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Biomedical subjects

F Focher

Publications and source records attributed to F Focher.

At least 37 records · Page 2Linked to original sources
PubMed1995

Late induction of human DNA ligase I after UV-C irradiation.

We have studied the regulation of DNA ligase I gene expression in UV-C irradiated human primary fibroblasts. An increase of approximately 6-fold both in DNA ligase I messenger and activity levels was observed 24 h after UV treatment, when nucleotide excision repair (NER) is no longer operating. DNA ligase I induction is serum-independent and is controlled mainly by the steady-state level of its mRNA. The activation is a function of the UV dose and occurs at lower doses in cells showing UV hypersensitivity. No increase in replicative DNA polymerase alpha activity was found, indicating that UV induction of DNA ligase I occurs through a pathway that differs from the one causing activation of the replication machinery. These data suggest that DNA ligase I induction could be linked to the repair of DNA damage not removed by NER.

Cells, Cultured↗
PubMed1995

Synthesis, properties, and pharmacokinetic studies of N2-phenylguanine derivatives as inhibitors of herpes simplex virus thymidine kinases.

Two series of selective inhibitors of herpes simplex virus types 1 and 2 (HSV1,2) thymidine kinases (TK) have been developed as potential treatment of recurrent virus infections. Among compounds related to the potent base analog N2-[m-(trifluoromethyl)phenyl]guanine (mCF3-PG), none was a more potent inhibitor than mCF3PG itself. Compounds related to the nucleoside N2-phenyl-2'-deoxyguanosine (PhdG), but with alkyl, hydroxyalkyl, and related substituents at the 9-position in place of the glycosyl group of PhdG, retained significant but variable inhibitory potencies against the HSV TKs. The most potent inhibitor of HSV1 TK among 9-substituted derivatives, 9-(4-hydroxybutyl)-N2-phenylguanine (HBPG), was a competitive inhibitor with respect to the substrate thymidine but was not itself a substrate for the enzyme. Water solubilities and 1-octanol:water partition coefficients for the 9-substituted N2-phenylguanines were linearly but oppositely related to the sum of hydrophobic fragmental constants (sigma f) of the 9-substituents. Four of the inhibitors were given as solutions to mice by iv and ip routes, and the time course of their plasma concentrations was determined by HPLC analysis of the parent compounds. HBPG was completely absorbed by the ip route, and the plasma concentration could be prolonged by use of suspension formulations. HBPG is a candidate for animal trials of the ability of TK inhibitors to prevent recurrent herpes virus infections.

Antiviral Agents↗
PubMed1995

Lack of stereospecificity of some cellular and viral enzymes involved in the synthesis of deoxyribonucleotides and DNA: molecular basis for the antiviral activity of unnatural L-beta-nucleosides.

Among enzymes involved in the synthesis of nucleotides and DNA, some exceptions have recently been found to the universal rule that enzymes act only on one enantiomer of a chiral substrate and that only one of the enantiomeric forms of chiral molecules may bind effectively at the catalytic site, displaying biological activity. The exceptions include: herpes virus thymidine kinases, cellular deoxycytidine kinase and deoxynucloside mono- and diphosphate kinases, cellular and viral DNA polymerases, such as DNA polymerase alpha, terminal transferase and HIV-1 reverse transcriptase. The ability of these enzymes to utilize unnatural L-beta-nucleosides or -nucleotides as substrate may be exploited from chemotherapeutic point of view.

Antiviral Agents↗
PubMed1995

5-Iodo-2'-deoxy-L-uridine and (E)-5-(2-bromovinyl)-2'-deoxy-L-uridine: selective phosphorylation by herpes simplex virus type 1 thymidine kinase, antiherpetic activity, and cytotoxicity studies.

