RESUMEN
Benzoyl peroxide (BP), a tumor promoter, has been shown to cause free-radical-induced lipid peroxidation and membrane damage at toxic concentrations. However, its effects on lipid metabolism at concentrations that were not overtly toxic have not been investigated. The purpose of the current study was to examine the effects of BP and its final degradation product, benzoic acid (BA), on lipid metabolism. Two cell lines, hamster cheek pouch (HCP) and human monocytes (THP-1), were used to determine the effects of BP, BA, and BP combined with FeCl2 on cell lipid metabolism. Cells were exposed to BP and 14C acetate for 24 h, or cells with prelabeled lipids were harvested, and the lipids were extracted and separated with the use of thin-layer chromatography. Lipid metabolism of some neutral lipids such as triglycerides was altered for both cell types in response to BP. Also, cholesterol content was reduced in THP-1 cells and a phospholipid, phosphatidylethanolamine (PE), was reduced in HCP cells. The final degradation product of BP, BA, failed to elicit any response in lipid metabolism. Subtoxic concentrations of BP induced changes in neutral lipids such as triglycerides and cholesterol. The metabolism of major phospholipids except PE remained unchanged. The effects were related to BP and its degradation and varied with the cell type.
Asunto(s)
Peróxido de Benzoílo/farmacología , Queratolíticos/farmacología , Metabolismo de los Lípidos , Animales , Ácido Benzoico/farmacología , Recuento de Células , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cricetinae , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Monocitos/efectos de los fármacos , Monocitos/metabolismoRESUMEN
A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.
Asunto(s)
Carcinoma/metabolismo , Neoplasias de la Boca/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Transporte Activo de Núcleo Celular , Carcinoma/patología , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/fisiología , Cinética , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/patología , Proteína smad3 , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta1 , Células Tumorales CultivadasRESUMEN
The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The IE protein of 1487 amino acids contains a serine-rich tract (SRT) between residues 181 and 220. Deletion of the SRT decreased transactivation activity of the IE protein. Previous results from investigation of the ICP4 protein, the IE homolog of herpes simplex virus 1 (HSV-1), revealed that a domain containing a serine-rich tract interacts with EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein), a 15-kDa nucleolar-ribosomal protein (R. Leopardi, and B. Roizman, Proc. Natl. Acad. Sci. USA 93, 4572-4576, 1996). DNA binding assays revealed that (i) glutathione S-transferase (GST)-EAP disrupted the binding of HSV-1 ICP4 to its cognate DNA in a dose-dependent manner, (ii) GST-EAP interacted with the EHV-1 IE protein, but did not disrupt its binding to its cognate site in viral DNA. GST-pulldown assays indicated that the SRT of the IE protein is required for physical interaction with EAP. The IE protein and EAP colocalized in the cytoplasm of the infected equine ETCC cells at late times of the infection cycle. This latter finding may be important in EHV-1 gene regulation since late viral gene expression is greatly influenced by the EICP0 trans-activator protein whose function is antagonized by the IE protein.
Asunto(s)
Herpesvirus Équido 1/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Citoplasma/metabolismo , ADN Viral/metabolismo , Eliminación de Gen , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/genética , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/genética , Ratones , Proteínas Recombinantes de Fusión/metabolismo , Serina/química , Serina/genética , Activación TranscripcionalRESUMEN
Green tea polyphenols are known to induce apoptosis in certain types of tumor cells. However, the mechanism(s) that enables normal cells to evade the apoptotic effect is still not understood. In this study, Western blot analysis combined with cycloheximide treatment was used to examine the effects of green tea polyphenols on the expression levels of p57, a cyclin-dependent kinase and apoptosis inhibitor, in normal human keratinocytes and in the oral carcinoma cell lines SCC25 and OSC2. The results showed that the most potent green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), induced p57 in normal keratinocytes in a dosage- and time-dependent manner, while the levels of p57 protein in oral carcinoma cells were unaltered. The differential response in p57 induction was consistent with the apoptosis status detected by annexin V assay. The data suggest that the chemopreventive effects of green tea polyphenols may involve p57-mediated cell cycle regulation in normal epithelial cells.
