RESUMEN
Skin cancer is the most frequently diagnosed cancer globally and is preventable. Various risk factors contribute to different types of skin cancer, including melanoma, basal cell carcinoma, and squamous cell carcinoma. These risk factors encompass both extrinsic, such as UV exposure and behavioral components, and intrinsic factors, especially involving genetic predisposition. However, the specific risk factors vary among the skin cancer types, highlighting the importance of precise knowledge to facilitate appropriate early diagnosis and treatment for at-risk individuals. Better understanding of the individual risk factors has led to the development of risk scores, allowing the identification of individuals at particularly high risk. These advances contribute to improved prevention strategies, emphasizing the commitment to mitigating the impact of skin cancer.
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COVID-19 , Eritema Multiforme , Vacunas contra la COVID-19 , Humanos , SARS-CoV-2 , VacunaciónRESUMEN
We report a case of a patient with erythema multiforme major following COVID-19 (coronavirus disease 2019) vaccination. Lesions on skin and mucous membranes developed 48â¯h after the second dose of the mRNA-vaccine BNT162b2 (Tozinameran, Comirnaty®). Under the application of external glucocorticoids complete resolution was achieved within 3 weeks.
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COVID-19 , Eritema Multiforme , Vacuna BNT162 , Vacunas contra la COVID-19 , Eritema Multiforme/inducido químicamente , Eritema Multiforme/diagnóstico , Humanos , SARS-CoV-2 , Vacunación/efectos adversosRESUMEN
A novel handheld optical sensor for quantification of fluorescent microarrays, the so-called portMD-113 has been developed. On the surface of a planar waveguide, the spots of different fluorescently labeled biological complexes are excited by the evanescent field of the guided light. The emitted fluorescence signals of the spots are independently and simultaneously detected applying our system, which consists of a pinehole array, a microlens array, an interference filter and a detector array. As it is demonstrated in comparative measurements, the detection limit of this sensor is close to that of commercial top microarray readers, e.g. of modern laser scanners, while it has remarkable and important advantages over them. Namely, the device comprises only a few low-cost, lightweight and small components without applying any moving or energy-intensive elements, which results in turn in a commercially competitive, handheld and compact design and in the possibility to be supplied simply by a battery or a personal computer. These advantageous properties open prospects e.g. for point-of-care medical checks, as well.
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Diseño de Equipo , Fluorescencia , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Técnicas Biosensibles/instrumentación , Humanos , Rayos Láser , Luz , Sistemas de Atención de PuntoRESUMEN
The polled locus has been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. As an intermediate step in our efforts to identify the polled locus and the underlying causative mutation for the polled phenotype, we have constructed a BAC-based physical map of the interval containing the polled locus. Clones containing genes and markers in the critical interval were isolated from the TAMBT (constructed from Angus and Longhorn genomic DNA) and CHORI-240 (constructed from horned Hereford genomic DNA) BAC libraries and ordered based on fingerprinting and the presence or absence of 80 STS markers. A single contig spanning 2.5 Mb was assembled. Comparison of the physical order of STSs to the corresponding region of human chromosome 21 revealed the same order of genes within the polled critical interval. This contig of overlapping BAC clones from horned and polled breeds is a useful resource for SNP discovery and characterization of positional candidate genes.
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Bovinos/genética , Mapeo Contig , Cuernos , Animales , Cromosomas Artificiales Bacterianos , Cromosomas de los Mamíferos , Mapeo Contig/veterinaria , Humanos , FenotipoRESUMEN
OBJECTIVE: Glaucoma is a neurodegenerative disease. Since vascular dysregulation is supposed to be a risk factor for the development of glaucomatous damage, the preventive treatment might slow down the disease development. The efficiency of the therapeutic treatment depends particularly on a drug efflux pump regulated by ABC transporters. ABC 1 is also known to participate on the vascular regulation. This study was focused on the comparative analysis of ABC 1 expression levels in circulating leukocytes of non-glaucomatous individuals and glaucoma patients. RESULTS AND CONCLUSIONS: The expression rates of ABC 1 were significantly increased in leukocytes of glaucoma patients compared to non-glaucomatous individuals. The expression level of ABC 1 was, furthermore, highly homogeneous in glaucoma patients. In contrast, these expression levels in non-glaucomatous individuals were extremely heterogeneous. This transporter acts as the energy-dependent unidirectional transmembrane cholesterol efflux pump and can export a wide range of hydrophobic drugs. Additionally an observed enhanced ABC 1 expression in circulating leukocytes may be implicated in the vascular regulation mechanisms of glaucoma. We proposed the enhanced expression of ABC 1 in leukocytes as a potential marker for the diagnostics and ex vivo molecular monitoring of glaucoma.
