RESUMEN
Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Antivirales/química , Fragmentos de Péptidos/inmunología , Proteínas de la Matriz Viral/inmunología , Latencia del Virus/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/aislamiento & purificación , Clonación Molecular , Mapeo Epitopo/métodos , Epítopos/genética , Epítopos/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Humanos , Inmunización , Ratones , Fragmentos de Péptidos/genética , Biblioteca de Péptidos , Dominios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas de la Matriz Viral/genéticaRESUMEN
A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia pastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day).
Asunto(s)
Antídotos , Butirilcolinesterasa/biosíntesis , ADN/biosíntesis , Butirilcolinesterasa/química , Butirilcolinesterasa/genética , Clonación Molecular , ADN/genética , Humanos , Organofosfatos/química , Organofosfatos/toxicidad , Péptidos/química , Pichia/enzimología , Pichia/genética , Prolina/químicaRESUMEN
Recombinant plasmids containing the mellitin gene under the control of the lac and the tetM gene promoters were used for studying cytotoxic activity of mellitin in cells of mollicutes (mycoplasmas) and Escherichia coli. After transformation of Acholeplasma laidlawii and Mycoplasma hominis cells with recombinant plasmid DNAs by electroporation, cell growth was suppressed. The expression of the mellitin gene in A. laidlawii and M. hominis cells was demonstrated using the reverse transcription polymerase chain reaction (RT-PCR). The possibility of using the mellitin gene in the recombinant vector as a potential antimycoplasmic gene-therapeutic agent with its selective expression in target cells is discussed.
Asunto(s)
Acholeplasma laidlawii/genética , Vectores Genéticos , Meliteno/farmacología , Mycoplasma hominis/genética , Secuencia de Aminoácidos , Secuencia de Bases , Supervivencia Celular/efectos de los fármacos , ADN Recombinante , Electroporación , Meliteno/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Gene therapy of chronic infectious diseases of urogenital tract represents a new perspective field in the modern biological and medical sciences. In the review discuss one of the new directions in gene therapy of urogenital infections caused by Mycoplasma: inhibition of mycoplasmal infection after administration of recombinant plasmid vectors, expressed the genes of cytotoxic peptides.
Asunto(s)
Citotoxinas/genética , Enfermedades Urogenitales Femeninas/terapia , Terapia Genética , Enfermedades Urogenitales Masculinas , Infecciones por Mycoplasma/terapia , Péptidos/genética , Antígenos Bacterianos/genética , Enfermedad Crónica , Citotoxinas/metabolismo , Enfermedades Urogenitales Femeninas/microbiología , Vectores Genéticos , Humanos , Péptidos/metabolismoRESUMEN
Mycoplasma hominis was transformed by electroporation with plasmid pAM120 containing the transposon Tn916 that carried the tetM gene responsible for the resistance to tetracycline. The frequency of transformation was 10(-7)-10(-8) colony-forming units (CFU) per 10 micrograms of plasmid DNA. The PCR analysis of transformed DNA confirmed the transposon integration into the mycoplasma genome.
Asunto(s)
Mycoplasma hominis/genética , Plásmidos , Transformación Genética , Secuencia de Bases , Cartilla de ADN , Elementos Transponibles de ADN , ADN Bacteriano , Electroporación , Genoma Bacteriano , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Resistencia a la Tetraciclina/genéticaRESUMEN
The DNA probes--pA6-CSF-1 and pA2-CSF-1 for the alternative splicing region of the 6 exon human CSF-1 gene were prepared using PCR and subsequent subcloning in pUC19 plasmid at the XmaI/BamHI sites. Due to the insert sequencing and blotting of human leukocytes DNA, the DNA probes obtained can be useful for screening of mutations in the human CSF-1 gene.