Asunto(s)
Anticarcinógenos/farmacología , Flavonoides , Proteínas Nucleares/biosíntesis , Fenoles/farmacología , Polímeros/farmacología , Té , Anciano , Carcinoma de Células Escamosas/metabolismo , Catequina/análogos & derivados , Catequina/farmacología , Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Masculino , Neoplasias de la Boca/metabolismo , Células Tumorales CultivadasRESUMEN
OBJECTIVES: Diacylglycerol-kinase (DAG-kinase) is an enzyme that phosphorylates diacylglycerol (DAG) to phosphatidic acid (PA), which serves as a precursor to phosphoglycerides involved in cell signaling or as cell membrane structural components. DAG-kinase can be inhibited by diacylethylene glycols (DAEG). We hypothesize that 2-hydroxyethyl methacrylate (HEMA) may alter phosphorylation of DAG to PA following intracellular formation of DAEG. METHODS: Cultured rabbit kidney (RK13) epithelial cells were treated with HEMA, EG, or known inhibitors of DAG-kinase for 24 h, then exposed to [32P]O4- in the presence of a synthetic diacylglycerol for 2 h. Other cultures were radiolabeled with [3H]-oleic acid for 24 h, then exposed to HEMA for an additional 24 h. The cells were harvested and the lipids extracted. Radioactive lipids were separated by thin layer chromatography, located by autoradiography, and quantitated as cpm/ug protein. Cell cultures treated with HEMA were homogenized and the DAG-kinase activity was assayed and expressed as cpm/ug protein. Data were analyzed by one-way ANOVA and Newman-Keuls Multiple Comparison Test. RESULTS: Cultures exposed to HEMA or known DAG-kinase inhibitors exhibited reduced incorporation of radioactivity in the PA fraction compared to control cultures. Direct assays of DAG-kinase activity from cells exposed to HEMA demonstrated decreased enzyme activity. Evaluation of cell phospholipid synthesis showed altered formation of phosphatidylethanolamine and phosphatidylcholine. SIGNIFICANCE: Results suggest that HEMA impairs formation of PA, possibly by acylation of EG released by hydrolysis of the HEMA and resultant production of the inhibitor DAEG. The decreased availability of PA may alter PA-dependent cell structural lipid pathways and lipid-dependent signaling pathways, altering cell growth.
Asunto(s)
Recubrimientos Dentinarios/farmacología , Metacrilatos/farmacología , Ácidos Fosfatidicos/metabolismo , Análisis de Varianza , Animales , Autorradiografía , Células Cultivadas , Cromatografía en Capa Delgada , Diacilglicerol Quinasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Hidrólisis , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Ácido Oléico/metabolismo , Ácidos Fosfatidicos/antagonistas & inhibidores , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosforilación , Conejos , Radiofármacos , Estadística como Asunto , TritioRESUMEN
The EICP22 protein (EICP22P) of Equine herpesvirus 1 (EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Herpesvirus Équido 1/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Virales , Animales , Western Blotting , Células Cultivadas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/metabolismo , Herpesvirus Équido 1/genética , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/genética , Ratones , Microscopía Confocal , Pruebas de Precipitina , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Components of dental resins such as dimethylaminoethyl methacrylate (DMAEMA) can alter cell lipid composition, presumably by esterase-mediated hydrolysis. The resulting dimethylethanolamine is incorporated into cell phospholipids, while the methacrylic acid may alter several metabolic pathways. We hypothesize that HEMA is cleaved in a similar manner and the released ethylene glycol is incorporated into cell lipids, yielding phosphatidylethylene glycol (PtEG), and the methacrylic acid alters other lipid pathways in a manner similar to that of methacrylic acid released from hydrolysis of DMAEMA. Cultures of hamster buccal pouch (HCP) and rabbit kidney (RK13) epithelial cells were exposed to subtoxic concentrations of HEMA in the presence of [14C]-acetate or [3H]-oleic acid. Other cultures were prelabeled with [14C]-acetate followed by exposure to various concentrations of HEMA. Cell lipids were extracted by the method of Bligh and Dyer and separated by thin layer chromatography on silica gel K-6 plates or SG-81 silica gel loaded chromatography paper. The fate of the ethylene glycol was traced using [14C]-ethylene glycol. Radioactive lipids were located using autoradiography and known standard lipids and quantitated by liquid scintillation spectrometry. In the presence of HEMA several classes of lipids were altered. Among the neutral lipids, the most notable changes involved sterol precursors, triglycerides, fatty acids, and cholesterol esters, while phosphatidylcholine was affected among the phospholipids. The results differed quantitatively between the two cell types. Results also suggest that EG, including that released by hydrolysis of HEMA, is incorporated into cell phospholipids, producing PtEG. The changes in neutral lipid labeling may occur by alteration of lipid synthetic pathways utilizing acetyl Co-A as well as inhibition of enzymes involved in synthesis of cholesterol from sterol precursors and hydrolysis of cholesterol esters. Synthesis of PtEG may take place via phospholipase D-mediated headgroup exchange. Alterations in the cellular lipids may affect cell membrane properties and associated cell functions.
