RESUMEN
OBJECTIVE: To evaluate tubal patency using hysterosalpingography after clinical treatment of tubal pregnancy. METHOD: Of 80 patients who underwent hysterosalpingography after clinical treatment of tubal pregnancy from April 1994 to February 2002, 30 were treated with a single 50 mg/m(2) dose of methotrexate intramuscularly (n=30) and 50 were followed up expectantly. RESULTS: Patency of the ipsilateral tube was 84% after methotrexate treatment and 78% after expectant management. In addition, contralateral tubal patency was 97% after methotrexate treatment and 92% after expectant management. There were no statistically significant differences between the groups. CONCLUSION: Findings from this study suggest similar tubal patency rates after methotrexate treatment and expectant management.
Asunto(s)
Trompas Uterinas/fisiología , Embarazo Tubario/cirugía , Abortivos no Esteroideos/uso terapéutico , Femenino , Humanos , Histerosalpingografía , Metotrexato/uso terapéutico , Embarazo , Embarazo Tubario/tratamiento farmacológico , Estudios Prospectivos , Recuperación de la FunciónRESUMEN
The IGF system is one of the most important endocrine and paracrine growth factor systems that regulate fetal and placental growth. We hypothesized that intrauterine growth restriction (IUGR) in guinea pigs is mediated by the altered expression of IGFs and/or IGF binding protein (BP) mRNAs in tissues and is related to growth of specific tissues. IUGR was induced by unilateral uterine artery ligation on day 30 of gestation, and fetal plasma, amniotic fluid and tissue samples were collected at 55-57 days (term about 68 days) from paired IUGR and control fetuses (n=6). Western ligand blotting and immunoblotting were used to compare IGFBP levels in plasma and amniotic fluid. Total RNA was extracted from placenta and fetal tissues, and the relative abundance of IGF-II and IGFBP-1-6 mRNA was determined by Northern blotting, using species-specific probes where available. IUGR fetuses had decreased (P<0.01, by Student's t-test) placental weight and body weight with an increase in the brain:liver weight ratio. The principal IGFBPs in fetal plasma migrated at 40-35, 30 and 25 kDa and were identified as IGFBP-3, -2 and -4 respectively. IUGR was associated with elevated plasma IGFBP-2 and IGFBP-4 and reduced IGFBP-3 levels. IGFBPs were detected at low levels in amniotic fluid of control fetuses but at higher levels in IUGR fetuses. In IUGR placentae, there was a small increase in IGFBP-4 mRNA (P<0.05). IGFBP-2 mRNA increased (P<0.001) in liver of IUGR fetuses. IGF-II and IGFBP mRNA expression did not change in fetal muscle. The results are consistent with reduced IGF action, directly or through inhibition by IGFBPs, particularly by circulating and tissue IGFBP-2, as a potential causal factor in decreased growth of the placenta and certain fetal tissues.
Asunto(s)
Retardo del Crecimiento Fetal/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Amnios/química , Animales , Northern Blotting/métodos , Western Blotting/métodos , Femenino , Sangre Fetal/química , Edad Gestacional , Cobayas , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Factor II del Crecimiento Similar a la Insulina/genética , Hígado/química , Modelos Animales , Placenta/química , Embarazo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The human macrophage colony stimulating factor (hM-CSF) in its monomeric form has been over-produced in BmN cells and in silkworm larvae infected with the recombinant baculovirus Bm284M-CSF. The recombinant monomeric M-CSF (rhM-CSF) exhibited the activity of 8-14 x 10(4) units/ml of cell culture medium. When the insect larvae were infected with the recombinant virus, the maximum rhM-CSF was expressed 4-5 days post infection with an activity of 3 x 10(6) units/ml hemolymph. The monomeric rhM-CSF was purified to homogeneity through three steps of purification. A pilot purification yielded 1 mg of homogeneous monomeric rhM-CSF from 10 larvae. The purified rhM-CSF monomers gradually dimerized in vitro. In contrast, the crude or the semi-purified monomers did not dimerize in vitro, indicating that the presence of an unknown moiety in the rhM-CSF preparations obtained from hemolymph interfered with dimerization.
