RESUMEN
Human adenoviruses have often been used as surrogates for testing broad-spectrum virucidal efficacy of biocides. However, recent studies have shown that members of this group of viruses have quite different chemical sensitivities and only serotypes 5 and 44 can be recommended as model viruses. In this study, the hexon protein of the serotypes 1, 2, 5, 6 and 8 was exposed to biocides and subsequently detected by western blotting and the RPS Adeno Detector. Only peracetic acid (PAA) at a relatively high concentration of 0.5% led to complete denaturation of hexon protein within 60 min. This effect was uniform for all adenoviruses tested and was not observed after exposure to 0.05-2.5% povidone-iodine (PVP-I) or 0.7% formaldehyde. However, viral infectivity and genome integrity were influenced by PVP-I and formaldehyde and lower concentrations of PAA. In conclusion, the hexon protein of human adenoviruses shows an unexpectedly high and uniform resistance to chemical biocides. The different chemical sensitivities of adenoviruses cannot be explained by the sensitivity of this main structural compound, but the present findings provide new insights into the virucidal action of disinfectants.
Asunto(s)
Adenoviridae/efectos de los fármacos , Antivirales/farmacología , Cápside/efectos de los fármacos , Desinfectantes/farmacología , Desinfección/métodos , Adenoviridae/fisiología , Proteínas de la Cápside/análisis , Proteínas de la Cápside/efectos de los fármacos , Formaldehído/farmacología , Humanos , Ácido Peracético/farmacología , Povidona Yodada/farmacologíaRESUMEN
Human adenoviruses are model viruses for testing the virucidal efficacy of disinfectants. However, a recent study has shown that the chemical sensitivity of adenovirus serotypes varies significantly, possibly due to the composition of the viral capsid and/or the resistance of nucleic acids. We have investigated the effect of molecular changes in the viral genome of the human adenovirus subgenera C and D. A common oligonucleotide fragment within the hexon gene was amplified by polymerase chain reaction (PCR) after exposure to liposomal povidone-iodine (PVP-I), peracetic acid (PAA), and formaldehyde. The findings were compared with infectivity in cell cultures. PVP-I (0.125%) destroyed the infectivity of most serotypes within 60 min. However, PCR revealed no destruction of the adenoviral genome in most serotypes, even after exposure to higher PVP-I concentrations. PAA (0.5%) failed to inactivate the hexon gene of adenovirus types 22 and 44. Furthermore, the hexon gene of adenovirus type 22 was not altered by 0.7% formaldehyde. In conclusion, the genomes of human adenoviruses show considerably more chemical resistance than the complete viral particle. The molecular resistance of individual serotypes also varies. However, there was no clear correlation between the differences in the effect of disinfectants on infectivity of the complete viral particle and destruction of the viral genome.
Asunto(s)
Adenovirus Humanos/efectos de los fármacos , Adenovirus Humanos/genética , Desinfectantes/farmacología , Formaldehído/farmacología , Genoma Viral/genética , Ácido Peracético/farmacología , Povidona Yodada/farmacología , Desinfectantes/clasificación , Genoma Viral/efectos de los fármacos , Humanos , Control de Infecciones , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , SuspensionesRESUMEN
With the introduction of varicella vaccination, surveillance of varicella-zoster virus (VZV) strains occurring in cases of chickenpox or zoster should be considered. Differentiating Oka vaccine strain from wild-type VZV can be achieved only using molecular genotyping. In the present study, the VZV genotype was examined in 53 VZV strains isolated from patients with varicella or zoster and in 73 samples from skin eruptions, cerebrospinal fluid, and throat swabs obtained from patients with VZV infections in Germany. The polymerase chain reaction and restriction fragment length polymorphisms analysis using DNA fragments of the open reading frames 38, 54, 62, and the R5 repeat region were used. Whereas all VZV isolates could be typed, direct genotyping of viral DNA in patients' samples was achieved in 63 of 73 cases (86.3%). The dominant genotype of VZV found in 88.8% of 116 patients had the wild-type pattern PstI(+) BglI(-) R5A followed by the wild-genotype PstI(+) BglI(+) R5A in 6.0%, the wild-genotype PstI(+) BglI(-) R5B in 3.4%, the wild-genotype PstI(+) BglI(-) R5C and the Oka vaccine genotype PstI(-) BglI(+) R5B in 0.9% of patients each. BglI(-) wild-types were found in 90.7% of patients with zoster and in 9.3% of patients with varicella. By contrast, the BglI(+) wild-type was diagnosed in five patients with varicella and in two patients with zoster. In conclusion, VZV strains found in Germany are similar to strains circulating in the United States and the United Kingdom. VZV wild-type strains containing a BglI restriction site in ORF 54 as well as Oka vaccine strains can rarely be detected.
