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Y Langelier

Publications and source records attributed to Y Langelier.

35 records · Page 2Linked to original sources
PubMed1989

Retention and expression of the left end subfragment of the herpes simplex virus type 2 BglII N DNA fragment do not correlate with tumorigenic conversion of NIH 3T3 cells.

Cotransfection experiments have been carried out using recombinant plasmids pAG60, conferring resistance to antibiotic G418, and pXho3 which contains the left end subfragment (map coordinates 0.583 to 0.596) of the transforming herpes simplex virus type 2 BglII N DNA fragment and encodes the 36K polypeptide associated with the viral ribonucleotide reductase activity. Several NIH 3T3 cell clones resistant to G418 and having morphological changes commonly observed for transformed NIH 3T3 cells were isolated and examined for the presence and stable retention of the viral sequences. Seven of the clones that retained the transfected viral sequences were analysed for the expression of the 36K polypeptide and the tumorigenic phenotype. The results gathered from these studies show that neither the retention of the viral DNA nor the expression of the 36K polypeptide correlated with tumorigenic conversion of these cells.

Animals↗
PubMed1988

Mechanism of inhibition of herpes simplex virus (HSV) ribonucleotide reductase by a nonapeptide corresponding to the carboxyl terminus of its subunit 2. Specific binding of a photoaffinity analog, [4'- azido-Phe6] HSV H2-6(6-15), to subunit 1.

Herpes simplex virus (HSV) ribonucleotide reductase activity is specifically inhibited by a synthetic peptide, Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (HSV H2-(7-15], corresponding to the carboxyl terminus of its subunit 2 (H2). In order to elucidate the mechanism of action of the nonapeptide a photoreactive analog, [4'-azido-Phe6]HSV H2-(6-15), was synthesized. The photoaffinity probe inhibits HSV ribonucleotide reductase activity, and when radioiodinated, it specifically labeled three viral proteins of 144, 95, and 85 kDa. We demonstrated by immunoprecipitation of the 144- and 95-kDa photolabeled proteins with antibodies specific to subunit 1 (H1) of HSV ribonucleotide reductase that the nonapeptide interacts with H1 and probably with its degradation products. Moreover, we obtained evidence that this specific binding is directly responsible for the ribonucleotide reductase inhibition.

Affinity Labels↗
PubMed1987

Structure-activity studies on synthetic peptides inhibiting herpes simplex virus ribonucleotide reductase.

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) ribonucleotide reductase is formed by the association of two nonidentical subunits. A peptide corresponding to the COOH terminus of the subunit 2, Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(7-15)), has been shown to completely inhibit the reductase activity (IC50 = 36 microM) without affecting the host isoenzyme. In order to study the relationship between chemical requirements and inhibitory potencies, a series of peptides, including fragments and analogs of H2-(7-15), were synthesized. The minimum active core can be assigned to the Val-Val-Asn-Asp-Leu sequence (IC50 = 760 microM). N alpha-Extended peptides, such as Ser-Thr-Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(4-15)) and Glu-Cys-Arg-Ser-Thr-Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu (H2-(1-15) ), respectively, have inhibitory potencies 2.1- and 1.4-fold greater than the nonapeptide H2-(7-15). N alpha-Deamination or acetylation of H2-(7-15) increases its potency by 1.8- and 3.0-fold, respectively, whereas amidation of the alpha-carboxylic function diminishes its activity by 3.2-fold. These results indicate that the alpha-amino group is not essential for maximum potency but suggest that a free carboxylic function is required. Substitution of Tyr7 or Ala8 by their respective D-isomer leads to a decrease of potency, suggesting that a specific conformation of the NH2-terminal portion is required to have a maximum activity. Monosubstitution in positions 11, 13, 14, and 15, by L-alanine completely abolishes activity stressing the importance of each amino acid residue contained in the minimum active core. Finally, nonapeptides corresponding to the COOH-terminal portion of the subunit 2 of Epstein-Barr and varicella-zoster virus ribonucleotide reductases also inhibit the HSV-1 reductase activity. The varicella-zoster virus nonapeptide is 4.0 times more potent than H2-(7-15), whereas the Epstein-Barr virus nonapeptide is 3.1 times less potent. These results should help us to design a new generation of potent inhibitors of herpes virus ribonucleotide reductases.