5-Iodo-2'-deoxy-L-uridine (L-IdU) and (E)-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU) have been prepared and found to inhibit herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) with activities comparable to those of their analogs with the natural D-sugar configuration. The mechanism of inhibition is purely competitive for L-IdU (Ki = 0.24 microM) and mixed-type for L-BVdU (Ki = 0.13 microM). High performance liquid chromatographic analysis of the reaction products demonstrated that the viral enzyme phosphorylates both L-enantiomers to their corresponding monophosphates with efficiency comparable to that for D-enantiomers. Neither L-enantiomer inhibits the human cytosolic TK. In contrast to their D-enantiomers, L-IdU and L-BVdU have no effect on human thymidylate synthase, either in HeLa cells or in TK-deficient HeLa cells transformed with the HSV-1 TK gene. Both L-enantiomers (i) have no effect on HeLa cell growth, (ii) are 1000-fold less cytotoxic toward TK-deficient HeLa cells transformed with the HSV-1 TK gene than are their D-enantiomers, (iii) in contrast to their D-enantiomers, are fully resistant to hydrolysis by nucleoside phosphorylase, and, (iv) in spite of their much lower cytotoxicity, most probably due to the very low affinity of L-BVdU monophosphate and L-IdU monophosphate for thymidylate synthase, are only 1 or 2 orders of magnitude less potent than their D-enantiomers in inhibiting viral growth, with potency comparable to that of acyclovir.

Antineoplastic Agents↗
PubMed1994

Kinetic studies with N2-phenylguanines and with L-thymidine indicate that herpes simplex virus type-1 thymidine kinase and thymidylate kinase share a common active site.

It is known that the Herpes simplex virus type 1 (HSV-1)-encoded thymidine kinase (TK) co-purifies with an associated thymidylate kinase (TMPK) activity and that thymidylate (TMP) inhibits the phosphorylation of thymidine by the HSV-1 TK. Here we demonstrate that: (i) TMP phosphorylation catalysed by the viral TMPK is competitively inhibited by thymidine (TdR) with a Ki equal to its Km as substrate for the viral TK; (ii) L-thymidine (L-TdR), the enantiomer of the naturally occurring D-TdR and a substrate for the HSV-1 TK [Spadari, Maga, Focher, Ciarrocchi, Manservigi, Arcamone, Capobianco, Caruso, Colonna, Iotti and Garbesi (1992) J. Med. Chem. 35, 4214-4220], is a powerful inhibitor of the HSV-1 TMPK activity with a Ki value identical with its Km as a substrate for the viral TK; (iii) both viral TK and TMPK activities are inhibited, in a competitive way and with identical Ki values, by novel, non-substrate inhibitors of HSV-1 TK, N2-phenylguanines; (iv) L-TdR is phosphorylated to L-TMP by the viral TK, but L-TMP is not phosphorylated to L-TDP by the viral TMPK activity; and (v) L-TMP inhibits competitively and with identical potencies the phosphorylation of TdR and TMP catalysed respectively by the HSV-1 TK and TMPK activities. In conclusion, our data demonstrate that both TK and TMPK activities encoded by HSV-1 share a common active site which is very tolerant in accepting modified nucleosides, but cannot readily accommodate modified nucleoside monophosphates.

Binding Sites↗
PubMed1994

Inhibitor analysis of calf thymus DNA polymerases alpha, delta and epsilon.

Quantitative effects of inhibitors of the replicative DNA polymerases (pol) alpha, delta and epsilon from calf thymus are reported under similar assay conditions. Carbonyldiphosphonate was a competitive inhibitor of pols delta and epsilon, with 4- to 6-fold selectivity compared to pol alpha. Aphidicolin inhibited pols alpha and delta with 6- to 10-fold selectivity compared to pol epsilon. The 'butylphenyl' nucleotides, BuPdGTP and BuAdATP, inhibited pol alpha with at least 1000-fold selectivity compared to pols delta and epsilon. The use of these inhibitors under similar assay conditions permits the discrimination of the three enzymes.

Animals↗
PubMed1994

Identification, partial purification and inhibition by guanine analogues of a novel enzymic activity which phosphorylates guanosine to GMP in the protozoan parasite Eimeria tenella.

From oocysts of the protozoan parasite Eimeria tenella, responsible for avian coccidiosis, we have partially purified and characterized a novel enzymic activity which specifically phosphorylates guanosine to GMP. The enzyme is able to use several phosphate donors, in the order: acetyl phosphate (Ac-P) > ATP > UTP > CTP > phosphoribosyl pyrophosphate (PRPP) > dUTP > or = dATP. The low specificity of this enzyme for the phosphate donor suggested that it be named guanosine phosphotransferase (GPTase). This enzyme is biochemically distinct from the previously described adenosine kinase (AK) and hypoxanthine/xanthine/guanine phosphoribosyltransferase (HXGPRTase), and may enable the parasite to synthesize guanine nucleotides under conditions of imbalance between adenine and guanine nucleotides. Because of its possible role in the purine salvage pathways, we have studied the effect of several guanine and guanosine analogues, recently synthesized in our laboratory, on the activity of GPTase in vitro. GPTase is specifically inhibited in the micromolar range by several substituted N2-phenylguanine bases. These results indicate that, as previously found for AK and HXGPRTase, GPTase could be a potential target for antiparasitic chemotherapy.