Asunto(s)
Vacuna contra la Varicela , Varicela/epidemiología , Herpesvirus Humano 3/clasificación , Herpesvirus Humano 3/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Varicela/virología , Niño , Preescolar , Femenino , Genotipo , Alemania/epidemiología , Herpes Zóster/epidemiología , Herpes Zóster/virología , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
To study molecular aspects of cytotoxicity of the anticancer drug beta-D-glucose-ifosfamide mustard we investigated the potential of the agent to induce apoptosis and DNA breakage. Since beta-D-glucose-ifosfamide mustard generates DNA interstrand crosslinks, we used as an in vitro model system a pair of isogenic Chinese hamster V79 cells differing in their sensitivity to crosslinking agents. CL-V5B cells are dramatically more sensitive (30-fold based on D(10) values) to the cytotoxic effects of beta-D-glucose-ifosfamide mustard as compared to parental V79B cells. After 48 h of pulse-treatment with the agent, sensitive cells but not the resistant parental line undergo apoptosis and necrosis, with apoptosis being the predominant form of cell death (70 and 20% of apoptosis and necrosis, respectively). Apoptosis increased as a function of dose and was accompanied by induction of DNA double-strand breaks in the hypersensitive cells. Furthermore, a strong decline in the level of Bcl-2 protein and activation of caspases-3, -8 and -9 were observed. The resistant parental cells were refractory to all these parameters. Bcl-2 decline in the sensitive cells preceded apoptosis, and transfection-mediated overexpression of Bcl-2 protected at least in part from apoptosis. From the data we hypothesize that non-repaired crosslinks induced by beta-D-glucose-ifosfamide mustard are transformed into double-strand breaks which trigger apoptosis via a Bcl-2 dependent pathway.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Reactivos de Enlaces Cruzados/farmacología , Daño del ADN , ADN/efectos de los fármacos , Mostazas de Fosforamida/farmacología , Animales , Caspasas/metabolismo , Cricetinae , Activación Enzimática/efectos de los fármacos , Glucosa/análogos & derivados , Ifosfamida/análogos & derivadosRESUMEN
A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Glucósidos/síntesis química , Guanina/análogos & derivados , Guanina/síntesis química , Monosacáridos/química , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Sistema Libre de Células , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucósidos/química , Glucósidos/farmacología , Guanina/química , Guanina/farmacología , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , O(6)-Metilguanina-ADN Metiltransferasa/química , Solubilidad , Relación Estructura-ActividadRESUMEN
The DNA-repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a decisive determinant of resistance of tumor cells to methylating and chloroethylating anti-cancer drugs. Therefore, selective inhibition of MGMT in tumors is expected to cause tumor sensitization. Several inhibitors of MGMT have been developed which function in both tumors and normal tissue. To deplete MGMT preferentially in tumors, strategies to target the inhibitor to the tumor tissue need to be developed. Here, we report on the properties of glucose-conjugated MGMT inhibitors that might be useful for tumor targeting since tumor cells frequently over-express glucose transporter. O(6)-Benzylguanine (O6BG), 8-aza-O(6)-benzylguanine, O(6)-(4-bromothenyl)-guanine (O6BTG) and the corresponding spacer-linked beta-D-glucose conjugates were analyzed comparatively for MGMT-inhibitory activity. Substitution at the N9 position of the purine moiety resulted generally in a reduction in the efficiency with which the inhibitors blocked MGMT. However, the inhibitory activity of the O6BTG conjugates increased with increasing spacer length, and O6BTG conjugated with a C8 spacer with beta-D-glucose was nearly as effective as O6BTG on its own. MGMT was inhibited by the conjugates both in crude cell extracts and upon treatment of intact HeLa cells, indicating efficient uptake of the glucose conjugates into cells. Since the O6BTG-C8-D-glucose conjugate 8-[O(6)-(4-bromothenyl)-guan-9-yl]-octyl-beta-D-glucoside was highly efficient at MGMT inhibition in a non-toxic concentration range, the drug might be a useful tool for specific tumor sensitization.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Guanina/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Glucosa/química , Guanina/análogos & derivados , Células HeLa/efectos de los fármacos , Humanos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