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Transportadoras de Casetes de Unión a ATP/biosíntesis , Glaucoma/metabolismo , Leucocitos/metabolismo , Regulación hacia Arriba , Anciano , Biomarcadores/metabolismo , Glaucoma/diagnóstico , Glaucoma/patología , Humanos , Leucocitos/patología , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: Vascular disorder leading to local ischemia/reperfusion has been shown to play an important role in the glaucomatous damage. A decreased expression level of XPGC-gene has been found in circulating leukocytes of normal-tension glaucoma patients. Although decreased activity of XPGC-gene leads to insufficient DNA-repair, no leukopenia has been observed in glaucoma. Molecular mechanisms ensuring cell survival have not been elucidated yet for glaucoma with vascular disorder. MATERIAL AND METHODS: Using the ex vivo optical imaging method of alkaline "comet assay" comparative quantification of DNA breaks was performed in circulating leukocytes of non-glaucomatous non-vasospastic and vasospastic individuals as well as both normal-tension and high-tension glaucoma patients. Relative expression levels of the anti-apoptotic factors P21(WAF1/CIP1) and 14-3-3 sigma were investigated in all groups tested. RESULTS AND CONCLUSIONS: The quantification of P21(WAF1/CIP1) showed the highest expression rates in high-tension glaucoma patients which were significantly higher than those in all other groups tested. The highest expression rates of 14-3-3 sigma were found in both groups of glaucoma patients. These expression levels correlated well with DNA breaks measured. Since the expression of P21(WAF1/CIP1) in leukocytes was shown to be crucial for their survival under stress conditions, we suppose further that the up-regulation of this gene is the key event in the survival mechanisms of leukocytes in glaucoma accompanied with vascular disorder. The p21(WAF1/CIP1) gene should be further taken into consideration as a potential marker, the up-regulation of which in circulating leukocytes of vasospastic individuals may indicate an increased risk for the developing glaucoma.
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Proteínas 14-3-3/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Daño del ADN , Glaucoma/metabolismo , Leucocitos/metabolismo , Regulación hacia Arriba , Adulto , Anciano , Anciano de 80 o más Años , Glaucoma/patología , Humanos , Leucocitos/patología , Masculino , Persona de Mediana Edad , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patologíaRESUMEN
Ocular ischemia resulting from perfusion disturbances may play a major role in initiation of glaucoma. Possibly secondary to ischemia autoimmunogenic events are activated in glaucoma patients with increased prevalence of systemic autoimmune diseases. The determination of potential molecular markers in blood leukocytes could be useful for early noninvasive diagnostics of glaucoma. Our study using subtractive hybridization showed altered gene expression in leukocytes of glaucoma patients in comparison to age and sex matched healthy subjects. Subtracted genes encoding lymphocyte IgE receptor (Fc epsilon RII/CD23), T cell-specific tyrosine kinase, thromboxan A2 receptor, alkaline phosphatase and Na(+)/K(+)-ATPase are differentially expressed in circulating leukocytes of glaucoma patients. These genes show expression profiles characteristic for adherent leukocytes which could be an important contributor to blood-brain barrier breakdown which has been found in glaucoma patients.
