RESUMEN
We study angular and frequency-angular distributions of the terahertz (THz) emission of the low-frequency region (0.3-3 THz) from a two-color femtosecond plasma spark experimentally and in three-dimensional numerical simulations. We investigate the dependence of the angular shapes of the THz radiation on focusing conditions and pulse durations by using two laser facilities (pulse durations 35 and 150 fs) for different focusing geometries. Our experiments and simulations show that decrease in the numerical aperture from NA ≈0.2 to NA ≈0.02 results simultaneously in (I) squeezing of the THz angular distribution and (II) formation of the bright conical emission in the THz range. The moderate focusing NA ≈0.05, which forms the relatively narrow unimodal THz angular distribution, is identified as optimal in terms of angular divergence. Numerical simulations with carrier wave resolved show that bright THz ring structures appear at the frequencies ≥2 THz for longer focuses (NA ≈0.02), while for optimal focusing conditions NA ≈0.05 the conical emission develops at THz frequencies higher than 10 THz.
RESUMEN
The polymeric immunoglobulin receptor (pIgR) is a type I transmembrane protein that is expressed on the surfaces of glandular and intestinal epithelial cells. The extracellular portion of the pIgR is composed of six different domains. Domain 6 is involved in the enzymatic cleavage and release of the pIgR into the intestinal lumen as a free secretory component (fSC). A highly conserved 9-amino acid sequence is present in this region in various species. Although mutations in domain 6 are associated with particular diseases, such as IgA nephropathy and Epstein-Barr virus-related nasopharyngeal cancer, and the glutamic acid residues in the conserved 9-amino acid sequence are expected to be indispensable for the secretion of fSC, the importance of these residues has not been examined. In the present study, we attempted to examine the role of these residues in the enzymatic cleavage of the pIgR. The enzymatic cleavage of the pIgR was not affected by the presence of an alanine to valine substitution at position 580 or glutamine to alanine substitutions at positions 606 and/or 607, or the deletion of the whole 9-amino acid conserved sequence. Intriguingly, the 10 amino acid sequences flanking the N- and C-terminal ends of the conserved 9-amino acid sequence had opposite effects on pIgR cleavage. Namely, the N-terminal and C-terminal sequences enhanced and reduced pIgR cleavage efficiency, respectively. These results indicated that the pIgR can be divided into several functionally distinct regions.
Asunto(s)
Sustitución de Aminoácidos , Proteínas Mutantes/genética , Receptores de Inmunoglobulina Polimérica/genética , Eliminación de Secuencia , Alanina/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Western Blotting , Células CHO , Cricetinae , Cricetulus , Glutamina/genética , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Receptores de Inmunoglobulina Polimérica/metabolismo , Transfección , Valina/genéticaRESUMEN
BACKGROUND: The beta chain of the interleukin 2/15 receptor (IL-2/15Rbeta) is induced by the expression of the EWS-WT1. A case of desmoplastic small round cell tumour (DSRCT) expressing only an unusual EWS-WT1 treated by us is reported here. AIM: To characterise an unusual form of EWS-WT1. METHODS: Frozen tissue sections of the axillary tumour were examined using a laser-assisted microdissection technique and reverse transcriptase polymerase chain reaction. RESULTS: The novel fusion of exon 8 of EWS and the defective exon 10 of WT1 (-KTS) was detected. Although it was an unusual form, the coexpression of the present EWS-WT1, IL-2/15Rbeta and Janus kinase (JAK1) mRNA was detected in the tumour cells. IL-2 and signal transducers and activators of transcription (STAT5) mRNA were detected in both tumour and stromal cells. CONCLUSION: The induction of the IL-2/15 receptor signalling pathway may contribute to tumorigenesis in DSRCT through a paracrine or an autocrine system, even though the EWS-WT1 was an unusual form.