c-Fos and p53 are DNA damage-inducible proteins that are involved in gene regulation, cell cycle checkpoint control and cell proliferation following exposure to genotoxic agents. To investigate comparatively the role of c-Fos and p53 in the maintenance of genomic stability and the induction of apoptosis, we generated mouse fibroblast cell lines from knockout mice deficient for either c-fos (fos -/-) or p53 (p53-/-) or for both gene products (fosp53-/-). The sensitivity of these established cell lines was compared with the corresponding wild-type cells as to the cytotoxic, clastogenic and apoptosis-inducing effects of ultraviolet (UV-C) light and methyl methanesulfonate (MMS). Additionally, we analysed the frequency of apoptosis of the cell lines after treatment with ionizing radiation (IR). We observed c-fos-/-, p53-/- and fosp53-/- cells to be more sensitive than wild-type cells with respect to cell death, as measured in a cytotoxicity (MTT) assay. Regarding apoptosis, all deficient cell lines displayed hypersensitivity to UV-C light, MMS and IR. With chromosomal aberrations as the endpoint, the sensitivity of the double-knockout cells was between wild-type and single-knockouts. The results indicate that both c-Fos and p53 play an important role in protecting fibroblasts against a broad range of genotoxic agents. The results also show that, in fibroblasts, apoptosis induced by UV-C light, MMS and IR does not require p53 and that, in this cell type, p53 rather protects against DNA damage-induced apoptotic cell death.
Asunto(s)
Apoptosis , Aberraciones Cromosómicas , Daño del ADN , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Metilmetanosulfonato/toxicidad , Mutágenos , Radiación Ionizante , Rayos Ultravioleta , Animales , Northern Blotting , Western Blotting , Línea Celular , Supervivencia Celular , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Necrosis , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Factores de Tiempo , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Striated muscle of the esophagus was until recently considered to consist of "classical" skeletal muscle fibers innervated by cholinergic vagal motoneurons. The recently described co-innervation originating from enteric neurons expressing nNOS, VIP, NPY, and galanin added a new dimension of complexity. The aim of this study was to summarize current knowledge about, and to get further hints as to the possible function of enteric co-innervation of striated esophageal muscle fibers. Aldehyde fixed rat esophagi were processed for immunocytochemistry for CGRP or VAChT (to demonstrate vagal motor terminals), nNOS/NADPH-d, VIP, NPY, and galanin (to demonstrate enteric terminals), met-enkephalin, mu opiate receptor, muscarinic receptors m1-3, soluble guanylyl cyclase, and cGMP dependent kinase type I and II. Motor endplates were visualized using fluorochrome tagged alpha-bungarotoxin to label nicotinic receptors, or with AChE histochemistry. Besides light and confocal laser scanning microscopy, immuno electron microscopy was also employed. Up to 80% of motor endplates were co-innervated. In addition to nNOS, VIP, NPY, and galanin, many enteric terminals in esophageal motor endplates expressed met-enkephalin. Some appeared to stain for the muscarinic m(2) receptor. There was prominent immunostaining for the micro opioid receptor in the sarcolemma at both junctional and extrajunctional sites. Immunostaining for soluble guanylyl cyclase was prominent immediately beneath the clusters of nicotinic receptors. Enteric varicosities and vagal terminals intermingled in motor endplates often without intervening teloglial processes. During ontogeny, initially high co-innervation rates were reduced to adult levels in a cranio-caudally progressing manner. We conclude that, in addition to a possible nitrergic, VIP-, NPY-, and galaninergic modulation of neuromuscular transmission by enteric neurons, opioidergic mechanisms could play a role. On the other hand, cholinergic influence on enteric neurons may be exerted also by the nucleus ambiguus via motor endplates, in addition to the input from the dorsal motor nucleus. The observations that enteric nerve fibers contact striated muscle fibers at specialized sites, i.e., motor endplates, and that these contacts appear in an ordered cranio-caudal sequence after cholinergic motor endplates have been established point to a specific function in neuronal control of esophageal muscle rather than to be an unspecific "hangover" from the smooth muscle past of this organ.
