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J M Colacino

Publications and source records attributed to J M Colacino.

At least 37 records · Page 2Linked to original sources
PubMed1994

Mammalian DNA polymerases alpha, beta, gamma, delta, and epsilon incorporate fialuridine (FIAU) monophosphate into DNA and are inhibited competitively by FIAU Triphosphate.

Fialuridine [FIAU, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- iodouridine] was used in clinical trials for chronic hepatitis B virus infection and was extremely toxic. Evidence suggested targets of FIAU toxicity included mitochondria, but toxic mechanisms were unclear. Since FIAU is a thymidine analog, we reasoned that triphosphorylated FIAU (FIAUTP) could be incorporated into mitochondrial DNA by DNA pol-gamma and into genomic DNA by DNA polymerases alpha, beta, delta, and epsilon. All five purified mammalian DNA polymerases incorporated FIAUMP into the nascent DNA chain during in vitro DNA synthesis. When FIAUTP was substituted for dTTP, oligonucleotide products were generated efficiently by DNA pol-gamma and were similar to those generated in the presence of the four normal dNTPs. In contrast, oligonucleotide products generated by the four nuclear DNA polymerases in the presence of FIAUTP were significantly reduced in length relative to those generated in the presence of dTTP. In parallel kinetic assays, FIAUTP competitively inhibited the accumulation of radiolabeled dTTP into DNA by DNA pol-gamma. The Ki with DNA pol-gamma was 0.04 microM, the lowest Ki among the mammalian DNA polymerases. Competition between FIAUTP and dTTP and the relative ease of accumulation of FIAUMP in mitochondrial DNA by DNA pol-gamma in vitro together may relate to clinical FIAU toxicity.

Animals↗
PubMed1994

The in vitro anti-hepatitis B virus activity of FIAU [1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil] is selective, reversible, and determined, at least in part, by the host cell.

A human hepatoblastoma cell line was stably transfected with a head-to-tail dimer of the Hepatitis B virus (HBV), subtype adw, genome to generate a cell line which produces HBV. FIAU [1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil] inhibited viral replication in these cells with an IC50 of 0.90 microM, as determined by PCR analysis of extracellular Dane particle DNA, and displayed a 50% cytotoxic concentration (TC50) of 344.3 microM, as determined using the MTT assay. The selectivity index of FIAU (TC50/IC50) was 382.6. In cells incubated for 10 days with FIAU (100 microM) and then incubated with drug-free media with daily media changes for 7 days, viral DNA replication was markedly inhibited but resumed within 24 h after drug removal, demonstrating that the in vitro anti-HBV activity of FIAU is reversible. Both the antiviral activity and cytotoxicity of FIAU were reversed by the addition of equimolar to 10-fold excess molar concentrations of thymidine. The de-iodinated metabolite of FIAU, FAU, had only marginal anti-HBV activity at 100 microM, indicating that this metabolite does not contribute significantly to the activity of FIAU. The examination of intracellular viral DNA replicative intermediates revealed that FIAU was 2000-fold more active against duck HBV DNA replication in human hepatoma cells (IC50 = 0.075 microM) than against this same virus in chicken liver cells (IC50 = 156 microM). FIAU was anabolized to a 25-fold greater extent in human hepatoma cells than in chicken cells, indicating that the anti-HBV activity of this nucleoside analog is dependent, in part, on its phosphorylation by the host cell.

Antiviral Agents↗
PubMed1994

Effect of fialuridine on replication of mitochondrial DNA in CEM cells and in human hepatoblastoma cells in culture.

Fialuridine (FIAU) is a nucleoside analog with potent activity against hepatitis B virus in vitro and in vivo. In this report, the effect of FIAU on mitochondrial DNA (mtDNA) replication in vitro was investigated. CEM cells, a cell line derived from human T cells, were incubated for 6 days in up to 20 microM FIAU. Total cellular DNA was isolated, normalized for the number of cells, and slot hybridized to a probe specific for mtDNA sequences. Treatment of CEM cells with FIAU did not result in a dose-dependent decrease in the amount of mtDNA. In contrast, dideoxycytidine (ddC) inhibited mtDNA replication by 50% at a concentration of approximately 0.1 microM. After 6 days of incubation, both compounds displayed a 50% toxic dose at a concentration of approximately 2 microM in CEM cells and approximately 34 microM in human hepatoblastoma cells (HepG2). In further experiments, CEM cells were incubated for 15 days in up to 2.5 microM FIAU, and again, no inhibition of mtDNA was observed. Over a 6-day incubation, FIAU, at concentrations of up to 200 microM, also failed to inhibit mtDNA replication in either HepG2 or HepG2 cells which constitutively replicate duck hepatitis B virus. In contrast, ddC inhibited mtDNA replication in these cells with a 50% inhibitory concentration of approximately 0.2 microM over a 6-day incubation. Treatment of cells with either FIAU or ddC resulted in a dose-dependent increase in lactate levels in the cell medium, indicating that any effect of FIAU on mitochondrial function may not be related to inhibition of mtDNA replication on the basis of the in vitro data. Alternative explanations for mitochondrial toxicity are considered.