Asunto(s)
Carcinoma de Células Pequeñas/metabolismo , Subunidad beta del Receptor de Interleucina-2/biosíntesis , Neoplasias Pulmonares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Adulto , Secuencia de Bases , Resultado Fatal , Humanos , Subunidad beta del Receptor de Interleucina-2/genética , Masculino , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , ARN Mensajero/genética , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 microM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0. 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.
Asunto(s)
Proteínas/química , Puromicina/química , Secuencia de Aminoácidos , Sitios de Unión , Sistema Libre de Células , Escherichia coli , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas/metabolismo , Puromicina/metabolismoRESUMEN
Neurinomas developed in an African lungfish (Protopterus annectens), living in an aquarium in Western Japan. The 2 tumors, measuring 7.5 X 9.0 X 6.5 and 13 X 4 X 6 cm, were located on the skin. As shown by light microscopy, tumor cells were composed of spindle-shaped cells with huge pleomorphic nuclei, which were arranged in parallel rows or whorls in interlacing connective tissue. Long-term culture of these tumor cells was achieved in vitro at 25 degrees C with use of conditioned medium over a period of more than 4 months. The nuclear DNA contents of erythrocytes (normal diploids, 2C) and tumor cells dispersed from the fixed tumor tissues were measured by 4',6-diamidino-2-phenylindole hydrochloride-DNA microfluorometry by using mouse cerebellar small granule cells (normal diploids, 2C) as a reference. The 2C value of the lungfish was approximately 28-fold greater than that of the mouse. Furthermore, consistent with the nuclear pleomorphism observed by light microscopy, the nuclear DNA contents of tumor cells showed a wide distribution from hypo-2C to hyper-4C. DNA repair synthesis was measured autoradiographically in organ cultures of the tumor, lung, and skin, exposed to chemical carcinogens or UV radiation. Considerable repair was observed in the tumor and skin cells exposed to 1-methyl-1-nitrosourea (CAS: 684-93-5), N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 70-25-7), or 254-nm or sunlamp UV light. Only traces of repair synthesis were detected in lung exposed to 1-methyl-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. 4-Hydroxyaminoquinoline 1-oxide (CAS: 4637-56-3) did not induce repair in any of the three tissues. The observed values for repair, relative to the amount of DNA, were similar to those in other fishes.
Asunto(s)
Reparación del ADN , Enfermedades de los Peces/patología , Neurilemoma/veterinaria , Neoplasias Cutáneas/veterinaria , 4-Hidroxiaminoquinolina-1-Óxido/toxicidad , Animales , Células Cultivadas , ADN/análisis , Reparación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Enfermedades de los Peces/metabolismo , Peces , Neurilemoma/metabolismo , Neurilemoma/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
The metabolism of benzo(a)pyrene (BaP) by rat liver microsomes was examined in the presence and absence of uridine 5'-diphosphoglucuronic acid (UDPGA). BaP metabolites were separated by high-pressure liquid chromatography. The normal chromatographic patterns of the metabolities were altered by the addition of UDPGA. At concentrations of UDPGA at which the elutions of dihydrodiol components were unchanged, phenol and quinone elution profiles were selectively decreased. The decreased activities of microsomal mixed-function oxidases by UDPGA were also observed with the use of the aryl hydrocarbon hydroxylase assay. The decrease may not be due to inhibition of those enzymes, but rather to formation of glucuronide conjugates with oxygenated BaP metabolites. These results suggest that glucuronidation may be important in the detoxification of BaP.