Adenosine Kinase↗
PubMed1993

Lack of stereospecificity of suid pseudorabies virus thymidine kinase.

We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine >> L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs.

Binding, Competitive↗
PubMed1993

Herpes simplex virus type 1 uracil-DNA glycosylase: isolation and selective inhibition by novel uracil derivatives.

We have purified Herpes simplex type 1 (HSV1) uracil-DNA glycosylase from the nuclei of HSV1-infected HeLa cells harvested 8 h post-infection, at which time the induction of the enzyme is a maximum. The enzyme has been shown to be distinct from the host enzyme, isolated from HeLa cells, by its lack of sensitivity to a monoclonal antibody to human uracil-DNA glycosylase. Furthermore, several uracil analogues were synthesized and screened for their capacity to discriminate between the viral and human uracil-DNA glycosylases. Both enzymes were inhibited by 6-(p-alkylanilino)uracils, but the viral enzyme was significantly more sensitive than the HeLa enzyme to most analogues. Substituents providing the best inhibitors of HSV1 uracil-DNA glycosylase were found to be in the order: p-n-butyl < p-n-pentl = p-n-hexyl < p-n-heptyl < p-n-octyl. The most potent HSV1 enzyme inhibitor, 6-(p-n-octylanilino)uracil (OctAU), with an IC50 of 8 microM, was highly selective for the viral enzyme. Short-term [3H]thymidine incorporation into the DNA of HeLa cells in culture was partially inhibited by OctAU, whereas it was unchanged when 6-(p-n-hexylanilino)uracil was present at concentrations that completely inhibited HSV1 uracil-DNA glycosylase activity. These compounds represent the first class of inhibitors that inhibit HSV1 uracil-DNA glycosylase at concentrations in the micromolar range. The results suggest their possible use to evaluate the functional role of HSV1 uracil-DNA glycosylase in viral infections and re-activation in nerve cells.

Cell Nucleus↗
PubMed1993

Myoclonus epilepsy and ragged-red fibers: blood mitochondrial DNA heteroplasmy in affected and asymptomatic members of a family.

By a rapid PCR-based method to assess the 8344 mtDNA mutation associated with MERRF disease, we have studied DNA from blood samples of 10 individuals belonging to a family spanning four generations in which one patient showed the complete MERRF phenotype, three other members were less severely affected, while the remaining were unaffected. The percentage of mutant mtDNA was quantified by laser-densitometric scanning of the negative photographic sheets of the agarose gels. The results showed that the MERRF patient had 53% of mutated mtDNA while the two less affected patients had 62% and 14% of mutated mtDNA, respectively. However, a high percentage of mutated genomes (up to 64%) was also found in some unaffected relatives. These results show that although on one hand the mutation is probably the primary cause of the disease, on the other hand the relative amount of mutated mtDNA in blood samples is not indicative of its clinical expression.

Adolescent↗
PubMed1992

DNA ligase I gene expression during differentiation and cell proliferation.

We have studied the regulation of mammalian DNA ligase I gene by using a cDNA probe in Northern blot experiments with RNA extracted from several cell types in different growth conditions. DNA ligase I mRNA is detected in all analysed cell systems, regardless of their proliferation state, including mature rat neurons. A significant increase in DNA ligase I mRNA level is observed when cells are induced to proliferate, in agreement with the raise of DNA joining activity found in the same cell systems. The increase parallels the start of DNA synthesis, but the messenger remains at high level beyond the end of the S phase and is detected also in the presence of aphidicolin. A decrease in DNA ligase I mRNA is observed in HL-60 and NIH-3T3 cells after differentiation. The high stability of DNA ligase I mRNA in both resting and proliferating human fibroblasts suggests a cell proliferation dependent rate of transcription. On the other hand the presence of a basal level of DNA ligase I in nondividing cells, strongly suggests an involvement of this enzyme in DNA repair. This conclusion is supported by a threefold increase in DNA ligase I observed 24 h after UV irradiation of human confluent primary fibroblasts.