Antiviral Agents↗
PubMed1994

Priming of duck hepatitis B virus reverse transcription in vitro: premature termination of primer DNA induced by the 5'-triphosphate of fialuridine.

Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.

Animals↗
PubMed1993

Inhibition of duck hepatitis B virus replication by hypericin.

Hypericin was found to be active against a member of the hepatitis B virus family, duck hepatitis B virus (DHBV). After a single 1 h incubation with hypericin, cells stably-transfected with a clone of DHBV stopped producing infectious virus for several days, though virus-like particles continued to be released into the culture medium. Characterization of these virions revealed a buoyant density characteristic of infectious virus preparations and lower than that of virus cores, suggesting that the particles were enveloped. Western blot analysis suggested, however, that the viral preS protein in surface antigen particles and, by inference, in virions, was present in covalently cross-linked aggregates. Evidence of a similar level of aggregation of the core subunit of virion nucleocapsids was not found, nor was there evidence of a similar high level of aggregation of cell-associated core and preS proteins. Hypericin was only slightly virucidal against DHBV and culture medium from treated cultures did not block initiation of infection when added to DHBV susceptible cultures prior to a challenge with infectious DHBV. Thus, the primary antiviral activity of hypericin against DHBV replication appears to be exerted at a late step in viral morphogenesis.

Animals↗
PubMed1992

Alteration of hemoglobin function by diadenosine 5',5'''-P1,P4-tetraphosphate and other alarmones.

Heat-shocked organisms are known to produce not only "heat shock proteins" but also diadenosine tetraphosphate (Ap4A) and related compounds that may act as "alarmones" that alert the cell to the onset of metabolic stress. We found that Ap4A is synthesized in chicken erythrocytes and that the Ap4A level in the whole blood of heat-stressed birds increases about 10-fold. In searching for alarmone receptors, we found that the diadenosine polyphosphates bind preferentially with high affinity to the deoxy conformation of hemoglobin in a ratio of one/tetramer. The binding affinity of this new class of effectors of hemoglobin function is directly related to the number of phosphates which bridge the nucleotide moieties, with the most dramatic in vitro effect on oxygen affinity being shown by Ap6A. Decreasing effects are brought about by diadenosine penta-, tetra-, tri-, di-, and monophosphates. The association constant for Ap4A binding to deoxygenated human hemoglobin at pH 7.25 is 26 microM-1, close to that for 2,3-diphosphoglycerate. At 100-fold excess over heme, Ap4A increases the P50 of stripped Hb A in 0.05 M HEPES buffer at pH 7.25, 20 degrees C, from 0.85 to 6.03 mm Hg. The binding, which markedly enhances the Bohr effect, involves the beta chain anion-binding site. The kinetics of both ligand binding and dissociation are affected, with a greater quantitative effect on the oxygen dissociation process. Although the low concentration of the diadenosine polyphosphates in red cells precludes a physiologically significant modulation of oxygen delivery, competition with the ATP- and NAD(P)H-binding sites on hemoglobin or regulatory enzymes may prove to be of adaptive significance.

Animals↗
PubMed1990

Cytopathogenicity, drug susceptibility, and thymidine kinase activity of a retinovirulent herpes simplex virus type 2.

We investigated some of the biological and biochemical characteristics of a neuroinvasive, retinovirulent herpes simplex virus type 2 strain SL (HSV-2[SL]) and compared them with those of a neurovirulent, nonretinovirulent HSV-2 (186). HSV-2(SL) was shown to spread rapidly and produce large syncytium in vitro. HSV-2(SL) and HSV-2(186) were equally susceptible to acyclovir (ACV) and thymine arabinoside (Ara-T). However, HSV-2(SL) was fourfold and 44-fold more susceptible than HSV-2(186) to iododeoxyuridine (IUdR) and bromovinyldeoxyuridine (BVDU), respectively. In addition, cytosolic TK from HSV-2(SL)-infected cells phosphorylated 4, 20, and 23,000 times more IUdR, iododeoxycytidine (IdCyD), and Ara-T than the TK of HSV-2(186), respectively. Further, HSV-2(186) TK did not phosphorylate Ara-T, but HSV-2(186) replication was inhibited by Ara-T. These studies indicate that the retinovirulent HSV-2(SL) has a syn phenotype and a TK with broad substrate activity.