Asunto(s)
Benzopirenos/metabolismo , Microsomas Hepáticos/metabolismo , Uridina Difosfato Ácido Glucurónico/farmacología , Azúcares de Uridina Difosfato/farmacología , Animales , Cromatografía Líquida de Alta Presión , Glucuronatos/metabolismo , Glicoles/metabolismo , Técnicas In Vitro , Masculino , Microsomas Hepáticos/efectos de los fármacos , Oxigenasas de Función Mixta/metabolismo , Fenoles/metabolismo , Quinonas/metabolismo , RatasRESUMEN
Genetic differences in metabolism of benzo(a)pyrene (BP) were investigated using cultured epidermal keratinocytes and dermal fibroblasts isolated from four inbred strains of mice: C3H/HeJms and C57BL/6J that are responsive; and DBA/2 and AKR/Jms that are nonresponsive in terms of inducibility of aryl hydrocarbon hydroxylase (AHH) activity. Primary cultures of epidermal and dermal cells isolated from newborn mice were treated with benz(a)anthracene for 24 hr. In both types of cells, AHH activity was induced in all four strains of mice, irrespective of their responsiveness in vivo. In the epidermal cells, basal AHH activity varied within a relatively small range of 4.6 to 8.8 pmol per mg protein per hr, and the activity was increased 4.4 to 8.7 times by benz(a)anthracene treatment. There was no difference in the extent of the induction in different strains of mice. In dermal cells, the basal level of AHH activity was in the range of 3.6 to 9.1 pmol per mg protein per hr, and benz(a)anthracene treatment induced AHH, but to various degrees depending on the responsiveness in vivo. Epidermal cells of the responsive mice metabolized more than 90% of the added BP in 48 hr, while those of nonresponsive mice metabolized only 60 to 70%. Dermal cells also metabolized BP, but to a lesser extent than did epidermal cells, and there was no strain difference in its metabolism. Time-course studies revealed that epidermal cells of responsive mice metabolized BP more rapidly than did those of nonresponsive mice. The activities of epidermal and dermal cells to metabolize BP were further demonstrated by a cell-mediated mutation assay. Consistent with the results of time-course analysis of the metabolites, shorter treatments with BP (less than 24 hr) showed a clear difference in BP metabolism associated with responsiveness.
Asunto(s)
Benzopirenos/metabolismo , Ratones Endogámicos/genética , Piel/metabolismo , Animales , Hidrocarburo de Aril Hidroxilasas/biosíntesis , Benzo(a)pireno , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Inducción Enzimática , Epidermis/metabolismo , Hígado/enzimología , Ratones , Ratones Endogámicos/metabolismoRESUMEN
Induction of aryl hydrocarbon hydroxylase (AHH) was studied in mouse hepatocytes in primary culture and compared with that in rat hepatocytes. Enzyme activity in hepatocytes from C57BL/6 mice was found to increase dose dependently after treatment with benz(a)anthracene. However, the induction was strictly dependent on culture medium. Although appreciable levels of AHH activity were inducible in Sprague-Dawley rat hepatocytes cultivated in either Dulbecco's minimal essential medium (DMEM) or Waymouth's medium, C57BL/6 mouse cells cultivated in DMEM responded to the inducer only very slightly, whereas those in Waymouth's or Ham's F-12 medium demonstrated a marked increase. Proline, but not glutamic acid or cysteine, all of which were lacking in DMEM but present in Waymouth's and Ham's F-12 medium, restored the potential for response to the cells in DMEM. While increased amounts of P450 mRNA in C57BL/6 cells cultivated in DMEM were transient and decreased after a peak observed at 24 h, levels of mRNA in Waymouth continued to demonstrate an increase at 48 h. Addition of proline to mouse hepatocytes in DMEM increased the generation of transcripts without, however, influencing the decrease observed from 24 h to 48 h. Timing of treatment with benz(a)anthracene and incubation in Waymouth greatly influenced the eventual AHH activity. Thus, while enzyme activities measured at 48 h were in the same range after treatment with benz(a)anthracene for either the whole period or only for the initial 24 h, and prominent induction was observed with cells in Waymouth for 24 to 48 h regardless of whether they were at first cultivated in DMEM or Waymouth, levels remained low if the cells were incubated in DMEM during the 24- to 48-h period. These observations suggest that induction of AHH in mouse hepatocytes is regulated by both transcriptional and posttranscriptional events and that proline-dependent events are required for expression of the enzyme activity.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , Hígado/enzimología , Prolina/farmacología , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Benzo(a)Antracenos/farmacología , Northern Blotting , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Inducción Enzimática , Femenino , Genes/efectos de los fármacos , Cinética , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Transcripción Genética/efectos de los fármacosRESUMEN