Asunto(s)
Empalme Alternativo , Exones , Enfermedades Hematológicas/genética , Factor Estimulante de Colonias de Macrófagos/genética , Polimorfismo Genético , ARN Mensajero/genética , Secuencia de Bases , Donantes de Sangre , Clonación Molecular , Sondas de ADN , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
For investigation of FMS gene polymorphism and mutations that reveal functionally meaning in leukemia and myelodysplastic disorders the overlapping recombinants lambda-clones inserted by FMS gene fragments have been obtained from human leukocyte genomic library in the EMBL 3A phage by using oligonucleotide prode (27 nucleotides) based on 12 exon of the FMS gene. 15 DNA probes were prepared by subcloning the lambda-clones obtained in the pBSKS+ plasmid. The probes obtained allow to analyse extracellular, transmembrane and tyrosine kinase regions of the FMS gene independently.
Asunto(s)
Biblioteca de Genes , Reordenamiento Génico , Leucocitos/fisiología , Polimorfismo Genético , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Mapeo Restrictivo , Clonación Molecular , Exones , Amplificación de Genes , Vectores Genéticos , Humanos , Leucemia/genética , Mutación , Sondas de Oligonucleótidos , Preleucemia/genéticaRESUMEN
The polypeptide with a mobility of the tryptophanyl-tRNA-synthetase subunit can be labeled in bovine pancreas extracts from [gamma-32P]ATP. Immunoprecipitation analysis with monospecific polyclonal antibodies against the enzyme as well as identification of [32P]phosphoamino acids in the immunoprecipitate revealed that in bovine pancreas extracts tryptophanyl-tRNA-synthetase undergoes phosphorylation at serine residues. The level of phosphorylation does not change in the presence of activity modulators of cAMP-, cGMP- and Ca2(+)-dependent protein kinases, decreases after addition of phosphoseryl/phosphothreonyl-protein phosphatase inhibitors and increases in the presence of their activators. It was supposed that phosphorylation of tryptophanyl-tRNA-synthetase catalyzed by seryl/threonyl-specific protein kinase depends on the activity of phosphoseryl/phosphothreonyl-phosphatase.
Asunto(s)
Páncreas/enzimología , Extractos Pancreáticos/química , Triptófano-ARNt Ligasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Catálisis , Bovinos , Polipéptido Pancreático/biosíntesis , Fosforilación , Pruebas de Precipitina , Serina/químicaRESUMEN
Karyological and morphological analysis of the wild type MDBK cell line (spontaneously transformed bovine kidney cells) was undertaken. The results were compared with the same data obtained with resistant lines derived from the wild type line after prolonged cultivation with increasing quantities of tryptophanol and tryptamine, competitive analogues of tryptophan. Tetraploids are much more abundant in the resistant lines than in the initial one. In tryptamine-resistant cells a large marker acrocentric chromosome is duplicated in 96% of cells and elongated, due to appearance of an additional segment. In the population of resistant cells bi- and multinuclear cells are abundant as well as giant cells; the nuclei are enlarged and the number of nucleoli is increased. A hypothesis is proposed that resistance to tryptophan analogues is associated with amplification of tryptophanyl-tRNA synthetase gene.
Asunto(s)
Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Triptófano-ARNt Ligasa/antagonistas & inhibidores , Triptófano/análogos & derivados , Animales , Bovinos , Línea Celular , Resistencia a Medicamentos , Marcadores Genéticos , Cariotipificación , Riñón/citología , Ploidias , Triptaminas/farmacologíaRESUMEN
The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.
Asunto(s)
Nicotiana/microbiología , Plantas Tóxicas , ARN Viral/biosíntesis , Virus del Mosaico del Tabaco/fisiología , Replicación Viral , Peso Molecular , Nicotiana/metabolismo , Virus del Mosaico del Tabaco/metabolismo , Virus del Mosaico del Tabaco/patogenicidadRESUMEN
Electron microscopic examination of the oesophageal squamous cell carcinoma revealed the presence in the tumour of both undifferentiated and differentiated squamous cells. Keratinocytes appeared and the signs of keratinization were more pronounced in the cytoplasm of differentiated cells. Dysplastic changes were observed at the periphery of the primary node. Two main variants of dysplasia (dark-cell and clear-cell) were distinguished, this at the ultrastructural level being the reflection of the direction of differentiation and the degree of cell maturity in the dysplastic foci. With the exception of few cases with a cell polymorphism in the foci of a severe dysplasia, dysplastic changes of the squamous epithelium were characterized by a monotonous ultrastructural cell composition. The dysplastic cells were distinguished by a degree of differentiation and high synthetic activity.