Asunto(s)
Materiales Biocompatibles/farmacología , Células Epiteliales/efectos de los fármacos , Metabolismo de los Lípidos , Metacrilatos/farmacología , Animales , Materiales Biocompatibles/farmacocinética , Células Cultivadas , Mejilla , Cricetinae , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Glicol de Etileno/metabolismo , Hidrólisis , Riñón , Ensayo de Materiales , Metacrilatos/farmacocinética , Fosfolípidos/metabolismo , ConejosRESUMEN
Dimethylaminoethylmethacrylate (DMAEMA), a commonly-used component of visible-light polymerized dental resins, has the potential to elute and interact with tissue cells to cause cytotoxicity or sublethal metabolic changes. Short-term exposure of cultured oral epithelial cells to sublethal DMAEMA concentrations has been shown previously to affect cell neutral lipid and phospholipid metabolism, resulting in accumulation of significant quantities of dimethylphosphatidylethanolamine (DMPE). In non-treated cells, DMPE is a transient intermediate in phospholipid metabolism and is not detectable by standard methods. In the current study, the effects of prolonged exposure of cells to DMAEMA, and the mechanisms for formation of DMPE in the presence of DMAEMA were examined. Exposure of a keratinizing hamster buccal cheek pouch cell line (HCP cells) to 0.8 mM DMAEMA for 2, 3, 7, and 14 days resulted in reduced incorporation of [14C]acetate into several classes of phospholipids. DMPE was detectable at all time points in DMAEMA-exposed cultures and comprised between 12.48% and 18.33% of the total radiolabeled phospholipids. The results of short-term exchange experiments indicated that headgroup exchange was not the major reaction responsible for formation of DMPE in DMAEMA-treated cells; rather the formation appeared to occur through typical phospholipid metabolic pathways. The cells appeared able to re-establish and maintain homeostasis in the presence of this altered cell lipid composition.
Asunto(s)
Materiales Biocompatibles/toxicidad , Células Epiteliales/efectos de los fármacos , Lípidos de la Membrana/metabolismo , Metacrilatos/metabolismo , Metacrilatos/toxicidad , Análisis de Varianza , Animales , Materiales Biocompatibles/metabolismo , Células Cultivadas , Mejilla , Cromatografía en Capa Delgada , Cricetinae , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Lípidos de la Membrana/análisis , Fosfatidiletanolaminas/metabolismo , Fosfolípidos/análisis , Fosfolípidos/metabolismo , Ácidos Polimetacrílicos/metabolismo , Ácidos Polimetacrílicos/toxicidad , Estadísticas no ParamétricasRESUMEN
The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.
Asunto(s)
Antígenos Virales/metabolismo , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Herpesvirus Équido 1/metabolismo , Proteínas Virales/metabolismo , Animales , Antígenos Virales/genética , Línea Celular , Cricetinae , Proteína Vmw65 de Virus del Herpes Simple/genética , Herpesvirus Équido 1/genética , Caballos , Sistemas de Lectura Abierta , ARN Mensajero/análisis , Proteínas Virales/genéticaRESUMEN
Methacrylates can affect cell functions by surfactant-like effects or by altering cell lipid composition. Dimethylaminoethyl methacrylate (DMAEMA), an activator widely used in visible-light polymerized dental resins has been shown to elute readily into aqueous environments. The current study examined the metabolism of this material by oral epithelial cells (HCP) and its subsequent effects on cell lipids. Cells were plated in culture medium, then exposed to DMAEMA in the presence of 14C-acetate, a precursor which labeled the cell lipids. Other cultures were prelabeled with radioisotope, then exposed to DMAEMA. After incubation, the cell lipids were extracted and separated by TLC. Radioactive lipids were located and quantitated. Exposure of the cells to DMAEMA resulted in decreased synthesis of cholesterol with a concomitant increase in sterol precursors. Cholesterol esters and triacylglycerides also increased. Among the polar lipids, phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) decreased in response to DMAEMA. However, dimethylphosphatidyl ethanolamine (DMPE), a precursor of PC not detectable in control cultures, accumulated to a significant extent in cells exposed to DMAEMA. Furthermore, changes in PC and DMPE levels persisted in the cells for at least 48 h after removal of the DMAEMA. The results indicate that DMAEMA produces alterations in the relative amounts of several cellular neutral and polar lipids. Such alterations, especially of the normal phospholipid composition, along with an alteration in cellular cholesterol, could result in altered membrane-associated cell functions.