Acyclovir↗
PubMed1990

Virucidal activity of hypericin against enveloped and non-enveloped DNA and RNA viruses.

Hypericin is a polycyclic anthrone first isolated from the plant St. Johnswort and was shown to have dramatic anti-retroviral activity against Friend leukemia virus and radiation leukemia virus in mice. Hypericin displayed marginal activity (IC50 = 6 micrograms/ml) against Moloney murine leukemia virus (Mo-MuLV) in vitro. Hypericin did not display selective antiviral activity against herpes simplex virus, influenza A, adenovirus, or poliovirus. The 50% cytotoxic concentration was approximately 25 micrograms/ml. When virus was incubated with hypericin before infecting cells, the drug was virucidal to all enveloped viruses tested (herpes simplex, influenza virus A, and Mo-MuLV) at concentrations of 1.56 micrograms/ml to 25 micrograms/ml. Hypericin was not virucidal to the non-enveloped viruses tested (adenovirus and poliovirus). These data indicate that the mechanism of viral inactivation for hypericin is dependent upon the presence of a viral lipid envelope. In vivo, hypericin (50 mg/ml) was effective against FLV or HSV-1 if incubated with the virus for 1 h at 37 degrees C before infecting mice, but was not effective if pre-incubated with virus for 1 h at 4 degrees C or if administered concurrently with virus.

Animals↗
PubMed1990

Evaluation of the anti-influenza virus activities of 1,3,4-thiadiazol-2-ylcyanamide (LY217896) and its sodium salt.

1,3,4-Thiadiazol-2-ylcyanamide (LY217896) and its sodium salt were shown to be effective against influenza A and B viruses in vitro and in the mouse model. In nondividing confluent MDCK cells, the 50% inhibitory concentration of LY217896 ranged from 0.37 to 1.19 micrograms/ml against various strains of influenza A virus and from 0.75 to 1.54 micrograms/ml against various strains of influenza B virus, with no apparent cytotoxicity. However, at a concentration of 0.31 microgram/ml, LY217896 inhibited the replication of dividing MDCK cells. LY217896 (9 mg/m2 of body surface area per day) administered in the diet, in the drinking water, by oral gavage, by intraperitoneal injection, or by aerosolization was well tolerated and protected CD-1 mice infected with a lethal dose of influenza A or B virus. Effective administration of the compound could be delayed for up to 96 h postinfection. Virus titer was reduced by 1 to 2 log10 units in lungs of mice given LY217896 in the drinking water. Mice treated initially with protective levels of LY217896 were resistant to a subsequent challenge of influenza virus in the absence of the compound, indicating that the animals were able to develop immunity to the initial infection. Administration of LY217896 to uninfected mice did not induce interferon-like activity or interfere with natural killer cell function. In the ferret, LY217896 was effective in preventing fever induced by influenza virus.

Animals↗
PubMed1989

Detection of thymidine kinase activity using an assay based on the precipitation of nucleoside monophosphates with lanthanum chloride.

A new assay method for the measurement of thymidine kinase (TK) is described. Cytosols were prepared from TK- and TK+ cells and evaluated for TK activity using an assay which is based on the phosphorylation of [125I]-iododeoxyuridine, [125I]-iododeoxycytidine, or [3H]thymidine and the precipitation of the monophosphates of these nucleosides by lanthanum chloride. The specificity, reproducibility, sensitivity, and convenience of this assay are demonstrated.

Bromodeoxycytidine↗
PubMed1989

Physical structure and molecular cloning of equine cytomegalovirus DNA.