3T3 Cells↗
PubMed1992

Uracil-DNA glycosylases preferentially excise mispaired uracil.

We have investigated the substrate specificity of human, viral and bacterial uracil-DNA glycosylases employing as substrate double-stranded oligonucleotides containing in the same position of the 5'-32P-labelled strand an uracil residue facing, on the complementary strand, guanine (mimicking cytosine deamination) or adenine (mimicking dUTP misincorporation). The enzyme removal of uracil was monitored and quantified by the generation of alkali-sensitive apyrimidinic sites. All three uracil-DNA glycosylases excise uracil from mispaired oligonucleotides (U/G) more efficiently than from paired oligonucleotides (U/A). The enzymes also remove uracil from single-stranded oligonucleotide with an efficiency similar to that observed with U/A paired oligonucleotide. The efficient recognition of U/G mispair by uracil-DNA glycosylase is important in minimizing miscoding transcripts and C/G-->T/A transitions in proliferating cells.

Animals↗
PubMed1992

L-thymidine is phosphorylated by herpes simplex virus type 1 thymidine kinase and inhibits viral growth.

We have demonstrated that herpes simplex 1 (HSV1) thymidine kinase (TK) shows no stereospecificity for D- and L-beta-nucleosides. In vitro, L enantiomers are not recognized by human TK, but function as specific substrates for the viral enzyme in the order: L-thymidine (L-T) >> 2'-deoxy-L-guanosine (L-dG) > 2'-deoxy-L-uridine (L-dU) > 2'-deoxy-L-cytidine (L-dC) > 2'-deoxy- L-adenosine (L-dA). HSV1 TK phosphorylates both thymidine enantiomers to their corresponding monophosphates with identical efficiency and the Ki of L-T (2 microM) is almost identical to the Km for the natural substrate D-T (2.8 microM). The L enantiomer reduces the incorporation of exogenous [3H]T into cellular DNA in HeLa TK-/HSV1 TK+ but not in wild-type HeLa cells, without affecting RNA, protein synthesis, cell growth, and viability. L-T markedly reduces HSV1 multiplication in HeLa cells. Our observations could lead to the development of a novel class of antiviral drugs characterized by low toxicity.

Cell Division↗
PubMed1992

Quantitative structure-activity relationships of N2-phenylguanines as inhibitors of herpes simplex virus thymidine kinases.

Quantitative structure-activity relationships of the Hansch-type were developed to account for inhibition of thymidine kinases from Herpes simplex viruses types 1 and 2 (HSV1,2) by N2-phenylguanines. Derivatives with meta and/or para substituents on the phenyl ring display a wide range of overlapping, but not identical, potencies as inhibitors of the enzymes. IC50 values for 36 (HSV1) and 35 inhibitors (HSV2) were used to develop equations using hydrophobic (pi), electronic (sigma, R), and group size (MR) parameters. Equations 1 and 2 with correlation coefficients of 0.797 and 0.805, respectively, were obtained for inhibitors of the types 1 and 2 enzymes. Potencies were correlated positively with pi values of meta substituents but negatively with pi values of para substituents in the phenyl ring. Positive correlations were also obtained with the resonance parameter R of para substituents and with sigma constants of meta substituents. The most potent inhibitor of both enzymes was N2-[m-(trifluoromethyl)phenyl]guanine, although HSV2 thymidine kinase was more sensitive to certain compounds than the HSV1 enzyme.

Antiviral Agents↗
PubMed1992

Uracil in OriS of herpes simplex 1 alters its specific recognition by origin binding protein (OBP): does virus induced uracil-DNA glycosylase play a key role in viral reactivation and replication?