Asunto(s)
Metabolismo de los Lípidos , Metacrilatos/farmacología , Mucosa Bucal/efectos de los fármacos , Sustancias Reductoras/farmacología , Acetatos/farmacología , Análisis de Varianza , Animales , Radioisótopos de Carbono , Células Cultivadas , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Cromatografía en Capa Delgada , Cricetinae , Materiales Dentales/normas , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Indicadores y Reactivos/farmacología , Marcaje Isotópico , Mucosa Bucal/citología , Mucosa Bucal/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Triglicéridos/metabolismoRESUMEN
The ability of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of the UL41 gene product, the virion-associated host shutoff (vhs) protein. To date, UL41 homologs have been identified in the genomes of three other alphaherpesviruses: equine herpesvirus 1 (EHV-1), varicella-zoster virus, and pseudorabies virus, but very little is known about the putative products of these homologous genes. Our earlier observations that no rapid early host protein shutoff occurred in EHV-1-infected cells led us to test EHV-1 vhs activity more thoroughly and to examine the expression and function of the EHV-1 UL41 homolog, ORF19. In the present study, the effects of EHV-1 and HSV-1 infections on cellular protein synthesis and mRNA degradation were compared at various multiplicities of infection in several cell types under an actinomycin D block. No virion-associated inhibition of cellular protein synthesis or vhs-induced cellular mRNA degradation was detected in cells infected with any of three EHV-1 strains (Ab4, KyA, and KyD) at multiplicities of infection at which HSV-1 strain F exhibited maximal vhs activity. However, further analyses revealed that (i) the EHV-1 vhs homolog gene, ORF19, was transcribed and translated into a 58-kDa protein in infected cells; (ii) the ORF19 protein was packaged into viral particles in amounts detectable in Western blots (immunoblots) with monoclonal antibodies; (iii) in cotransfection vhs activity assays, transiently-expressed ORF19 protein had intrinsic vhs activity comparable to that of wild-type HSV-1 vhs; and (iv) this intrinsic vhs activity was ablated by in vitro site-directed mutations in which either the functionally inactive HSV-1 vhs1 UL41 mutation (Thr at position 214 replaced by Ile [Thr-214-->Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 192-->Phe). From these results we conclude that EHV-1's low vhs activity in infected cells is not a reflection of the ORF19 protein's intrinsic vhs activity but may be due instead to the amount of ORF19 protein associated with viral particles or to modulation of ORF19 protein's intrinsic activity by another viral component(s).
Asunto(s)
Herpesvirus Équido 1/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Expresión Génica , Herpesvirus Équido 1/genética , Humanos , Datos de Secuencia Molecular , ARN Viral/análisis , Conejos , Ribonucleasas , Homología de Secuencia de Aminoácido , Células Vero , Proteínas Virales/genética , Virión/metabolismoRESUMEN
Although glass ionomer cements are generally considered to be tissue-compatible, it has been suggested that unreacted components or setting reaction by-products can affect cell metabolism. The current study examined the effects of constituents leached out of three glass ionomer cements on growth and metabolism of oral epithelial cells. Aseptically prepared discs of Ketac-Cem Radiopaque (KCR), Ketac-Cem Maxicap (KCM) and Fuji I were incubated in Dulbecco's medium for 10 d, with daily medium changes. Cultures of hamster cheek pouch (HCP) cells, a line of hamster buccal pouch epithelial cells, were incubated in control or eluate-containing media for 24 h. Viable cell numbers were determined by the colorimetric MTS assay, and DNA and RNA syntheses were assessed using [3H]thymidine and [3H]uridine incorporation, respectively. Responses to materials were determined by comparison of cell numbers and radioisotope incorporation (counts per minute (cpm) per 1000 cells). Results were analysed by ANOVA and Duncan's multiple range test, then converted to percent control for comparison. The eluates of all three materials from the first 24 h of soaking inhibited HCP cell growth. The number of cells in cultures exposed to Fuji were 88% of control cultures, while those exposed to KCR and KCM were 58% and 59% of control, respectively. The difference between Fuji-exposed and control cultures was significant (P < 0.05). The two Ketac cements were different from Fuji-exposed and control cultures (P < 0.05) but not from each other. All of the materials caused significant increases in labelling of DNA compared to control cultures (P < 0.05) when calculated on a per cell basis, but the materials did not differ from each other. Both Ketac cements also significantly stimulated labelling of RNA per cell compared to control cultures (P < 0.05). All effects of the material decreased over time. Results suggest that leachable components of the materials may affect the rate of progression of HCP cells through the cell cycle, rather than overt toxicity that results in cell death.