Asunto(s)
Esófago/inervación , Músculo Esquelético/inervación , Plexo Mientérico/anatomía & histología , Animales , Animales Recién Nacidos , Esófago/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Placa Motora/citología , Placa Motora/metabolismo , Fibras Musculares Esqueléticas , Músculo Esquelético/metabolismo , Plexo Mientérico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , RatasRESUMEN
Hyperbaric oxygen (HBO) treatment of human subjects (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total period of 3 x 20 min) caused clear and reproducible DNA damage in lymphocytes, as detected with the comet assay (single cell gel electrophoresis). Induction of DNA damage was found only after the first HBO exposure and not after further treatments of the same individuals. Furthermore, blood taken 24 h after HBO treatment was significantly protected against the induction of DNA damage by hydrogen peroxide (H(2)O(2)) in vitro, indicating that adaptation occurred due to induction of antioxidant defenses. The cells were not significantly protected against the genotoxic effects of gamma-irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA or an inducible change in the levels of free transition metals. We now demonstrate increased levels of heme oxygenase-1 (HO-1) in lymphocytes 24 h after HBO treatment of volunteers. Under the same conditions, superoxide dismutase, catalase and the DNA repair enzymes apurinic endonuclease and DNA polymerase beta were not enhanced in expression. We also show that protection against the induction of DNA damage by H(2)O(2) in lymphocytes even occurs with a shortened HBO treatment which did not induce significant DNA damage by itself. Our results suggest that increased sequestration of iron as a consequence of induced HO-1 might be involved in the adaptive protection after HBO treatment and that the induction of DNA damage is not the trigger for adaptive protection.
Asunto(s)
Adaptación Fisiológica/fisiología , Daño del ADN , Hemo Oxigenasa (Desciclizante)/biosíntesis , Oxigenoterapia Hiperbárica , Estrés Oxidativo/fisiología , Adulto , Liasas de Carbono-Oxígeno/biosíntesis , Catalasa/biosíntesis , ADN Polimerasa beta/biosíntesis , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Inducción Enzimática , Hemo-Oxigenasa 1 , Humanos , Peróxido de Hidrógeno/toxicidad , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Proteínas de la Membrana , Superóxido Dismutasa/biosíntesisRESUMEN
BACKGROUND: The herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system. As effective antiviral drugs are available, rapid and reliable diagnosis has become important. OBJECTIVES: To evaluate retrospectively the usefulness of polymerase chain reaction (PCR) as well as serological procedures for the diagnosis of HSE. STUDY DESIGN: 631 cerebrospinal fluids (CSF) from patients with clinical suspicion of encephalitis were tested for type-specific herpes simplex virus (HSV) DNA using PCR. Virus-specific antibodies including their intrathecal synthesis were measured in 624 CSF and 2409 serum samples of 2711 patients suspected of having encephalitis. RESULTS: Positive results were obtained by PCR in eight patients (1. 3%) for HSV-1 and in seven (1.1%) for HSV-2. Intrathecal antibody synthesis was estimated in 24 (3.8%) patients. In general, no intrathecal antibodies could be measured in patients with positive PCR results and vice versa the intrathecal immune response became positive when CSF was cleared from the HSV. Results of the antibody detection in serum specimens revealed an active HSV infection in 268 out of 2367 patients (11.3%). CONCLUSIONS: The detection of HSV-DNA by PCR is the method of choice for diagnosis of HSE in the early phase of the disease. During the later stage, it has to be diagnosed by the estimation of intrathecally synthesized antibodies.