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Barrera Hematoencefálica/fisiología , Perfilación de la Expresión Génica , Glaucoma/fisiopatología , Isquemia/metabolismo , Leucocitos/fisiología , Adulto , Anciano , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Barrera Hematoencefálica/fisiopatología , Femenino , Biblioteca de Genes , Humanos , Hibridación Genética , Leucocitos/citología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas/genética , Receptores de IgE/genética , Receptores de Tromboxano A2 y Prostaglandina H2/genética , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
We studied the localization and distribution of connective tissue growth factor (CTGF) in corneal scar tissue and membranes using in situ hybridization in 8 corneas from keratoplasty and 4 normal corneas. Identification of the cells was done with immunohistochemistry for SM-alpha-actin, vimentin, and Lu5. CTGF mRNA was found in activated corneal fibroblasts in 7 of 8 scars, 7 of 8 retrocorneal membranes and 2 subepithelial membranes, whereas the control corneas showed no CTGF mRNA expression. Vimentin was positive in all scars, retrocorneal and subepithelial membranes, SM-alpha-actin in 7 of 8 scars and 6 of 8 retrocorneal membranes. These results suggest that CTGF plays a crucial role in corneal wound healing and membrane formation.
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Proteínas Portadoras/metabolismo , Cicatriz/metabolismo , Córnea/metabolismo , Enfermedades de la Córnea/metabolismo , Lesiones Oculares Penetrantes/metabolismo , Sustancias de Crecimiento/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Actinas/metabolismo , Biomarcadores , Proteínas Portadoras/genética , Cicatriz/etiología , Cicatriz/patología , Factor de Crecimiento del Tejido Conjuntivo , Córnea/patología , Córnea/cirugía , Enfermedades de la Córnea/etiología , Enfermedades de la Córnea/patología , Lesiones de la Cornea , Lesiones Oculares Penetrantes/complicaciones , Lesiones Oculares Penetrantes/patología , Sustancias de Crecimiento/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Hibridación in Situ , Queratoplastia Penetrante , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vimentina/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
PURPOSE: To investigate the correlation between connective tissue growth factor (CTGF) mRNA expression and immunohistochemical characteristics of anterior subcapsular cataract (ASC) formation as well as posterior capsule opacification (PCO) development (expression of type I collagen, alpha-smooth muscle actin and tenascin) under in vivo and under in vitro conditions in human and porcine lens epithelial cells. METHODS: CTGF mRNA expression was investigated using in situ hybridization and RT-PCR. Expression of type I collagen, alpha-smooth muscle actin and tenascin was detected by immunohistochemical staining. RESULTS: CTGF mRNA was expressed in human cataractous plaques of ASC and human PCO membranes, and appeared simultanously with the expression of type I collagen, alpha-smooth muscle actin and tenascin. CONCLUSION: The predominant expression of CTGF mRNA in human ASC and human PCO membranes suggests a significant role of CTGF in the pathological course of these ocular disorders.
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Catarata/metabolismo , Sustancias de Crecimiento/genética , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intercelular , ARN Mensajero/metabolismo , Actinas/metabolismo , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo , Humanos , Inmunohistoquímica , Hibridación in Situ , Músculo Liso/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tenascina/metabolismoRESUMEN
BACKGROUND: Connective tissue growth factor (CTGF) is a novel, cysteine-rich secreted protein, which is implicated in fibrotic disorders and atherosclerosis. To elucidate the role of CTGF in fibrovascular proliferative retinopathy, we investigated the regulation of CTGF gene expression in a cell line of retinal vascular endothelial cells (RVEC) stimulated with fetal calf serum (FCS) and angiogenic growth factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF-BB), endothelial growth factor (EGF), transforming growth factor-beta 1 and -beta 3 (TGF-beta 1, TGF-beta 3), and insulin-like growth factor-I (IGF-I). METHODS: RVEC derived from Macaca mulatta (CRL-1780; ATCC) were stimulated with 10% FCS as well as with VEGF, bFGF, PDGF-BB, TGF-beta 1, TGF-beta 3, EGF, or IGF-I. Time-dependent CTGF gene expression was assessed by northern blot analysis. RESULTS: FCS, TGF-beta 1, TGF-beta 3, bFGF, and EGF induced an upregulation of CTGF gene expression in RVEC in a time-dependent manner. Highest expression was induced with TGF-beta 1. No response on CTGF gene expression could be detected to VEGF, PDGF-BB, or IGF-I. CONCLUSION: The present study demonstrates for the first time that CTGF mRNA is expressed at high levels in RVEC, and that the level of the temporal pattern of its expression is differentially regulated by angiogenic growth factors, indicating a significant role of CTGF in the pathological course of uncontrolled retinal angiogenesis.