Asunto(s)
Carcinoma de Células Escamosas/ultraestructura , Neoplasias Esofágicas/ultraestructura , Lesiones Precancerosas/ultraestructura , Transformación Celular Neoplásica/ultraestructura , Epitelio/ultraestructura , Esófago/ultraestructura , Humanos , Microscopía ElectrónicaRESUMEN
Informosome-like virus-specific ribonucleoprotein (vRNP) of tobacco mosaic virus (TMV) comprise a set of four major polypeptides having molecular weights of 17 500, 31 000, 37 000 and 39 000. Of the minor polypeptides, those of apparent molecular weights 25 000, 55 000, 68 000 and 70 000 had electrophoretic mobilities of polypeptides found in a ribonucleoprotein preparation from uninoculated plants. Polypeptide with mol.wt. 175 000 is TMV coat protein so far as: a) vRNP was precipitated with immunoglobulins against TMV and TMV coat protein; b) it had electrophoretic mobility similar to mobility of TMV coat protein; c) the peptide map of polypeptides with mol.wts 31 000, 37 000 and 39 000 are probably virus-specific-products. This is supposed because they are not present in cell informosomes protein, and they are not revealed in vRNP induced in cells after infection with potato virus X (PVX). Electrophoresis of vRNP-PVX protein reveals polypeptides of 23 000 (PVX coat protein), 55 000, 70 000, 78 000, 95 000, 120 000 and 145 000.
Asunto(s)
Nicotiana/microbiología , Péptidos/análisis , Virus de Plantas/análisis , Plantas Tóxicas , Proteínas/análisis , ARN Mensajero/análisis , Virus del Mosaico del Tabaco/análisis , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Sustancias Macromoleculares , Peso Molecular , Proteínas de Plantas/análisis , Virus de Plantas/genética , Proteínas/genética , ARN Mensajero/genética , Ribonucleoproteínas/análisis , Nicotiana/análisis , Nicotiana/genética , Virus del Mosaico del Tabaco/genética , Proteínas Virales/análisisRESUMEN
We have previously detected in TMV-infected cells virus-specific informosome-like ribonucleoproteins (vRNP) that differed in the CsCl buoyant density from mature TMV particles. It is shown in the present work that [3H]uridine-labelled TMV-specific structures, when fractionated in Cs2SO4, produce three types of structures, i. e. with a buoyant density of 1.23 g/cm3 (the so-called 1.23 material), 1.29 g/gm3 (mature virus) and 1.34--1.49 g/cm3 (vRNP). The 1.23 material has been investigated. The incorporation of [3H]palmitic acid and the sensitivity of this material to 0.1% Na dodecyl sulphate was interpreted to mean the presence of membrane components. Treatment of the 1.23 material Na dodecyl sulphate induces the release of the mature virus, vRNP and free viral RNA. vRNP was shown to contain genome TMV RNA (mol. weight, 2.0 x 10(6)) and a considerable amount of subgenomic TMV RNA (mol. weight, 1.1--1.3 x 10(6) and 0.6--0.8 x 10(6)). It is demonstrated that RNA isolated from vRNP codes for TMV-specific proteins and is able to hybridize with recombinant plasmid containing DNA-copy of 3'-end RNA TMV fragment (about one half of genome).