Restriction endonuclease (RE) mapping studies and molecular hybridization analyses were conducted to determine the molecular structure of the genome of equine cytomegalovirus (ECMV). The ECMV genome is a linear, double-stranded DNA with a molecular size of 126 +/- 0.6 MDa (189 kbp). A library of cloned BamHI, EcoRI, and HindIII fragments of the viral genome was used to construct RE maps. Individual 32P-labeled cloned DNA fragments were hybridized to Southern blots of viral genomic DNA digested to completion with BamHI, EcoRI, HindIII, or SalI. These analyses revealed that the ECMV genome consists of a 97-MDa unique long region which is bracketed by repeated sequences. At one terminus of the genome, a 21.3-MDa segment of repeated sequences with no apparent unique sequences was identified. At the other terminus, a 6-MDa unique region bracketed by 2.4-MDa repeat segments was identified. No submolar RE fragments were identified upon digestion of the ECMV genome with BamHI, EcoRI, HindIII, SalI, or other REs, including BclI, BglII, NruI, and XbaI. The genome possesses only two termini as judged by lambda exonuclease digestion and by T4 DNA polymerase end-labeling of the intact DNA followed by digestion with BamHI, EcoRI, HindIII, SalI, BclI, BglII, NruI, or XbaI. In addition, Southern blot analysis of DNA extracted from ECMV-infected rabbit kidney cells revealed that only one viral DNA fragment within the intracellular viral DNA pool contains fused genomic termini. Taken together, these observations indicate that the ECMV genome does not isomerize and suggest that the genome of ECMV may be unique among those of the herpesviruses and especially those of the betaherpesviruses (cytomegaloviruses) since it contains regions of extensive internal homology yet does not undergo isomerization. Lastly, the relatively small size of the viral genome indicates an evolutionary diversification among the cytomegaloviruses.

Animals↗
PubMed1987

A model for oxygen storage by hemoglobin.

An oxygen store based on the reversible combination of oxygen with hemoglobin or related hemoproteins could be of considerable value to organisms or tissues faced with intermittent hypoxia or anoxia. It would be of interest, therefore, to know the physical and chemical requirements and limitations of useful storage. A model is presented which predicts storage time from physical, chemical, and geometric properties of the store. Storage time is arbitrarily defined as the time for 50% of the stored O2 to diffuse from a fully-loaded store when the store is exposed to a zero O2 environment. Results are presented in the form of simple approximate equations and in graphical form and indicate that: (1) a wide range of storage times can be obtained with reasonable biological parameters; (2) storage times approaching 1,000 s can be achieved even with stores of microscopic dimension; (3) biological O2 storage is predominantly diffusion-limited, although reaction-limited behavior is not impossible; and (4) the major effect of co-operativity appears to be on the form of O2 release: hemoglobins without co-operativity release O2 at a constantly declining rate, whereas those with high co-operativity release O2 at a constant rate. The model is applicable not only to hemoglobin-based O2 storage, but also to any reaction-diffusion system in which one substance can be "stored" by reversible combination with some other substance which acts as the store.

Allosteric Regulation↗
PubMed1985

Antiviral activity of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine against human cytomegalovirus in human skin fibroblasts.

2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine (FIAC) was shown to be a selective anti-human cytomegalovirus agent in vitro with a 50% antiviral effective dose of 0.6 microM (J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother. 26:505-508, 1983) and a 50% cell growth inhibitory dose of 8 microM. Antiviral activity was more readily reversed with 10-fold excess thymidine, whereby the 50% effective dose was increased to 11.3 microM. FIAC-induced cytotoxicity was more readily reversed with 10-fold excess of deoxycytidine, whereby the 50% inhibitory dose was increased to greater than 100 microM. Thymidine was unable to reverse completely the antiviral activity of FIAC. Although, the extent of phosphorylation of thymidine, deoxycytidine, and deoxyuridine was 6-, 4-, and 4-fold greater, respectively, in human cytomegalovirus-infected cell lysates than in uninfected cell lysates, the extent of phosphorylation of FIAC was only 1.3-fold greater in human cytomegalovirus-infected cell lysates than in uninfected cell lysates. By comparison, the extent of FIAC phosphorylation was 500 times greater in herpes simplex virus type 1-infected cells than in uninfected cell lysates. Methotrexate was 400 times more effective against human cytomegalovirus replication than it was against herpes simplex virus type 1 replication, indicating that thymidylate synthetase may be important for human cytomegalovirus replication. However, 10 microM FIAC did not inhibit thymidylate synthetase activity in uninfected or virus-infected cells as determined by their metabolism of [6-3H]deoxyuridine in the presence or absence of drug. FIAC at 1 microM suppresses and FIAC at 10 microM completely inhibits human cytomegalovirus DNA replication as indicated by Southern blot analysis. This inhibition was reversible. FIAC incorporation into the DNA of human cytomegalovirus strain AD169-infected cells was stimulated relative to that in nondividing, uninfected cells.

Antiviral Agents↗
PubMed1984

Metabolic competition studies of 2'-fluoro-5-iodo-1-beta-d-arabinofuranosylcytosine in vero cells and herpes simplex type 1-infected vero cells.