Amino Acid Sequence↗
PubMed1987

Identification of viral polypeptides involved in pseudorabies virus ribonucleotide reductase activity.

We studied pseudorabies virus-induced ribonucleotide reductase and found that it exhibited biochemical properties very similar to those of herpes simplex virus reductase. A polyclonal rabbit antiserum (P9) directed against the carboxy terminus of subunit H2 polypeptide (38,000 daltons) of herpes simplex virus reductase neutralized the pseudorabies virus reductase, as well as the herpes simplex virus isozyme. This serum recognized two pseudorabies virus-specified polypeptides of 34,000 and 110,000 daltons, which may represent the two subunits of the enzyme. Furthermore, as already shown for herpes simplex virus reductase (E. A. Cohen, P. Gaudreau, P. Brazeau, and Y. Langelier, Nature [London] 321:441-443, 1986), we show that the nonapeptide itself specifically inhibited pseudorabies reductase activity.

Herpesvirus 1, Suid↗
PubMed1987

Activities of both ribonucleotide reductase subunits, M1 and M2, decrease upon serum starvation of baby hamster kidney 21/C13 cells.

Ribonucleotide reductase from mammalian cells is composed of two nonidentical subunits M1 and M2 which are both required to form the catalytic site. The level of ribonucleotide reductase activity is cell cycle controlled and several reports suggest that this control is achieved mainly by the regulation of M2 subunit synthesis. In the present study, we have found that the activities of both subunits decreased markedly upon serum starvation in the Syrian baby hamster kidney 21/C13 cell line. These decreases did not seem to be correlated with the appearance of an inhibitory factor in serum-starved cells. Quantification of the amount of the M1 subunit protein (89,000 molecular weight) by [32P]dTTP photoaffinity labelling revealed that the decrease in M1 activity was not due to variation in M1 protein level. Therefore, a posttranslational mechanism probably exists which inactivates M1 subunit when cells stay in the quiescent (G0) state and this mechanism could play an important role in the control of ribonucleotide reductase activity.

Animals↗
PubMed1986

5-Fluorouracil-metronidazole combination therapy in metastatic colorectal cancer. Clinical, pharmacokinetic and in vitro cytotoxicity studies.

We have investigated the role of metronidazole (MND) combined with 5-fluorouracil (5-FU) in the treatment of metastatic colorectal cancer. MND (750 mg/m2) was administered i.v. 1 h before 5-FU (600 mg/m2) i.v., daily for 5 consecutive days. Treatment was repeated every 4 weeks until disease progression or prohibitive toxicity occurred. Of the 27 patients entered in the study, 4 (15%) had an objective complete or partial response lasting an average of 7 months. 5-FU toxicity was greatly enhanced by the administration of MND, however, 74% of patients having granulocytopenia (less than 1500/microliter). We investigated the possible mechanisms underlying this enhanced 5-FU toxicity by examining whether MND modified 5-FU pharmacokinetics or whether the two drugs had a synergistic effect in vitro against the HCT-8 colon cancer cell line. While the in vitro studies failed to reveal any synergism between 5-FU and MND, pharmacokinetic evaluation revealed that 5-FU clearance was significantly reduced (26.9%, P less than 0.001) by prior MND administration. MND reduces 5-FU's therapeutic index in the treatment of colorectal cancer by impairing its clearance, which leads to increased toxicity without enhanced therapeutic efficacy.

Adult↗
PubMed1986

Neutralization of herpes simplex virus ribonucleotide reductase activity by an oligopeptide-induced antiserum directed against subunit H2.

Herpes simplex virus type 1 ribonucleotide reductase is associated with two polypeptides of apparent molecular weights 136,000 and 38,000. The two polypeptides form a tight complex and, therefore, are often coprecipitated by monoclonal antibodies. We report here that immunoglobulins G purified from polyclonal rabbit antisera (P9) raised against a nonapeptide corresponding to the carboxy terminus of the 38,000-dalton polypeptide specifically neutralize the herpes simplex virus ribonucleotide reductase activity. We suggest that the P9 immunoglobulin G neutralizes the reductase activity by impairing the association of the two subunits (H1 and H2) of the enzyme.