Asunto(s)
Nicotiana/microbiología , Plantas Tóxicas , ARN Viral/análisis , Ribonucleoproteínas/análisis , Virus del Mosaico del Tabaco/genética , Proteínas Virales/análisis , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Biosíntesis de Proteínas , Nicotiana/genéticaRESUMEN
Electron microscopic examinations of the ultrastructure of normal esophageal epithelium of an adult man revealed submicroscopic features of each epithelial layer. A specific structure of epithelial cells is represented by tonofibrils. Glycogen is found only in the prickle-cell and superficial layers. In the latter there were some signs of incomplete keratinization. Hyperplasia of the prickle-cell and superficial layers epithelium was accompanied by accumulation of glycogen and marked parakeratosis with disintegration of the nucleus, formation of keratin fibrils, and destruction of desmosomal bonds. Dysplastic changes in the esophageal epithelium were heterogenous and presented a whole set of ultrastructural rearrangements. We distinguish two variants of dysplasia. In the "dark-cell" variant there were hypertrophy and hyperchromia of the nucleus, deep invaginations of the nuclear membrane, swelling of the mitochondria, appearance of myelin-like structures in the matrix, increased number of free ribosomes, polysomes, and lipids, formation of tonofibrillar-keratohyaline complexes, increased number of cytoplasmic processes. The "clear-cell" variant was characterized by nuclear hypertrophy, marked dilation of cisterns of the rough endoplasmic reticulum, extensive vesicle formation, keratohyaline granules, few cytoplasmic outgrowths, dilation of the intercellular space.
Asunto(s)
Neoplasias Esofágicas/ultraestructura , Esófago/ultraestructura , Lesiones Precancerosas/ultraestructura , Adulto , Epitelio/ultraestructura , Humanos , Hipertrofia , Microscopía ElectrónicaRESUMEN
A high incidence of pathology in the esophagus was established in patients with chronic, particularly atrophic, gastritis (esophagitis, leukoplakia, dysplasia, fibrosis of submucosa, early forms of cancer, etc.). Most patients with chronic gastritis revealed esophagitis (71%) with concomitant leukoplakia (70.6%) or dysplasia (49%) which can be identified as precancer. Therefore, patients suffering from severe chronic gastritis, involving lesions of glands and atrophy of mucosa, living in areas with highly-endemic esophageal cancer should be referred to those at high risk for cancer of the esophagus. Timely examination for and therapy of chronic esophagitis in patients with a long history of severe chronic gastritis should be indispensable to prevent esophageal cancer.
Asunto(s)
Reservorios de Enfermedades , Enfermedades del Esófago/epidemiología , Neoplasias Esofágicas/epidemiología , Gastropatías/epidemiología , Adulto , Autopsia , Enfermedades del Esófago/patología , Neoplasias Esofágicas/patología , Esófago/patología , Etnicidad , Femenino , Humanos , Kazajstán , Masculino , Persona de Mediana Edad , Estómago/patología , Gastropatías/patologíaAsunto(s)
Gastritis Atrófica/diagnóstico , Gastritis/diagnóstico , Lesiones Precancerosas/diagnóstico , Neoplasias Gástricas/diagnóstico , Adulto , Biopsia , Citodiagnóstico , Femenino , Mucosa Gástrica/patología , Gastritis Atrófica/patología , Gastroscopía , Humanos , Kazajstán , Masculino , Persona de Mediana Edad , Pólipos/diagnóstico , Pólipos/patología , Lesiones Precancerosas/patología , Neoplasias Gástricas/patologíaAsunto(s)
Ácido Aurintricarboxílico/farmacología , Ácidos Ciclohexanocarboxílicos/farmacología , Genes Virales/efectos de los fármacos , ARN Viral/biosíntesis , Virus del Mosaico del Tabaco/efectos de los fármacos , Peso Molecular , Especificidad de la Especie , Estimulación Química , Virus del Mosaico del Tabaco/metabolismoRESUMEN
Studies on some morphological features of the squamous epithelium of esophageal mucosa in dysplastic processes and cancer in situ as well as quantitative studies of the DNA content have shown that changes in the DNA content, increased polyploidy and genetic heterogenicity of the cell elements correspond to the degree of epithelial cells dedifferentiation and might be taken into account in diagnosis of early malignification of esophageal squamous epithelium.