Asunto(s)
Encefalitis por Herpes Simple/diagnóstico , Encefalitis por Herpes Simple/virología , Herpesvirus Humano 1/aislamiento & purificación , Herpesvirus Humano 2/aislamiento & purificación , Adolescente , Adulto , Anciano , Anticuerpos Antivirales/sangre , Líquido Cefalorraquídeo/inmunología , Líquido Cefalorraquídeo/virología , Niño , Preescolar , ADN Viral/análisis , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/inmunología , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Especificidad de la EspecieRESUMEN
This study was carried out to determine the prevalence of antibodies to herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) in selected German populations, such as blood donors, hospital patients, and human immunodeficiency virus (HIV)-seropositive individuals. Serum samples collected between 1996 and 1998 were tested by enzyme immunoassays using monoclonal antibody-selected native gG1 and gG2 as antigens and an immunoblot using type-specific recombinant glycoproteins. Equivocal results were resolved by an "in-house" Western blot assay. The prevalence of HSV-1 antibodies increased steadily with age and reached high levels of >/=88% among subjects 40 years of age or older. In the sample of patients and blood donors, the HSV-2 seroprevalence was 12.8% (95% CI = 11.9-13.8%). About 81% of the HSV-2 seropositive subjects were coinfected with HSV-1. When adjusted for age, there was no difference in the HSV-2 seroprevalence between hospital patients and blood donors. The HSV-2 seroprevalence was significantly higher among women (15%) than among men (10.5%), yielding a female : male odds ratio of 1.5 for hospital patients and of 1.67 for blood donors. Among the HIV-infected population, 91.1% were seropositive for HSV-1 and 47.9% for HSV-2. HIV-infected women have a significantly higher risk of HSV-2 infection than men (odds ratio [OR] = 3.22; 95% confidence ratio [CI] 1.99-5.20). In conclusion, although the rate of infections with HSV-2 is relatively low in the German population, attention should be given to the further development in adolescents, especially in view of a possible decrease of HSV-1 seroprevalence in childhood.
Asunto(s)
Herpes Genital/epidemiología , Herpes Simple/epidemiología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 2/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Antivirales/sangre , Western Blotting , Niño , Preescolar , Femenino , Alemania/epidemiología , Seropositividad para VIH/epidemiología , Seropositividad para VIH/inmunología , Herpes Genital/sangre , Herpes Simple/sangre , Humanos , Incidencia , Lactante , Masculino , Persona de Mediana Edad , Factores de Riesgo , Estudios SeroepidemiológicosRESUMEN
Virological diagnosis of zoster should be rapid when effective antiviral chemotherapy is being considered. In the present study, vesicle specimens of 100 patients with zoster were analysed by detecting viral DNA using polymerase chain reaction (PCR). The findings were compared with those obtained by traditional virological and serological methods. PCR results confirmed the clinical diagnosis of zoster in 95%. Primers selected from varicella-zoster virus (VZV) gene 28 proved to be most sensitive. The sensitivity of virus culture was 20% (specificity 100%), of direct immunofluorescent VZV-specific antigen staining in vesicle samples 82% (specificity 76%), and in 48% there was a serological response to specific IgM and IgA antibodies within 4 days after the onset of rash. These findings suggest that PCR is the method of choice for rapid laboratory diagnosis of zoster.
Asunto(s)
Herpes Zóster/diagnóstico , Herpes Zóster/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Niño , Cartilla de ADN , ADN Viral/análisis , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/inmunología , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is inducible by genotoxic stress. MGMT induction results from transcriptional activation of the MGMT gene which is a specific response to DNA damage. A possible factor involved in triggering MGMT induction might be p53, because both p53 and MGMT are activated by DNA breaks. To study the effect of p53 on induction of the MGMT gene, we compared the presence of functional wild-type (wt) and mutant p53 with MGMT expression level in various mouse fibroblasts and rat hepatoma cell lines upon genotoxic treatment. Cells which responded to ionizing radiation (IR) by MGMT induction displayed functional p53, whereas in cells not expressing wt p53, MGMT induction was not observed. Also, the cloned MGMT promoter was inducible by IR upon transfection into p53 wt cells, but not in cells deficient for p53. Thus, expression of wt p53 appears to be required for induction of MGMT mRNA and protein by IR. On the other hand, transfection of a MGMT-promoter-CAT construct together with p53 (either wt or mutant) in cells expressing wt p53 markedly reduced the basal activity of the MGMT promoter whereas cotransfection with a p53 antisense construct slightly increased MGMT promoter activity. Furthermore, cotransfection of MGMT promoter with wt or mutant p53 in p53 wt cells reduced radiation evoked MGMT promoter induction. Thus, transfection mediated high level expression of p53 has inhibitory effect both on basal MGMT promoter activity and its activation by IR. The results give evidence for involvement of p53 in DNA damage-induced MGMT promoter activation.