We have recently demonstrated that mammalian uracil-DNA glycosylase activity is undetectable in adult neurons. On the basis of this finding we hypothesized that uracil, derived either from oxidative deamination of cytosine or misincorporation of dUMP in place of dTMP during DNA repair by the unique nuclear DNA polymerase present in adult neurons, DNA polymerase beta, might accumulate in neuronal DNA. Uracil residues could also arise in the herpes simplex 1 (HSV1) genome during latency in nerve cells. We therefore suggest a role for the virus encoded uracil-DNA glycosylase in HSV1 reactivation and in the first steps of DNA replication. We show here 1) that the viral DNA polymerase incorporates dUTP in place of dTTP with a comparable efficiency in vitro; 2) that virus specific DNA/protein interactions between the virus encoded origin binding protein and its target DNA sequence is altered by the presence of uracil residues in its central region TCGCA. Thus uracil, present in viral OriS or other key sequences could hamper the process leading to viral reactivation. Hence, HSV1 uracil-DNA glycosylase, dispensable in viral proliferation in tissue culture, could be essential in neurons for the "cleansing" of the viral genome of uracil residues before the start of replication.

Base Sequence↗
PubMed1992

DNA repair mechanisms in neurological diseases: facts and hypotheses.

DNA repair mechanisms usually consist of a complex network of enzymatic reactions catalyzed by a large family of mutually interacting gene products. Thus deficiency, alteration or low levels of a single enzyme and/or of auxiliary proteins might impair a repair process. There are several indications suggesting that some enzymes involved both in DNA replication and repair are less abundant if not completely absent in stationary and non replicating cells. Postmitotic brain cell does not replicate its genome and has lower levels of several DNA repair enzymes. This could impair the DNA repair capacity and render the nervous system prone to the accumulation of DNA lesions. Some human diseases clearly characterized by a DNA repair deficiency, such as xeroderma pigmentosum, ataxia-telangiectasia and Cockayne syndrome, show neurodegeneration as one of the main clinical and pathological features. On the other hand there is evidence that some diseases characterized by primary neuronal degeneration (such as amyotrophic lateral sclerosis and Alzheimer disease) may have alterations in the DNA repair systems as well. DNA repair thus appears important to maintain the functional integrity of the nervous system and an accumulation of DNA damages in neurons as a result of impaired DNA repair mechanisms may lead to neuronal degenerations.

Animals↗
PubMed1991

HP 0.35, a cephalosporin degradation product is a specific inhibitor of lentiviral RNAses H.

Penicillins, cephalosporins and other betalactam antibiotics are widely used antibacterial drugs. Recently it was found that some of them also have effects on proliferating eukaryotic cells (Neftel, K.A. and Hübscher, U. (1987) Antimicrob. Agents Chemother. 31, 1657-1661), and one such effect was shown to be the inhibition of DNA polymerase alpha (Huynh Do,U., Neftel, K.A., Spadari, S. and Hübscher, U. (1987) Nucl. Acids Res. 15, 10495-10506). The data suggested that degradation products of betalactam antibiotics were responsible for the inhibitory effect on DNA polymerase alpha. There is some confirmation at the structural level, since we found that penicillin binding proteins, the natural target of the cephalosporins, share amino-acid homologies to DNA polymerases and also to reverse transcriptase from HIV1 (Hafkemeyer, P., Neftel, K.A. and Hübscher, U. Meth. Find. Exp. Clin. Pharmacol. 12, 43-46, 1990). We have purified and determined the structure of one product from the cephalosporin Ceftazidim and found one molecule (HP 0.35) that did not interfere with eukaryotic cell proliferation but rather had a specific inhibitory effect on the RNase H activity of human immunodeficiency virus 1 (HIV1) and feline immunodeficiency virus (FIV) reverse transcriptases, while the DNA polymerising activity of these enzymes was not affected. RNases H from HeLa cells, calf thymus and Escherichia coli on the other hand were much less affected by HP 0.35. The inhibitory concentration of 50% (IC50) was more than 10 times lower compared to those of all cellular RNases H. We therefore tested the effect of HP 0.35 on in vitro lentivirus infection as exemplified by FIV-infection of CD(4+)-cat lymphocytes in cell culture. Under conditions where cell proliferation was absolutely unaffected, HP 0.35 was able to inhibit FIV-infection in CD(4+)-cat lymphocytes. Moreover, preincubation of these lymphocytes with HP 0.35 rendered the cells completely unsusceptible to FIV-infection. These data suggest that a degradation product of a clinically used betalactam antibiotic might represent an effective inhibitor class for lentiviral RNase H.

Animals↗