Asunto(s)
Materiales Biocompatibles/farmacología , Cementos de Ionómero Vítreo/farmacología , Mucosa Bucal/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Mejilla , Cricetinae , ADN/biosíntesis , Células Epiteliales , Epitelio/efectos de los fármacos , Fluoruros/farmacología , Mucosa Bucal/citología , Mucosa Bucal/metabolismo , SolubilidadRESUMEN
Substances that elute from denture base resins may inhibit cell growth and disrupt various metabolic processes. This study investigated the effects on cell lipid metabolism of eluates from several denture base resins. Cultured oral epithelial cells were exposed in vitro to eluates of discs made from several denture base resins. Lipid metabolism of the cells was measured using isotopic labeling with 14C-acetate. Results demonstrated that the metabolism of several lipid classes found mainly in the cell membrane was altered by the resin eluates. Eluate from one resin caused the appearance of two previously unrecognized classes of lipids. The alterations of the cell lipids and the presence of the previously unrecognized lipids may be the basis for some clinically evident cytotoxic and allergic reactions.
Asunto(s)
Bases para Dentadura/efectos adversos , Metabolismo de los Lípidos , Resinas Sintéticas/toxicidad , Resinas Acrílicas/toxicidad , Análisis de Varianza , Animales , Membrana Celular/efectos de los fármacos , Células Cultivadas , Mejilla , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Cricetinae , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Lípidos/análisis , Lípidos de la Membrana/metabolismo , Mucosa Bucal/citología , Mucosa Bucal/efectos de los fármacos , Fosfatidilcolinas/metabolismo , Resinas Sintéticas/química , Esteroles/metabolismo , Triglicéridos/metabolismoRESUMEN
The IR4 gene (inverted repeat gene 4) of equine herpesvirus type 1 (EHV-), the homolog of the herpes simplex virus type 1 ICP22 gene, is differentially expressed as a 1.4-kb early transcript and a 1.7-kb late transcript that encode a series of proteins that migrate between 42 to 47 kDa, localize to the nucleus of EHV-1-infected cells, and become packaged within EHV-1 virions (V. R. Holden, G. B. Caughman, Y. Zhao, R. N. Harty, and D. J. O'Callaghan, J. Virol. 68, 4329-4340, 1994). To assess the role of the IR4 protein in EHV-1 gene regulation, an IR4 expression vector was cotransfected with EHV-1 chimeric promoter-CAT reporter constructs and EHV-1 effector plasmids to determine the effects of the IR4 protein on the expression of immediate-early (IE), early, and late promoters. These studies revealed that the IR4 protein: (i) minimally trans-activates EHV-1 promoters, (ii) acts synergistically with the UL3 (ICP27) gene product to trans-activate the IE promoter, (iii) does not interfere with the trans-repression of the IE promoter by the IE protein, (iv) enhances transactivation of early promoters by the IE protein, (v) enhances the transactivation of both early and late promoters by the IE and UL3 proteins, and (vi) interacts synergistically with the IE protein to trans-activate the heterologous HSV-1 ICP4 promoter. These data suggest that the IR4 gene product plays a significant role in EHV-1 gene regulation.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Équido 1/genética , Proteínas Virales/fisiología , Línea Celular , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Activación Transcripcional/genética , Transfección , Regulación hacia Arriba , Proteínas Virales/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro-translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.