The expression of P1450 and P3450 genes isolated from C57BL26 mouse liver in mouse hepatocytes from responsive (BALB/c, C3H/He, C57BL/6, and CBA) and nonresponsive (AKR, DBA/2, NZB, and NZW) strains in primary culture after exposure to aromatic hydrocarbons was investigated with respect to aryl hydrocarbon hydroxylase (AHH) activity and corresponding P450 transcript levels. Constitutive AHH activity in all strains decreased with an increasing culture period. The AHH activity of cells treated with either benz(a)anthracene, benzo(a)pyrene, 3-methylcholanthrene, or TCDD continuously or 24 h before harvesting was measured during observation periods for up to 5 days. Slight induction of AHH activity by benz(a)anthracene, benzo(a)pyrene, and 3-methylcholanthrene was observed in hepatocytes from the CBA and C3H/He strains during the first half of the observation period, followed by a steep increase thereafter. Enzyme activities in hepatocytes from BALB/c mice were the same as or lower than the control values during the first half of the observation period, and in the C57BL/6 mouse high levels of AHH activities were observed even during this period. AHH activities in hepatocytes from the AKR, DBA/2, NZB, and NZW mice after treatment with aromatic hydrocarbons were lower than control levels during the first half of the observation period; however, significant induction was observed thereafter. P450 transcripts including both P1450 and P3450 RNA species were detected when the treatment was started during the early incubation period, whereas only P1450 RNA was found at the later periods. The amounts of P1450 transcript also correlated well with AHH activity, higher levels being found after starting treatment with benz(a)anthracene at day 3 or 4 than at day 1. Our observations indicate that, although AHH induction was genetically determined in each strain, activity can be induced in hepatocytes of so-called nonresponsive as well as responsive mouse strains by treatment with aromatic hydrocarbons after transfer of the cells to primary culture.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , Benzo(a)Antracenos/farmacología , Sistema Enzimático del Citocromo P-450/genética , Dioxinas/farmacología , Hígado/enzimología , Metilcolantreno/farmacología , Dibenzodioxinas Policloradas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Femenino , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos , ARN/biosíntesis , Especificidad de la Especie , Transcripción GenéticaRESUMEN
The metabolism of benzo(a)pyrene (BP) in epidermal keratinocytes and dermal fibroblasts of humans and mice was investigated with emphasis on variation among species, individuals, and cell types. Human epidermal and dermal cells were isolated from the skin of normal subjects by trypsinization at 4 degrees overnight, followed by separation of the epidermis from the dermis with forceps. In confirmation of previous studies metabolic activity of human epidermal cells on BP was consistently demonstrated by cell-mediated assay, in which V79 Chinese hamster cells were plated on top of sheets of epidermal cells and treated with BP for 48 hr. Mutation of the V79 cells, measured as ouabain resistance, was induced in a dose-related fashion, although the extent of induced mutation varied from 5 to 22 ouabain-resistant colonies per 10(6) survivors/10 microM BP in cultures derived from different individuals. The most striking observation was that human dermal fibroblasts did not activate BP to a form that was mutagenic to cocultured V79 cells. This was observed without exception in all nine cultures of dermal fibroblasts and the one culture of embryo fibroblasts (MR-90) tested. Analysis by high-pressure liquid chromatography indicated that human epidermal and dermal cells both metabolized BP, producing almost the whole series of known metabolites of BP. The amount of BP 7, 8-dihydrodiol, a proximate metabolite of BP, produced by human dermal cells varied from 0.2 to 2.7% of the total BP added and seemed to be enough to induce mutation. Furthermore, human dermal cells not necessarily activated exogenously added BP 7,8-dihydrodiol to a form being mutagenic to V79 cells. These observations suggest that further metabolism of BP 7,8-dihydrodiol is partially or entirely blocked in human fibroblasts. In contrast to human fibroblasts, mouse fibroblasts isolated from the dermis of embryos did activate BP and induced mutation in cocultured V79 cells to a higher extent than did mouse epidermal cells, indicating interspecies variation in metabolic activation of BP between human and mouse fibroblasts.
