RESUMEN
The effects of persimmon extract (Diospyros kaki) and related polyphenols on eukaryotic DNA polymerase alpha were examined. It was found that persimmon extract, epigallocatechin gallate, and epicatechin gallate strongly inhibited the activity of DNA polymerase alpha purified from calf thymus. Among these polyphenols, persimmon extract had the most potent effect on DNA polymerase alpha activity and the concentration of persimmon extract producing 50% inhibition of the activity was 0.191 microM. Persimmon extract showed a weaker effect on DNA polymerase beta and slightly inhibited primase and DNA polymerase I. The inhibition of DNA polymerase alpha by persimmon extract was competitive with the template-primer and noncompetitive with dTTP substrate. The Ki value of DNA polymerase alpha for persimmon extract was estimated to be 70 nM. Moreover, persimmon extract inhibited [3H]thymidine incorporation of human peripheral lymphocyte cells stimulated by PHA.
Asunto(s)
ADN Polimerasa I/antagonistas & inhibidores , Flavonoides , Fenoles/farmacología , Proteínas de Plantas/farmacología , Polímeros/farmacología , ADN Polimerasa I/sangre , ADN Polimerasa beta/antagonistas & inhibidores , ADN Polimerasa beta/sangre , ADN Primasa/antagonistas & inhibidores , ADN Primasa/sangre , Células Eucariotas/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , PolifenolesRESUMEN
Werner syndrome (WS) is a rare autosomal recessive disorder characterized by prematurely aged appearance. Genetic linkage analysis has placed the relevant gene in subchromosomal band 8p12. DNA polymerase beta gene has been mapped to chromosome 8p12-11 and thought to be involved in DNA repair and possibly in recombination. Somatic cells from WS patients exhibit chromosomal instability, a markedly reduced replicative life span and slow growth. The functions of DNA polymerase beta gene and its position prompted us to examine this gene in WS patients. We have found the novel DNA polymerase beta cDNA species in blood samples from WS patients, which contain 107 bp insertions or 87 bp deletions in the catalytic domain of DNA polymerase beta. These mutations change the structure of DNA polymerase beta and thus the capacity of the DNA repair system would be impaired, which may account for the high mutation rate observed in WS.
Asunto(s)
Cromosomas Humanos Par 8 , ADN Polimerasa I/genética , Mutación , ARN Mensajero/genética , Síndrome de Werner/enzimología , Síndrome de Werner/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Bandeo Cromosómico , Mapeo Cromosómico , Clonación Molecular , ADN/genética , ADN Polimerasa I/sangre , Cartilla de ADN , Elementos Transponibles de ADN , ADN Complementario/análisis , Exones , Femenino , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Eliminación de Secuencia , Síndrome de Werner/sangreRESUMEN
We have cloned and expressed in Escherichia coli three different parts of the HBx open reading frame, the N- and C-termini and the interior or central portion, using two vector systems. The sera of 43 hepatitis B virus patients representing three clinical categories--asymptomatic carriers, chronic active hepatitis, and hepatitis B patients with cirrhosis--known to be anti-HBx positive, were tested for reactivity against these constructs by Western blotting. The great majority of sera, regardless of the clinical categories, clearly recognise all three parts of HBx, strongly suggesting that the normal mechanism of biosynthesis of the HBx gene product is a straight-forward translation of the open reading frame starting from the first ATG. However, asymptomatic carriers show a marked, often almost exclusive, preference for recognition of the central portion of HBx, while patients with chronic hepatitis and patients with cirrhosis generally recognise all three parts of HBx to a similar extent.
Asunto(s)
Portador Sano/diagnóstico , Hepatitis B/genética , Transactivadores/genética , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Western Blotting , Clonación Molecular , ADN Polimerasa I/sangre , Escherichia coli/genética , Genes Virales , Hepatitis B/complicaciones , Antígenos de la Hepatitis B/química , Antígenos de la Hepatitis B/genética , Hepatitis Crónica/complicaciones , Hepatitis Crónica/genética , Humanos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/genética , Cirrosis Hepática/microbiología , Sistemas de Lectura Abierta , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Transactivadores/biosíntesis , Transactivadores/química , Proteínas Reguladoras y Accesorias ViralesRESUMEN
1. Spermine, spermidine and putrescine activated DNA-dependent DNA polymerase from human sera by 47-125% at the concentrations of 0.2, 3 and 30 mM, respectively. 2. The polyamines shifted the optimal MgCl2 concentration for the polymerase activity from 10 mM to more physiological 5 mM. 3. Histamine having amino and imino groups at both ends of the molecule also increased the DNA polymerase activity, while cyclopentylamine and n-butylamine showed no effects on the enzyme activity. 4. The stimulatory effect of polyamines on the DNA polymerase activity was more evident with poly(dC)p(dG) used as a template/primer than with poly(dA)p(dT).
