration purified antigen of T. vaginalis was utilized to detect secretory antibodies in cervico-vaginal secretions by ELISA. The test was developed under optimum conditions using a rabbit anti-Trichomonas serum. Conditions established for monitoring antibodies to trichomonas in immunized rabbits were equally effective for human secretions. The crude extract was capable of assessing secretory antibodies in 45% of women with acute trichomoniasis and 43% of healthy women. The purified antigen showed a marked antigenic activity and compared to the crude antigen, testing 6 samples from infected women, detected a false positive and reduced the O.D. value of another sample by 1/4.

Animals↗

PubMed1987

Specific serum IgA in rotavirus gastroenteritis.

Virus-specific serum IgM, IgA, and IgG were detected by ELISA in sera obtained from children with rotavirus gastroenteritis and fractionated by gel filtration. Specific IgM and IgG could be easily demonstrated, whereas IgA were very low. Moreover, polymeric IgA (p-IgA) were not present, whereas in the immune response against viruses causing systemic infections, they are synthetised in large amounts.

Antibodies, Viral↗

PubMed1986

[Effect of mercaptans on serum IgA].

Using reducing agents like dithiothreitol (DTT) 2.5 mM IgM lose their ability to bind antigens but they can be still detected by an anti-IgM antiserum. IgA are more resistant to reduction than IgM but using higher concentrations of DTT they lose both the ability to bind antigens and the possibility to be detected by an anti-IgA antiserum. Because of this fact IgA after mild reduction can result more or less inactivated according to the method employed to detect them. Using ELISA or similar methods that need an immunological detection of preformed immune complexes IgA-Ag, the loss of activity is more marked than using techniques such as HAI that detect them directly.

Antigen-Antibody Reactions↗

PubMed1985

Virus-specific polymeric immunoglobulin A antibodies in serum from patients with rubella, measles, varicella, and herpes zoster virus infections.

More than 85% of the immunoglobulin A (IgA) antibodies in normal adult serum are monomeric (m-IgA). By contrast, virus-specific IgA is mainly polymeric (p-IgA) in sera from patients with rubella, measles, and varicella. Specific m-IgA antibodies only reach quantitative significance in late convalescence. In patients with herpes zoster, on the other hand, a varying response was observed: in three of six sera, specific IgA was absent or at a very low titer, whereas in the remaining three cases, a high titer of both p-IgA and m-IgA was noted. These results suggest that in the initial response to rubella, measles, and varicella-zoster viruses, specific IgA first appears as p-IgA and only later becomes, or is replaced by, m-IgA.

Antibodies, Viral↗

PubMed1985

Production of monoclonal antibodies against the carbohydrate of group A streptococci.

Mouse hybridomas were isolated by fusing P3.X63.Ag8-653 myeloma cells with spleen cells from BDF1 mice repeatedly immunized with Streptococcus pyogenes type 3 ATCC 12384. Two out of 480 growing cultures secreted antibodies against the bacterial strain used as immunogen, as evaluated by an ELISA assay. After cloning the most representative continuously growing clone, 1-181, the binding specificity of these monoclonal antibodies was determined using either Streptococci with carbohydrate of different group or different bacteria strains. Binding properties suggest that the 1.181 monoclonal antibodies specifically recognize the carbohydrate moiety of Streptococci group A.

Animals↗