Asunto(s)
Daño del ADN , Reparación del ADN , Regulación Enzimológica de la Expresión Génica , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células 3T3 , Animales , Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/análisis , ADN sin Sentido , Genes p53 , Neoplasias Hepáticas Experimentales , Ratones , Ratones Noqueados , O(6)-Metilguanina-ADN Metiltransferasa/biosíntesis , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
The DNA repair protein O6-methylguanine-DNA methyl-transferase (MGMT) is a main determinant of resistance of cells to the cytostatic effects of O6-alkylguanine-generating alkylating agents. The purpose of our study was to assay MGMT activity in cells of lung cancers and to correlate MGMT levels with chemotherapy response to cyclophosphamide (CTX) and cisplatin (DDP). MGMT levels were determined in 14 human lung tumor xenografts. There was a wide variation of MGMT expression in these tumors, ranging from 10 to 984 fmol/mg protein. There was also a wide range in the sensitivity of the xenografts to CTX and DDP, as measured by specific growth delay. When the MGMT levels of the different xenograft lines were compared with the corresponding responses to CTX and DDP, a close correlation was found between MGMT activity and CTX (lin reg., r = -0.83, p < 0.05). The higher the MGMT activity, the less pronounced was the growth-inhibiting effect of CTX. With DDP, no such correlation was found. Our results indicate that the in vivo response of tumors to CTX is related to the level of MGMT expression.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Cisplatino/farmacología , Ciclofosfamida/farmacología , Neoplasias Pulmonares/enzimología , O(6)-Metilguanina-ADN Metiltransferasa/efectos de los fármacos , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Ratones Desnudos , Trasplante HeterólogoRESUMEN
Vagal afferent neurons contain a variety of neurochemical markers and neuroactive substances, most of which are present also in dorsal root ganglion cells. To test for the suitability of the calcium-binding protein calretinin as a specific marker for vagal afferent fibers in the periphery, immunocytochemistry for this protein was combined with retrograde tracing. Nerve fibers in the rat esophagus, as well as vagal and spinal sensory neurons innervating the esophagus, were investigated for co-localization of calretinin with calbindin, calcitonin gene-related peptide, and NADPH diaphorase. The results indicated that calretinin immunocytochemistry demonstrates neuronal structures known as vagal afferent from other studies, in particular intraganglionic laminar endings. A few enteric neurons whose distribution was unrelated to intraganglionic laminar endings also stained for calretinin. Strikingly, calretinin immunoreactivity was absent from spinal afferent neurons innervating the rat esophagus. In intraganglionic laminar endings and nodose ganglion cells calretinin was highly co-localized with calbindin but not with calcitonin gene-related peptide. On the other hand, calbindin was also found in spinal afferents to the esophagus where it was co-localized with calcitonin gene-related peptide. Vagal afferent neurons innervating the esophagus were never positive for NADPH diaphorase. Thus, calretinin appears to be a more specific marker for vagal afferent structures in the esophagus than calbindin, which is expressed by both vagal and spinal sensory neurons. Calretinin immunocytochemistry may be utilized as a valuable tool for investigations of subpopulations of vagal afferents in certain viscera.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Esófago/inervación , Ratas Sprague-Dawley/anatomía & histología , Médula Espinal/citología , Estilbamidinas , Nervio Vago/citología , Animales , Tronco Encefálico/química , Tronco Encefálico/citología , Tronco Encefálico/enzimología , Calbindina 2 , Calbindinas , Péptido Relacionado con Gen de Calcitonina/análisis , Femenino , Colorantes Fluorescentes , Ganglios Espinales/química , Ganglios Espinales/citología , Ganglios Espinales/enzimología , Masculino , NADPH Deshidrogenasa/análisis , Ganglio Nudoso/química , Ganglio Nudoso/citología , Ganglio Nudoso/enzimología , Ratas , Ratas Wistar , Proteína G de Unión al Calcio S100/análisis , Médula Espinal/química , Médula Espinal/enzimología , Vagotomía , Nervio Vago/química , Nervio Vago/enzimologíaRESUMEN
Nickel compounds are widespread carcinogens, and although only weakly mutagenic, interfere with nucleotide excision repair and with the repair of oxidative DNA base modifications. In the present study we investigated the effect of nickel(II) on the induction and repair of O6-methylguanine and N7-methylguanine after treatment with N-methyl-N-nitrosourea (MNU). We applied Chinese hamster ovary cells stably transfected with human O6-methylguanine-DNA methyltransferase (MGMT) cDNA (CHO-AT), and compared the results with the MGMT-deficient parental cell line. As determined by high-performance liquid chromatography/electrochemical detection (HPLC/ECD), there was a slight but mostly not significant reduction in the formation of both types of DNA lesions by MNU in the presence of nickel(II). Although nickel(II) did not markedly affect the repair of N7-methylguanine, it decreased the repair of O6-methylguanine in a dose-dependent manner, starting at concentrations as low as 50 microM. While the MGMT protein level was not altered in the presence of nickel(II), the MGMT activity was diminished as demonstrated in cell extracts form nickel-treated cells. This repair inhibition was accompanied by an increase in MNU-induced cytotoxicity in nickel-treated CHO-AT cells but not in MGMT-deficient control cells. There is strong evidence that O6-methylguanine is involved in tumour formation after exposure to alkylating agents. Thus, the finding that nickel(II) inhibits the repair of this lesion could be of major importance for risk assessment in case of combined exposures at work places and in the general environment.