Asunto(s)
Herpesvirus Équido 1/genética , Herpesvirus Équido 1/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Animales , Anticuerpos Monoclonales , Antígenos Virales/genética , Unión Competitiva , Línea Celular , Cricetinae , Cicloheximida/farmacología , Replicación del ADN , ADN Viral/biosíntesis , ADN Viral/genética , Dactinomicina/farmacología , Mapeo Epitopo , Expresión Génica , Herpesvirus Équido 1/inmunología , Proteínas Inmediatas-Precoces/inmunología , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Conejos , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/virología , TransfecciónRESUMEN
Changes in the lipid composition of a cell membrane due to the binding of one cell modulator may affect binding of a second modulator, whether that binding is receptor-mediated (specific) or non-receptor-mediated (nonspecific). Such altered binding interactions have been demonstrated in oral epithelial cells, wherein N-nitrosonornicotine (NNN), a nonspecific ligand, enhances phorbol ester binding. To characterize membrane changes that may be responsible for such an effect, the current study examined lipid changes in hamster oral epithelial (HCP) cells associated with NNN binding. HCP cultures at two cell densities, 5 x 10(6) cells/100 mm plate (subconfluent cultures) or 10 x 10(6) cells/100 mm plate (confluent cultures) were incubated in Keratinocyte-Serum-Free Medium and exposed to 10 microM NNN or DMSO (solvent control) for 48 h. Lipids were labeled with 14C-acetate, then extracted, separated by thin layer chromatography, and the 14C-lipids located by autoradiography and counted. Exposure of subconfluent cultures to NNN for 48 h, with 14C-acetate present during the final 24 h, resulted in altered phospholipid and fatty acid labeling. Phospholipid labeling increased slightly in the presence of NNN compared to controls, while fatty acid labeling showed a modest but significant decrease in the presence of NNN. Similar changes occurred in the confluent cultures. Prelabeling of lipids in subconfluent cultures, followed by exposure to NNN in the absence of radiolabel, resulted in significantly (P < 0.05) greater phospholipid labeling in the presence of NNN compared to control cultures. At the same time, fatty acid labeling decreased significantly.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Queratinocitos/efectos de los fármacos , Lípidos/biosíntesis , Nitrosaminas/farmacología , Animales , Línea Celular , Cricetinae , Marcaje Isotópico , LigandosRESUMEN
In previous studies of equine herpesvirus 1 (EHV-1) gene regulation, we observed an abundant early infected cell polypeptide (ICP), designated ICP130, which appeared in reduced amounts in cells infected with defective interfering particle-rich EHV-1 stocks compared to standard EHV-1-infected cells. To characterize this ICP further, a monoclonal antibody (MAb) was developed to EHV-1 ICP130 and used to (1) affinity purify ICP130, (2) examine ICP130's ability to bind DNA, and (3) define the synthesis and intracellular localization of ICP130 during productive EHV-1 infections. Although anti-ICP130 MAbs did not crossreact with any HSV-1 protein in immunoblots, a polyclonal antiserum against HSV-2 major DNA binding protein (ICSP11,12) did react with purified EHV-1 ICP130. DNA band shift assays indicated that (1) the mobility of shifted bands representing DNA/EHV-1-infected cell protein complexes was further decreased by the addition of either anti-ICP130 MAbs or anti-ICSP11,12, but not by the addition of irrelevant MAbs, (2) the ability of ICP130 to complex with DNA was not sequence dependent, (3) ICP130 associated with both single- and double-stranded oligomers, and (4) similar supershifted patterns were produced using affinity-purified ICP130 and anti-ICP130 MAbs. During productive infection, ICP130 initially localized rapidly and exclusively to the infected cell's nucleus in a generalized, fine granular pattern. Over the course of infection, this pattern typically progressed to include several large, intensely reactive intranuclear granules, and by 6 hr p.i. some cytoplasmic reactivity also was visible. In < 5% of the cells, a dense, fibrillar network surrounding the nucleus was observed instead. The progressive changes in nuclear localization depended upon the onset of viral DNA replication, and once the late pattern was established, ongoing DNA synthesis was required to maintain it. The results indicate that ICP130 is the previously reported EHV-1 counterpart of the HSV major DNA-binding protein and is similar, but not identical, in many aspects.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Herpesvirus Équido 1/metabolismo , Animales , Anticuerpos Monoclonales , Antígenos Virales/genética , Antígenos Virales/metabolismo , Secuencia de Bases , Células Cultivadas , Chlorocebus aethiops , Cricetinae , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/inmunología , Herpesvirus Humano 1/metabolismo , Ratones , Datos de Secuencia Molecular , Conejos , Fracciones Subcelulares/metabolismo , Células VeroRESUMEN
Oral complications of chronic graft-versus-host disease are well known and may be present in up to 80% of persons with the condition. A case involving a 29-year-old woman with chronic graft-versus-host disease after allogeneic bone marrow transplantation is presented to illustrate how oral mucosal lesions may be adversely affected by severe caries. A variety of extensive mucosal lesions were continually irritated by the sharp edges of the carious teeth. Full-mouth extractions facilitated the complete resolution of the oral lesions. The patient has had no recurrence of oral lesions 4 years after the extractions and has shown no adverse mucosal changes 3 1/2 years after rehabilitation with complete dentures.