Asunto(s)
Benzopirenos/metabolismo , Dihidroxidihidrobenzopirenos , Piel/metabolismo , Adolescente , Adulto , Animales , Benzo(a)pireno , Benzopirenos/farmacología , Células Cultivadas , Niño , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Femenino , Humanos , Pulmón , Masculino , Ratones , MutaciónRESUMEN
The expression of Cyp2e-1 mRNA and protein was investigated in the C57BL/6NCrj mouse hepatocytes in primary culture, as well as liver and kidney. The mRNA and protein expression in the liver was in the same range in both sexes and was not affected by orchiectomy or ovariectomy. The mRNA expression was enhanced in the kidney of ovariectomized mice, in which the protein contents were not influenced. Orchiectomy decreased the expression of both mRNA and protein. When the hepatocytes were transferred to primary culture, the amounts of the mRNA were not changed within 24 h and about half remained by day 3. However, the expression was low thereafter. The expression of the protein gradually decreased after the start of culture. Dexamethasone showed a potential as an inducer at more than 10(-8) M. Sex hormones increased the expression of this P-450 species a little in culture, but growth hormone did not. These observations indicated that glucocorticoid hormone plays a role in modifying expression of Cyp2e-1 and that the mouse hepatocyte culture is useful for examining its regulation mechanism.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Hígado/enzimología , Oxidorreductasas N-Desmetilantes/genética , Animales , Secuencia de Bases , Castración , Células Cultivadas , Citocromo P-450 CYP2E1 , Sistema Enzimático del Citocromo P-450/metabolismo , Cartilla de ADN/química , Dexametasona/farmacología , Estradiol/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica , Glucocorticoides/farmacología , Riñón/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/metabolismo , ARN Mensajero/genética , Testosterona/farmacologíaRESUMEN
Nakano et al. have recently reported a Japanese case of infantile sialic acid storage disease [C. Nakano, Y. Hirabayashi, K. Ohno, T. Yano, T. Mito, M. Sakurai, Brain Dev., 18 (1996) 153-156]. For further etiological analysis of this disease, we prepared the Epstein-Barr virus (EBV)-transformed cell line (LCL) from the peripheral lymphocytes of this patient and performed initial characterization of the cells. Electron microscopy of the cells showed that the cells contained many vacuoles and swelled lysosomes. Cytochemical staining with sialic acid-specific lectin, Limax flavus agglutinin (LFA), showed strong staining on membranes and subcellular organelles on the patient-derived cells, whereas LCL from a normal person was only weakly stained. The cells from the patient contained 5.5-7.3 nmol/107 cells of free N-acetyl neuraminic acid, whereas three strains of LCLs derived from normal persons contained 1 nmol/107 cells. The culture supernatant of LCL from the patient contained 144 nmol/ml of free N-acetyl neuraminic acid, whereas the LCL culture supernatant from normal persons contained 57-73 nmol/ml of free sialic acid, which was the same or only at a slightly higher level than the fresh medium. In addition, cellular acidic sialidase measured as 4-methylumbelliferyl sialidase was elevated (107 nmol 4-methylumbelliferon released/mg cellular protein/60 min). The EBV-LCL from an ISSD patient is considered to remain as the abnormality of the cell donor.