Asunto(s)
ADN Polimerasa I/sangre , Poliaminas/farmacología , ADN/biosíntesis , Activación Enzimática/efectos de los fármacos , Histamina/farmacología , Humanos , Cinética , Cloruro de Magnesio/farmacología , Poli dA-dT/metabolismo , Polidesoxirribonucleótidos/metabolismo , Putrescina/farmacología , Espermidina/farmacología , Espermina/farmacología , Moldes GenéticosRESUMEN
An activity gel method was used to analyze the catalytic polypeptides of polymerases alpha and beta in human acute myeloblastic and lymphoblastic leukemia. A 175 kDa alpha-polymerase was found in 85% of bone marrow and in 57% of peripheral blood samples. At variance, a 40 kDa beta-polymerase was found in 94% of peripheral blood and only in 12% of bone marrow samples. No difference in the pattern of polymerase expression was found according to the type of leukemia and the disease status. The role of these enzymes in leukemia and their implications in drug sensitivity are discussed.
Asunto(s)
Médula Ósea/enzimología , ADN Polimerasa II/metabolismo , ADN Polimerasa I/metabolismo , Leucemia Linfoide/enzimología , Leucemia Mieloide Aguda/enzimología , Adolescente , Adulto , Anciano , Niño , ADN Polimerasa I/sangre , ADN Polimerasa II/sangre , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Leucemia Linfoide/sangre , Leucemia Mieloide Aguda/sangre , Masculino , Persona de Mediana Edad , Peso MolecularRESUMEN
46 male chronic hepatitis B virus (HBV) carriers with active viral replication were randomised, with stratification for histology and sexual preference, to receive six months' lymphoblastoid interferon or no therapy. After nine to eighteen months' follow-up, HBeAg was no longer detectable and anti-HBe was present in 6 of the 23 treated patients. HBsAg was not detectable in 5 of these patients and 3 had anti-HBs. All of the controls remained positive for HBeAg and HBsAg. Seroconversion from HBeAg to anti-HBe was preceded in all cases by a pronounced increase in serum aspartate aminotransferase levels of more than ten times the upper limit of normal at eight to twelve weeks; this response was exclusively associated with interferon therapy. These results suggest that loss of HBsAg and a hepatitis-like illness in the third month of therapy are direct effects of interferon treatment.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/análisis , Hepatitis B/terapia , Hepatitis Crónica/terapia , Interferón Tipo I/uso terapéutico , Adulto , Aspartato Aminotransferasas/sangre , Ensayos Clínicos como Asunto , ADN Polimerasa I/sangre , Anticuerpos Antihepatitis/análisis , Hepatitis B/inmunología , Antígenos e de la Hepatitis B/inmunología , Hepatitis Crónica/inmunología , Humanos , Masculino , Distribución AleatoriaRESUMEN
Amounts of DNA polymerase alpha and beta were determined in extracts of chicken erythroid cells at various stages of development. Concentrations of both polymerase activities are high in erythroblasts which are still dividing, decline after the cells cease dividing and begin maturation, and become almost undetectable in the fully mature erythrocytes. While DNA polymerase alpha activity declines gradually, firmly bound DNA polymerase beta activity in the nuclei drops abruptly after the cells finish DNA synthesis and dividing. The amount of a low molecular weight DNA polymerase extractable with the cytoplasmic fraction, possibly DNA polymerase beta, is low in erythroblasts, increases in the more mature erythroid population and then declines to an undectable level in the fully mature erythrocytes.
Asunto(s)
ADN Polimerasa II/sangre , ADN Polimerasa I/sangre , ADN Polimerasa Dirigida por ADN/sangre , Eritropoyesis , Anemia Hemolítica/sangre , Animales , Médula Ósea/metabolismo , Núcleo Celular/metabolismo , Centrifugación por Gradiente de Densidad , Pollos , ADN Polimerasa I/aislamiento & purificación , ADN Polimerasa II/aislamiento & purificación , Eritroblastos/metabolismo , Eritrocitos/metabolismo , Peso MolecularRESUMEN
Avian erythroid cells were separated into five developmental stages by sedimentation on discontinuous isotonic albumin gradients. Solubilized enzyme activities from whole cells were partially purified and characterized by ion exchange and ion filtration chromatography and velocity sedimenttation analysis. Three nucleotide polymerase types were investigated: (a) DNA-dependent RNA polymerases; (b) RNA-dependent terminal ribonucleotidyltransferases, and (c) DNA-dependent DNA polymerases. The two characteristic forms of eucaryotic DNA-dependent RNA polymerases, polymerase I (nucleolar) and polymerase II (nucleoplasmic), were identified. Polymerase III was only marginally detectable even in the earliest developmental populations. At least two species of RNA-dependent terminal ribosyltransferases were present. One apparently was the poly(A) polymerase observed in other systems. The other terminal transferase was present in two chromatographic forms, required an RNA primer, and used UTP and/or CTP as particularly efficient substrates. Three DNA polymerase activities were resolved, two of which were characteristic of the alpha and beta DNA polymerases described in other eucaryotic systems. The third polymerase was not the gamma polymerase but a separate entity. Poly(dC)-dependent RNA polymerase activity, associated with the alpha polymerase, was relatively enriched in the third DNA polymerase species. The activity levels of the nucleotide polymerases were monitored as a function of red cell maturation. Characteristic declining patterns of activity were obtained for each enzyme which correlate well with the synthetic rates of their in vivo products where these are known. These results correlate well with the synthetic rates of their in vivo products where these are known. These results are consistent with the postulate that the general transcriptive and replicative control processes operating during development may involve changes in the level of the requisite polymerases.