Asunto(s)
Caries Dental/complicaciones , Enfermedad Injerto contra Huésped/complicaciones , Enfermedades de la Boca/etiología , Mucosa Bucal/lesiones , Adulto , Trasplante de Médula Ósea/efectos adversos , Candidiasis Bucal/complicaciones , Enfermedad Crónica , Dentadura Completa , Diagnóstico Diferencial , Femenino , Enfermedad Injerto contra Huésped/etiología , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/cirugía , Enfermedades de la Boca/diagnóstico , Enfermedades de la Boca/terapia , Extracción Dental , Xerostomía/complicaciones , Xerostomía/etiologíaRESUMEN
The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (IR4) as a 1.4-kb early mRNA and a 1.7-kb late mRNA which are 3' coterminal (V. R. Holden, R. R. Yalamanchili, R. N. Harty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intracellular localization of the IR4 protein were investigated. Antiserum raised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 150) was used to identify the IR4 protein. Western immunoblot analysis revealed that IR4 is expressed abundantly from an open reading frame composed of 293 codons as a family of proteins that migrate between 42 to 47 kDa. The intracellular localization of IR4 was examined by cell fractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantly in the nucleus and is dispersed uniformly throughout the nucleus. Interestingly, when IR4 is expressed transiently in COS-1 or LTK- cells, a punctate staining pattern within the nucleus is observed by indirect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhibited greatly reduced intranuclear accumulation of the IR4 protein, indicating that this domain possesses an important intranuclear localization signal. Western blot analysis of EHV-1 virion proteins revealed that IR4 proteins are structural components of the virions. Surprisingly, the 42-kDa species, which is the least abundant and the least modified form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.
Asunto(s)
Herpesvirus Équido 1/metabolismo , Proteínas Inmediatas-Precoces , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cricetinae , Sueros Inmunes , Cinética , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Pruebas de Precipitina , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Virales/genética , Proteínas Reguladoras y Accesorias Virales , ViriónRESUMEN
Transient expression assays measuring induction of an equine herpesvirus 1 (EHV-1) immediate early (IE) promoter construct, pIE beta gal, were used to examine trans-induction of the IE gene (IR1) promoter by superinfection with intact EHV-1 (Kentucky A strain), uv-inactivated EHV-1, or EHV-1 stocks highly enriched for defective interfering particles (DIPs), and by cotransfection with plasmids containing EHV-1 DNA fragments. Our results confirm reports by others in that the IE promoter can be induced by both intact and uv-inactivated EHV-1 and provide evidence that DIP-rich virus stocks also possess transinducing activity. Furthermore, IE promoter activity was induced by cotransfection of the promoter construct with either of two plasmids containing restriction enzyme DNA fragments that span ORF12, the putative EHV-1 homolog of the herpes simplex virus 1 (HSV-1) alpha-trans-inducing factor (alpha-TIF) gene, UL48, or by cotransfection with an isolated DNA fragment containing only ORF12, while no transactivation was associated with plasmids linearized by restriction enzyme digestion at a single site within ORF12. These data indicate that, despite its predicted lack of an acidic carboxy terminus (a region essential in HSV-1 alpha-TIF for trans-inducing activity), the EHV-1 ORF12 product is a functional alpha-TIF.