Antigen-Antibody Reactions↗
PubMed1986

Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells.

In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed.

Animals↗
PubMed1985

Increased mutation frequency after herpes simplex virus type 2 infection in non-permissive XC cells.

The effect of herpes simplex virus type 2 (HSV-2) infection on the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was studied in the non-permissive XC cell line. When the cells were infected with 20 to 800 p.f.u./cell, there was initially a lag in cell growth and cell death, but after 4 days there was no difference in growth rate between infected and control cultures. However, the mutation frequency, as determined by the number of 6-thioguanine-resistant colonies, was increased in infected cultures by factors ranging from 2.5 to 10.3. This effect was found to be dependent on the multiplicity of infection. The maximum effect was obtained between 20 and 100 p.f.u./cell while further increase in the amount of virus resulted in a drop in the yield of mutants. The optimum multiplicity of infection was a reproducible characteristic but was variable between viral stocks. When a number of mutant clones were examined they were found to have HGPRT activities ranging from undetectable to 6.9% of wild-type, indicating that the mutations were in the HGPRT gene. These results show that, in a non-productive infection, HSV-2 particles can increase the mutation frequency. The possible mechanisms by which this effect is brought about in the host genome are discussed.

Animals↗
PubMed1985

Herpes simplex virus ribonucleotide reductase induced in infected BHK-21/C13 cells: biochemical evidence for the existence of two non-identical subunits, H1 and H2.

In nearly all systems studied, ribonucleotide reductase consists of two non-identical subunits. We present here the results of our study on herpes simplex virus (HSV) ribonucleotide reductase in favour of the existence of two subunits, H1 and H2, different from the mammalian subunits, M1 and M2. First, although the viral subunits could not be separated by Blue Sepharose chromatography (unlike mammalian subunits), they seemed to dissociate at very low protein concentration as suggested by the non-linear relationship between activity and low protein concentration. Second, pyridoxal phosphate (Pyr.P)-NaBH4 treatment and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ) treatment of partially purified extract of mammalian ribonucleotide reductase which inactivated M1 and M2 respectively also inhibited the HSV ribonucleotide reductase. This activity could be restored by mixing Pyr.P-NaBH4-treated extracts with MAIQ-treated extracts of viral ribonucleotide reductase, suggesting that each treated extract contains one active subunit. Moreover, the addition of exogenous M1 or M2 subunits to one or the other of these two treated extracts did not produce any detectable reductase activity. Our interpretation of these results is that the two subunits H1 and H2 which could dissociate upon treatment did not form enzymically active hybrids with the mammalian subunits. Also, the higher degree of resistance to heat inactivation and to hydroxyurea of the viral reductase as compared to the mammalian enzyme suggests that H1 differs from M1 and H2 from M2.

Animals↗
PubMed1982

Bromodeoxyuridine resistance in CHO cells occurs in three discrete steps.

Four independent mutants were isolated from mutagenized cultures of CHO cells by sib selection on the basis of resistance to a low concentration (2.6 x 10(-5) M) of BrdU. All four lines were stable, but all had about 100% of the wild-type (WT) specific activity of thymidine kinase (TK). None of the four yielded derivatives resistant to a high level of BrdU (2 x 10(-4) M) in one step even after mutagenesis, but variants resistant to 4-6 x 10(-5) M BrdU could be isolated at frequencies of about 2 x 10(-5)/cell. At frequencies of 10(-4)-10(-5), the second-step mutants gave colonies resistant to 2 x 10(-4) M BrdU. The second and third steps of resistance were correlated with partial and complete reduction, respectively, in the specific activity of TK, suggesting that the variants may be genotypically heterozygous and homozygous-negative at the tk locus. The first step of BrdU resistance was dominant and appeared to result from a mutation in the gene from ribonucleotide reductase, since in vitro assays on partially purified preparations showed that the reductase activity in mutant cells was less sensitive to BrdUTP than in WT cells.

Animals↗
PubMed1981

Characterization of ribonucleotide reductase induction in BHK-21/C13 Syrian hamster cell line upon infection by herpes simplex virus (HSV).