2'-Fluoro-5-iodo-1-beta-D-arabinofuranosylcytosine (FIAC) is a potent antiviral agent with minimal cytotoxicity. In Vero cells, incorporation of labeled dCyd and dThd into the acid-insoluble DNA fraction was, respectively, competitively and noncompetitively inhibited by FIAC. In herpes simplex type 1 (HSV-1) infected Vero cells, these inhibition patterns became noncompetitive. The inhibition constants of FIAC on dThd and dCyd incorporation into the acid-insoluble fraction during a 15-min period were greater than 30 microM which were much higher than the antiviral concentration of FIAC (ED90 = 0.003-0.013 microM) for continuous exposure. Incorporation of dUrd into acid-insoluble DNA was inhibited by 10 microM FIAC in HSV-1-infected Vero cells, but not in uninfected cells. The radioactivity of [2-14C]FIAC was incorporated into the acid-insoluble DNA fraction, and this incorporation in uninfected cells was strongly inhibited by 10 microM dCyd but not by dThd. By contrast, the incorporation in HSV-1-infected Vero cells was strongly inhibited by 10 microM dThd but not by dCyd. These data indicate that FIAC behaves metabolically like dThd, dUrd, or 5-iodo-dUrd in HSV-1-infected cells but like dCyd in noninfected cells. Thus, combined use of dCyd and FIAC may reduce cytotoxicity of FIAC or incorporation of FIAC into host cell DNA without affecting its antiviral activity. This finding is of significance since, for practical reasons, incorporation of FIAC into host cell DNA needs to be reduced as much as possible.

Animals↗
PubMed1983

Efficacy and selectivity of some nucleoside analogs as anti-human cytomegalovirus agents.

1-(2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluridine (FMAU), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouridine (FIAU), and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluridine (FEAU) were evaluated for antiviral activities against human cytomegalovirus (HCMV) and compared with 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) and E-5-(2'-bromovinyl)-2'-deoxyuridine (BVDU). The relative anti-HCMV potencies of these compounds, as determined by calculating the dose of drug which inhibited 50% plaque formation, were in order of decreasing potency: FIAC greater than FIAU greater than FMAU greater than acyclovir greater than FEAU greater than BVDU. The antiviral activity of FIAC occurred at levels much lower than those that caused cytotoxic or cytostatic effects in uninfected fibroblasts. Neither thymidine nor deoxycytidine reversed the anti-HCMV activity of FIAC, indicating that this drug was not acting as an analog of the natural nucleosides. FIAC was not phosphorylated by cytosols of HCMV-infected cells to a greater extent that by those of uninfected cells, indicating that, unlike the antiviral activity against herpes simplex virus type 1, the selectivity of this drug is probably not based on a virus-specified pyrimidine kinase.

Antiviral Agents↗
PubMed1981

Infra-red technique for cerebral blood flow: comparison with 133Xenon clearance.

A rapid infra-red optical technique has been developed for the measurement of cerebral blood flow. The method measures optical density changes across the intact skull during the passage of a bolus of the dye. Cardio-Green (CG). The clearance curves obtained for CG boluses are very short (less than 30 sec) in comparison with those obtained with tracers such as 133Xenon (10-30 min) that distribute into cerebral tissue. The volume of distribution of CG is totally intravascular, and the dye is relatively slowly cleared from the body. The important advantages of this spectrophotometric technique are its speed, versatility, and the avoidance of radioactive materials. The differential spectrophotometer used in this study, with trivial modifications, has been used to monitor changes in brain blood volume, oxygen saturation of hemoglobin, and cortical mitochondrial respiratory function, which illustrate the versatility of the technique for neurological assessments.

Animals↗
PubMed1978

Cerebral blood flow in birds: effect of hypoxia.

The effect of hypoxia on cerebral blood flow in ducks was investigated by the rate at which arterially injected xenon-133 was cleared from the duck's brain. A two-component clearance curve resulted, which we have attributed to flow through the grey and white matter. Decreasing the arterial oxygen tension (PaO2) to 75 mmHg had no effect on cerebral blood flow. However, decreasing the PaO2 below 75 mmHg significantly increased blood flow to the fast-clearing compartment. The greatest increase in blood flow was seen when the arterial PO2 was below 50 mmHg. At an arterial PO2 of 30 mmHg, the cerebral blood flow to the fast-clearing compartment was increased more than 600% above the normoxic level. The magnitude of this increase is much greater in the duck than has been reported for mammals at roughly equivalent arterial oxygen tensions. The ability of avian cerebral blood flow to increase at moderate levels of hypoxia, plus the magnitude of the increase, may play a role in the exceptional tolerance of birds to hypoxia.

Animals↗