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Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/farmacología , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intercelular , ARN Mensajero/biosíntesis , Vasos Retinianos/metabolismo , Animales , Northern Blotting , Línea Celular Transformada , Factor de Crecimiento del Tejido Conjuntivo , Endotelio Vascular/efectos de los fármacos , Factor VIII/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Sustancias de Crecimiento/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Técnicas para Inmunoenzimas , Macaca mulatta , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Vasos Retinianos/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation.
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Citomegalovirus/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Aminoácidos/genética , Secuencia Conservada , Humanos , Datos de Secuencia Molecular , Mutación , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Alineación de SecuenciaRESUMEN
PURPOSE: Contraction of the capsule of the ocular lens is based upon proliferation and contraction of transformed lens epithelial cells. It is assumed that these processes can be assisted by postoperative intraocular inflammation. Previously, we reported that lens epithelial cell proliferation is enhanced by lymphocyte-conditioned medium (LCM). In this study we investigated the effect of LCM as well as of a culture medium conditioned by pigmented ciliary epitheilal cells (CBCM) on the expression of the smooth-muscle alpha-actin of the contractile cytoskeletal elements. METHODS: Explants of the anterior lens capsule of freshly enucleated bovine eyes were cultured in serum-free LCM and CBCM for 3 days, followed by fixation. Smooth-muscle alpha-actin was identified by indirect immunoflorescence. Explants cultured in serum-free bFGF-containing and TGF-beta containing medium served as control. RESULTS: Lens epithelial cells expressed smooth-muscle alpha-actin under the influence of LCM or TGF-beta. No smooth muscle alpha-actin could be detected under the influence of CBCM or bFGF. CONCLUSION: Our results demonstrate that secreted molecules of activated lymphocytes are able to induce the transformation of lens epithelial cells into contractile myofibroblasts and may be involved in the post-operative contraction of lens capsules.
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Actinas/análisis , Medios de Cultivo Condicionados , Cápsula del Cristalino/inmunología , Linfocitos/inmunología , Animales , Bovinos , Cápsula del Cristalino/patología , Activación de Linfocitos/inmunología , Microscopía Fluorescente , Músculo Liso/inmunología , Músculo Liso/patologíaRESUMEN
In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5' fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protein and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs. The results indicated the presence of an N-terminal region within pUL97 which changed the intracellular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matrix fraction. Expression of all pUL97 mutants could be confirmed by Western blot analysis. pUL97-associated ganciclovir (GCV) phosphorylation in rVV-infected cells, determined quantitatively by HPLC analysis, was abolished completely using individual UL97 deletion mutants. Phosphorylation of full-length and some of the mutated pUL97 was detected in cells infected with the rVVs. The UL97 constructs carrying point mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V, and the 4 aa deletion 590AACR593, also resulted in decreased but not abolished phosphorylation of GCV in the rVV system, whereas the phosphorylation of pUL97 itself was not influenced. The rVV system is a suitable method for quantitatively testing the functional relevance of pUL97 mutations.