Asunto(s)
Linfocitos B/metabolismo , Linfocitos B/patología , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedades por Almacenamiento Lisosomal/patología , Ácido N-Acetilneuramínico/metabolismo , División Celular , Línea Celular Transformada , Herpesvirus Humano 4 , Humanos , Lactante , Lisosomas/ultraestructura , Microscopía Electrónica , Neuraminidasa/metabolismo , Vacuolas/ultraestructuraRESUMEN
When goldfish erythrophores isolated from the skin by tissue digestion and centrifugation in a Percoll density gradient were transfected in a monolayer-culture with v-Ha-ras or v-src oncogene either singly or in combination with v-myc by means of calcium phosphate-DNA co-precipitation, there appeared a certain number of transformants manifesting a chromatoblast-like profile and tumorous phenotypes as seen in the capability for unlimited growth, and piling-up in a monolayer-culture or colony formation in semi-solid soft agar. After successive growth in vitro for longer than one month which was scarcely observed with the erythrophores, the vast majority of such transformants began to differentiate into erythrophores and ceased proliferation spontaneously. The onset of their differentiation was ascertained by the deposition of marker pteridine pigments. None of the transformants differentiated into melanophores or iridophores or other neural crest derivatives as seen in goldfish erythrophoroma cells. Little difference was observed in their transforming efficiency (0.2-0.3 transformants/micrograms DNA) between the combinations of oncogenes applied but a tendency was noted that cells transfected with ras or src in combination with myc developed the capacity to grow for a longer period and differentiated at a later stage than those transfected solely with ras or src. One cell line (ESM-1) derived from the erythrophores transfected with src/myc grew successively over nine months, indicating its acquisition of immortality. The expression of the transfected oncogenes in this cell line was examined in comparison with the erythrophoroma cells by Western and Northern blot analyses.
Asunto(s)
Transformación Celular Neoplásica/genética , Cromatóforos/patología , Carpa Dorada/anatomía & histología , Oncogenes/genética , Piel/citología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Cromatóforos/citología , Expresión Génica , Genes myc/genética , Genes ras/genética , Genes src/genética , Fenotipo , TransfecciónRESUMEN
Two poor metabolizer individuals of debrisoquine were identified among 215 healthy Japanese by a phenotyping test. Analysis of the CYP2D6 gene from one of two poor metabolizers, who was not homozygous for the previously described CYP2D6 variant alleles (CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*18), showed that this individual was heterozygous for a new allele, CYP2D6/C8 (CYP2D6*21). CYP2D6*21 had a single cytosine insertion at position 2661 in exon 5. This cytosine insertion generated a stop codon at the 17 bp downstream of this insertion site. A method to detect this allele was established with an allele specific-polymerase chain reaction. This method showed that another one of two poor metabolizers also possessed CYP2D6*21 allele heterozygously. In 318 healthy Japanese, five individuals carried this allele, heterozygously (0.81%, 5/636 chromosomes). Based on the present and our previous data, the poor metabolizer frequency in Japanese was estimated to be 0.39%, which accounted for approximately 45% of the individuals phenotyped as poor metabolizers by in-vivo tests.
Asunto(s)
Alelos , Citocromo P-450 CYP2D6/genética , Exones , Frecuencia de los Genes , Secuencia de Aminoácidos , Secuencia de Bases , ADN , Femenino , Humanos , Japón , Masculino , Datos de Secuencia Molecular , Farmacogenética , FenotipoRESUMEN
In vitro virus is a molecular construct for in vitro protein evolution, which requires some mechanism to link phenotype to genotype. The first in vitro virus was realized by bonding a nascent protein with its coding mRNA via puromycin in in vitro translation. We report a new construct of in vitro DNA virus. The virion was a covalent cDNA-protein fusion, and virion formation did not require any modification of mRNA. Due to intactness of mRNA, this type of in vitro DNA virus will take the next step toward in vitro autonomous evolution, just like in vivo viral evolution in a cellstat.