Ribonucleotide reductase is an essential enzyme in mammalian DNA replication. In quiescent BHK-21/C13 cells exhibiting a low level of ribonucleotide reductase activity, infection with herpes simplex virus (HSV) resulted in the early induction of an altered ribonucleotide reductase. The extent of the induction was dependent upon the m.o.i. and could be diminished or prevented by u.v. treatment of the viral stock, or by inhibitors of mRNA synthesis or protein synthesis. The induction followed the same course of synthesis as viral thymidine kinase and DNA polymerase, and could thus be classified with them as a beta polypeptide. These results suggested that the new activity was produced as a consequence of the virus genome expression. Comparisons of the properties of ribonucleotide reductase extracted from exponentially growing BHK-21/C13 cells showed that the HSV-induced enzyme differed from the cellular isozyme by its insensitivity to inhibition by dTTP, dATP or araATP and its resistance to high salt concentrations. On the other hand, the virus-induced enzyme and the cellular isozyme exhibited a similar sensitivity to hydroxyurea. Therefore, the reported inhibition of HSV DNA replication by hydroxyurea could be the result of inhibition of both HSV-induced and cellular reductase activities.

Ammonium Sulfate↗
PubMed1981

Analysis of deoxycytidine (dC) deaminase activity in herpes simplex virus-infected or HSV TK-transformed cells: association with mycoplasma contamination but not with virus infection.

Deoxycytidine (dC) deaminase activity has been previously reported to be induced in herpes simplex virus (HSV)-infected cells (Chan, 1977). In contrast, we report here that HSV infection of either hamster cells naturally deficient in this enzyme activity or mouse cells containing a low level of activity never resulted in appearance of stimulation of dC deaminase, whereas thymidine kinase (TK) was always induced. Surprisingly, dC deaminase activity, which differed by electrophoretic mobility from the mouse or human cell enzyme, was discovered in some cells selected for the presence of HSV TK after infection with u.v.-irradiated HSV. Evidence is presented which suggests that the appearance of this new enzyme was not due to the presence of virus genes but rather to mycoplasma contamination.

Animals↗
PubMed1978

Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus.

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.

Animals↗
PubMed1975

Detection of virus-specific DNA sequences by quantitative in situ hybridization during infection with SV40.

In situ hybridization has been used as a quantitative method to study the replication of SV40 during lytic infection. Various parameters have been defined such as fixation and denaturation of cytological preparations, concentration of complementary (3H)-cRNA and volume of buffer used for the hybridization reaction. When all these parameters are carefully controlled, a reproducibility of +/-10% can be obtained for the quantitative study of the system SV40-monkey kidney cells (CV-1). In this system, it is evident that the synthesis of viral DNA is not synchronized in confluent cells. Moreover, a proportion of 20 to 30% of cells are not labelled whatever the multiplicity of infection. In SV40-transformed cells, it was not possible to detect significantly the integrated genome due either to the small size of the genome or to the fact that the number of genome-equivalents integrated at the same place is too low to yield a radioactivitity superior to background level for long periods of exposure.

Animals↗
PubMedDate not supplied

Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2.

Ribonucleotide reductase, an essential enzyme for the synthesis of deoxyribonucleotides, is formed by the association of two nonidentical subunits in almost all prokaryotic and eukaryotic cells. The same model probably holds for the herpes simplex virus (HSV)-encoded ribonucleotide reductase; two polypeptides of relative molecular mass 136,000 (136K; H1) and 40K (H2) (referred to elsewhere as RR1 and RR2; see for example, Dutia et al.) have been associated with the viral enzyme by both genetic and immunological studies. Furthermore, DNA sequence analyses have shown significant stretches of amino-acid homology between these viral polypeptides and those of, respectively, subunit 1 (ref. 12) and subunit 2 (ref. 13) of the Escherichia coli and mammalian enzymes. To assess the involvement of the 40K polypeptide in reductase activity, we synthesized a nonapeptide corresponding to the sequence of its carboxy terminus with the intention of raising neutralizing antibodies specific for the viral activity (E.A.C. et al., in preparation). We report here the unexpected finding that the nonapeptide itself specifically inhibits the HSV ribonucleotide reductase activity in a reversible, non-competitive manner, and we suggest that it does this by impairment of the correct association of the two subunits. This phenomenon emphasizes the potential usefulness of synthetic peptides in probing critical sites involved in macromolecular interactions.

Amino Acid Sequence↗