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Antivirales/metabolismo , Antivirales/farmacocinética , Citomegalovirus/metabolismo , Ganciclovir/metabolismo , Ganciclovir/farmacocinética , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Transporte Biológico Activo , Línea Celular , Núcleo Celular/metabolismo , Citomegalovirus/genética , Cartilla de ADN/genética , Genes Virales , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Matriz Nuclear/metabolismo , Sistemas de Lectura Abierta , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Virus Vaccinia/genéticaRESUMEN
UNLABELLED: The purpose of the present study was to conduct a comparative evaluation of the effect of several serum-free culture conditions on adhesion, population doubling, cryopreservation and PDGF-induced effects on cell proliferation of bovine stromal fibroblasts (BSF). Additionally, these effects were compared to serum-containing cultures. METHODS: Only second-passage BSF were used. Cells were cultured using four different culture media (WM/F12, WM/F12 + FCS 1%, LR-1, DMEM). After 24 h, plating efficiency was determined using a cell-counter system. Subsequently, the cells were seeded at a density of 100 cells/mm2 and cultured for 10 days using the different culture media. Cell number was determined at day 2, 4, 7 and 10 after seeding. Furthermore, the effect of 50 ng/ml PDGF-BB on the proliferation of BSF was tested for these conditions. Cell vitality was determined after cryopreservation of two weeks for each culture medium. RESULTS: The plating efficiency of BSF ranged from 50.2 to 55.5% for the serum-free culture media in contrast to serum-containing conditions, where plating efficiency was 94.8%. With WM/F12 + FCS 1%, a population doubling of 1.27 was observed after an incubation period of 10 days. In contrast, cultivation under serum-free conditions caused neither significant cell proliferation nor cell loss. The stimulation of cell proliferation with PDGF-BB was shown to be 28% (LR1), 40% (WM/F12 + FCS 1%) 76% (WM/F12) and 95% (DMEM) compared to the control. While cell vitality after cryo-preservation was found to be 62.7% using WM/F12 + FCS 1%, vitality using serum-free media was 12.6-22.8%. CONCLUSIONS: The results of the present study demonstrate that with respect to optimal cell adhesion and cell vitality after cryo-preservation, serum-containing media should be used. BSF cultured under the serum-free conditions used in the present study can be maintained quiescent and vital for at least 10 days. Therefore, these serum-free media are useful for cell-culture studies (e.g., determination of proliferation and cytotoxicity).
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Sustancia Propia/citología , Medio de Cultivo Libre de Suero , Fibroblastos/citología , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Recuento de Células , División Celular/efectos de los fármacos , División Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Sustancia Propia/efectos de los fármacos , Criopreservación , Fibroblastos/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacologíaRESUMEN
BACKGROUND: Although platelet-derived growth factor (PDGF) has been thought to be critical in the wound-healing response of Tenon's capsule fibroblasts after glaucoma filtration surgery, no information is currently available concerning the proliferative effect of PDGF isoforms on this cell type. The aim of the present study was to evaluate the proliferative effect of PDGF-AB heterodimer and PDGF-AA and -BB homodimers on cultured human Tenon's capsule fibroblasts. METHODS: Human Tenon's capsule fibroblasts, cultured under serum-free conditions, were stimulated with PDGF-AA, -AB and -BB isoforms in concentrations ranging from 1 to 100 ng/ml. Cell numbers were determined on days 1, 3, 5 and 7, using a cell counter. RESULTS: Addition of PDGF-AB and -BB led to a dose-dependent increase in cell proliferation. A maximal response (79.9% over control) was obtained after 7 days with 30 ng/ml of PDGF-BB, with an EC50 of 8.9 ng/ml. The maximal increase in cell proliferation caused by PDGF-AB (30 ng/ml) was 54.9%, with an EC50 of 12.5 ng/ml. Stimulation with PDGF-AA revealed a significant effect only with concentrations higher than 30 ng/ml. CONCLUSION: Our results indicate that PDGF-AB and -BB isoforms are potent stimulators of proliferation of human Tenon's capsule fibroblasts, suggesting that PDGF-AB and -BB isoforms play an important role in the wound-healing response after glaucoma filtration surgery.