Asunto(s)
Reparación del ADN/efectos de los fármacos , Guanina/análogos & derivados , Níquel/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Animales , Western Blotting , Células CHO , Cromatografía Líquida de Alta Presión , Cricetinae , Femenino , Guanina/metabolismo , Humanos , Metilnitrosourea/farmacología , TransfecciónRESUMEN
When using proteases in direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymic peptide synthesis. Analysis of the equilibrium position reveals a favourable shift towards the peptide product if starting materials are largely undissolved in the reaction medium and the product precipitates. This approach enabled us to obtain high peptide yields in thermolysin-catalysed reactions in high-density aqueous media with an equimolar supply of substrates. The easy scale-up (up to mol-scale) of this approach is demonstrated by two examples. Z-His-Phe-NH2 and Z-Asp-Phe-OMe, precursors for cyclo-[-His-Phe-] and the low-calorie sweetener Aspartame, respectively, were synthesized in preparative yields of 84-88%.
Asunto(s)
Dipéptidos/síntesis química , Termolisina/química , Catálisis , Cromatografía Líquida de Alta PresiónRESUMEN
Recently we have demonstrated the advantage of solid-phase substrate pools mainly in equilibrium controlled protease-catalysed peptide syntheses. The extension of this approach to protease-catalysed acyl transfer reactions will be presented. The model reaction was systematically investigated according to both the influence of solid phases present in the system on enzyme activity as well as nucleophile concentration on peptide yield. The key parameter for obtaining high peptide yield via acyl transfer is the ratio between aminolysis and hydrolysis. We combined high nucleophile concentrations with solid-phase acyl donor pools. This approach enabled us to supply ester substrate and nucleophile in equimolar amounts in a high-density media without the addition of any organic solvent. Several multi-functional di- to tetrapeptides were obtained in moderate to high yields.
Asunto(s)
Oligopéptidos/síntesis química , Acilación , Aminas/química , Catálisis , Quimotripsina/química , Hidrólisis , Cinética , Reproducibilidad de los ResultadosAsunto(s)
Análisis Químico de la Sangre/instrumentación , Recolección de Muestras de Sangre/instrumentación , Células Sanguíneas , Separación Celular/instrumentación , Recuento de Eritrocitos , Estudios de Evaluación como Asunto , Geles , Humanos , Recuento de Plaquetas , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: We studied the possibility of detecting varicella-zoster virus in formalin-fixed tissue samples from a still-born infant with congenital anomalies in order to verify the relationship with maternal varicella. CASE: A girl with hypoplasia of extremities, skin lesions, and microphthalmos was stillborn at 34 weeks' gestation. Her mother had had chickenpox between the 13th and 15th gestational weeks. Varicella-zoster virus DNA could be detected in formalin-fixed tissue samples of lungs, spleen, adrenal glands, bulbus oculi, and placenta by polymerase chain reaction (PCR). The method, with the use of primers from gene 29 encoding the major DNA-binding protein, proved to be highly sensitive. In addition, varicella-zoster virus DNA/antigens were localized in some organs by in situ hybridization/monoclonal antibodies. CONCLUSION: The PCR method should be included in the diagnosis of congenital varicella syndrome. The varicella-zoster virus can be detected in formalin-fixed tissue samples using this technique.