Asunto(s)
Virus ADN , Evolución Molecular Dirigida , Biosíntesis de Proteínas , Proteínas/química , Cartilla de ADN/metabolismo , Virus ADN/genética , Virus ADN/metabolismo , ADN Complementario , Evolución Molecular , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Hibridación de Ácido Nucleico , Proteínas/genética , Puromicina/metabolismo , ARN Mensajero/metabolismo , ViriónRESUMEN
We have developed a new method for the C-terminus-specific fluorescence labeling of proteins. This method is based on the experimental finding that a fluorescent puromycin analogue at lower concentrations bonds efficiently to the C-terminus of mature proteins in cell-free translation systems using mRNA without a stop codon. This labeling is performed under moderate conditions and its labeling efficiency is in the range of 50-95%. Here we demonstrate a protein-protein interaction assay using fluorescence polarization measurement. This labeling method should also be useful for other rapid molecular interaction assays without purification of the labeled proteins, such as fluorescence correlation spectroscopy.
Asunto(s)
Fluoresceínas/química , Polarización de Fluorescencia/métodos , Puromicina/análogos & derivados , Puromicina/química , Sistema Libre de Células , Fluorescencia , Unión Proteica , Biosíntesis de ProteínasRESUMEN
Adequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of 'in vitro virus' was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was approximately 10%, thus indicating a population of the in vitro virus to have approximately 10(12) protein variants, this number being 10(4) that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions.
Asunto(s)
Biosíntesis de Proteínas , Puromicina/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Proteínas tau/biosíntesis , Animales , Bacteriófagos , Evolución Biológica , Sistema Libre de Células , Cartilla de ADN , Dimerización , Variación Genética , Genotipo , Humanos , Modelos Genéticos , Reacción en Cadena de la Polimerasa/métodos , Conejos , Reticulocitos/metabolismo , Virión , Virus , Proteínas tau/genéticaRESUMEN
Detection of protein-protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein-protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins.
Asunto(s)
Drosophila/metabolismo , Colorantes Fluorescentes/análisis , Cinesinas/metabolismo , Microtúbulos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Encéfalo/metabolismo , Cartilla de ADN/química , ADN Complementario , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/métodos , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Porcinos , Transcripción GenéticaRESUMEN
For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required. A direct over-expression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of 15N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis.
Asunto(s)
Cosintropina/química , Péptidos/química , Péptidos/genética , Tiorredoxinas/genética , Secuencia de Aminoácidos , Aminoácidos/análisis , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Escherichia coli/genética , Expresión Génica , Vectores Genéticos , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Isótopos de Nitrógeno , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
During pregnancy, the uterus shows marked morphological and physiological changes under the regulation of ovarian steroid. To elucidate the molecular cues of these changes, we tried to identify the transcripts differentially expressed in the pregnant rat uterus by using the suppression subtractive hybridization method. Seven independent clones were isolated and one of the up-regulated genes was secreted frizzled-related protein 4 (sFRP4). sFRP4 contains a Wnt-binding domain and belongs to the secreted frizzled protein family whose members are assumed to function as modulators of the Wnt signal. The expression level of sFRP4 mRNA reached a peak in the pregnant uterus on day 12, when uterine decidualization was almost complete in the rat. In situ hybridization histochemistry revealed that sFRP4 transcripts were observed in the decidual cells. In addition, proliferating cell nuclear antigen (PCNA)-positive cells were shown to be overlapped in decidua, suggesting that sFRP4 mRNA expression was accompanied by the late phase of decidual cell proliferation. Moreover, sFRP4 and estrogen receptor-alpha transcripts were co-localized. Furthermore, we analyzed the regulation of sFRP4 by estrogen using 17 beta-estradiol-treated ovariectomized rats. sFRP4 mRNA was detected in the uterus at 48 h after estrogen treatment, especially in endometrial stroma where PCNA-positive cells were also observed. The results in this study led us to the notion that sFRP4 mRNA may be up-regulated after estrogen treatment in the late phase of uterine cell proliferation.