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Anticoagulantes/farmacología , Células del Tejido Conectivo/citología , Fibroblastos/citología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Becaplermina , Recuento de Células , División Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Células del Tejido Conectivo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas c-sis , Proteínas RecombinantesRESUMEN
The temporal expression of the UL97 gene product during human cytomegalovirus (HCMV) infection of human foreskin fibroblasts (HFF) and subcellular localization of this protein were analyzed by using a polyclonal antiserum raised against a truncated UL97 protein of 47 kDa. The UL97 protein was detectable 16 h after infection by Western blot (immunoblot) analysis. Since only reduced UL97 expression occurred in the presence of two inhibitors of DNA replication, phosphonoacetic acid and ganciclovir, we conclude that UL97 is an early-late gene, requiring DNA replication for maximum expression. By indirect immunofluorescence, the protein could be visualized in the nuclei of virus-infected HFF 22 h after infection. Nuclear localization of the UL97 protein was also detected in thymidine kinase-deficient 143B cells infected with a recombinant vaccinia virus containing the entire UL97 open reading frame (ORF), as well as in HFF transiently expressing the entire UL97 ORF under the control of HCMV major immediate-early promoter. However, transiently expressed 5'-terminal deletion mutants of the UL97 ORF in addition showed a cytoplasmic localization of the UL97 protein, confirming the presence of a nuclear localization site in the N-terminal region of the protein. Our high-pressure liquid chromatography analyses confirmed the ganciclovir phosphorylation by the UL97 protein, but no specific phosphorylation of natural nucleosides was observed, indicating that the UL97 protein is not a nucleoside kinase. During plaque purification of recombinant UL97-deficient HCMV, this virus was growth defective; hence, we presume that UL97 may be essential for the viral life cycle.
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Núcleo Celular/metabolismo , Citomegalovirus/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Western Blotting , Núcleo Celular/virología , Células Cultivadas , Citomegalovirus/genética , Cartilla de ADN , Replicación del ADN , Fibroblastos , Técnica del Anticuerpo Fluorescente Indirecta , Genes Virales , Humanos , Cinética , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfotransferasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Piel , TransfecciónRESUMEN
Although ascorbic acid and heparin are used for local therapy of corneal wounds that heal poorly (e.g., after chemical burns), little has been known up to now about the mechanisms underlying their effectiveness at the cellular level. The aim of the present study was therefore to evaluate the effect of heparin and ascorbic acid on the growth behaviour of corneal cells in vitro. For this purpose cell cultures from a corneal epithelial cell line were used. Stimulation of the cells with heparin at concentrations ranging from 10 to 200 micrograms/ml for 6 days led to a dose-dependent rise in growth rate (population doublings per day) of 0.48 +/- 0.50 to 2.19 +/- 1.65 (mean value +/- standard deviation, n = 10) with an EC50 of 132 micrograms/ml. In contrast, the addition of ascorbic acid at concentrations ranging from 0.5 to 3.0 mM led on average to a 40% dose-dependent inhibition of cell proliferation after 6 days, with an IC50 of 0.7 mM as-corbic acid. On the basis of these results, the use of heparin at concentrations of 180-200 micrograms/ml appears advantageous. In contrast, the local application of ascorbic acid for chemical burns with no stromal involvement should be subjected to a critical reassessment.
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Ácido Ascórbico/farmacología , División Celular/efectos de los fármacos , Córnea/citología , Heparina/farmacología , Animales , Recuento de Células/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Epiteliales , Conejos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Although the selective loss of retinal pericytes has long been known to be one of the earliest histopathological findings in diabetic retinopathy, only limited information is available concerning their function and cell biology. Recently, it has been shown that the interaction of endothelial cells and pericytes plays an important role in the maintenance of vascular integrity. Additionally, it has been suggested that pericytes have a contractile function. Platelet-derived growth factor (PDGF), released from endothelial cells, has been shown to be a potent mitogen and vasoconstrictor. Cytosolic free calcium ([Ca2+]i) has been shown to play a key role as a second messenger for PDGF, involved in the regulation of various cellular functions, e.g. cell proliferation and vascular contractility. In order to characterize the effect of different PDGF homodimers on cultured bovine retinal pericytes, we investigated PDGF-AA- and -BB-dependent alterations in [Ca2+]i was determined with the Ca(2+)-sensitive fluorescent probe Quin-2. Basal levels were 118 +/- 30 nM. Stimulation with PDGF-BB in concentrations ranging from 5 to 20 ng/ml led to a dose-dependent increase of [Ca2+]i with an EC50 of 5.8 ng/ml. Maximum stimulation, to about 280% of basal levels, occurred after 3-4 min. In contrast, PDGF-AA was not effective. The results suggest that PDGF-BB may influence the integrity and contractility of the retinal microvasculature via modulation of the intracellular calcium homeostasis of pericytes. Additionally, it can be speculated that cultured retinal pericytes express mainly PDGF-beta-type receptors.