Asunto(s)
Baculoviridae/metabolismo , Bombyx/metabolismo , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Alquilación , Animales , Western Blotting , Bombyx/virología , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Cromatografía en Gel , Cromatografía por Intercambio Iónico , ADN Complementario/biosíntesis , Hemolinfa/química , Humanos , Larva/metabolismo , Factor Estimulante de Colonias de Macrófagos/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL , Receptor de Factor Estimulante de Colonias de Macrófagos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Compuestos de Sulfhidrilo/análisisRESUMEN
A method is presented for efficient and large-scale isolation of plasmid DNAs from bacterial cells. Based on the cooperativity of heat and alkali actions, the method provides DNA preparations with high quality and yield (about 2 micrograms of DNA/ml culture), which are completely digestable by restriction enzymes and have a high transformation efficiency. Furthermore, the DNA preparations are extremely stable, and even through 4-year storage at -20 degrees C, the electrophorogram and transformation efficiency remain as high as before. The factors affecting the stability of various DNA samples are discussed.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Plásmidos/aislamiento & purificación , 1-Propanol , Ácido Edético , Calor , Concentración de Iones de HidrógenoRESUMEN
A human truncated macrophage colony-stimulating factor (M-CSF) cDNA encoding amino acid residues from 3 to 149 of the native M-CSF was isolated by using the polymerase chain reaction. When introduced into Saccharomyces cerevisiae by a general secretion vector pVT 102u/alpha, it directs the expression of the biologically active dimeric form of M-CSF. Through the 3 stages of purification, i.e. concentration by DEAE-cellulose column chromatography, hydrophobic chromatography on phenyl-sepharose and Mono Q fast protein liquid chromatography, the recombinant truncated M-CSF was purified as to exhibit a specific activity of 1.02 x 10(7) units/mg of protein. SDS-PAGE of this purified truncated M-CSF showed that its apparent molecular mass is 22 kDa under reducing conditions.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/aislamiento & purificación , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Liquida , ADN Complementario/química , Electroforesis en Gel de Poliacrilamida , Humanos , Factor Estimulante de Colonias de Macrófagos/biosíntesis , Factor Estimulante de Colonias de Macrófagos/química , Factor Estimulante de Colonias de Macrófagos/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADNRESUMEN
1. The Chemical modifications of amino acids and their derivatives are mainly due to different post-translational enzymatic reactions. 2. The enzymatic reactions resulting in amino acids such as acetylation-, formylation, methylation-phosphorylation-, sulfation-, hydroxylation, ADP ribosylation-, carboxylation-, amidation-, adenylylation-, glycosylation-, ubiquitination-, prenylation and acylation are listed and analytical methods are reported and extensively reviewed. 3. The post-translationally modified cross-linking molecules after maturations such as desmosines, allo-desmosine, hydroxy-, lysylpyridinoline, 3-hydroxypyridinium derivatives, cyclopentenosine recently found in matured elastin, and in collagen, and pulcherosine a novel tyrosine-derived found in fertilization envelope of Sea Urchin embryo, di-tyrosine in resilin, gamma-glutamyl-lysine isopeptide cross-linking molecule etc. are listed and both physico-chemical and analytical methods are extensively reviewed and discussed. 4. Other consequences of post-translational modifications encountered in the analytical procedure such as N-terminal step-wise Edman degradation of glycosylated site(s), phosphorylated-site(s) and or sulfated-site(s) were also reported by us.
Asunto(s)
Aminoácidos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Humanos , Datos de Secuencia MolecularRESUMEN
Inter-alpha-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as H1 and H2, respectively. We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H3 inside pre-alpha-trypsin inhibitor (Enghild et al. (1991) J. Biol. Chem. 266, 747-751). Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type N-glycans in both H1 and H2 and that of O-glycans in H2. H1 is eluted from Con-A Sepharose by alpha-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates. The capacity of H2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI.
Asunto(s)
alfa-Globulinas/química , Inhibidores de Tripsina/química , Secuencia de Aminoácidos , Carbohidratos/análisis , Cromatografía de Afinidad , Concanavalina A/química , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Polisacáridos/análisis , Análisis de SecuenciaRESUMEN
1. The "code-sequence" of N-glycosylation site(s), the amino acids located around O-glycosylation site(s), the sequence motifs of several kinases, the sequence motifs of--sulfation, amidation, isoprenylation, myristoylation, palmitoylation and N-acetylation, Aspartic and Asparagine hydroxylation-site, gamma-carboxyglutamate domain, phosphopantetheine attachment site etc. are extensively listed, compared to those reported by "PROSITE" Computer Screen Center and discussed. 2. The structural aspects of protein-DNA recognition are quoted as discussion and conclusion.
Asunto(s)
Secuencia de Aminoácidos , Procesamiento Proteico-Postraduccional , Acetilación , Acilación , Animales , Glicosilación , Humanos , Hidroxilación , Datos de Secuencia Molecular , Panteteína/análogos & derivados , Panteteína/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
Multiple homeobox genes are expressed in haematopoietic cell lineages and their expression is cell-type specific. Thus we hypothesized that certain homeobox genes may play an important role in the process of haematopoiesis. To prove that issue, normal murine bone marrow cells were stimulated with appropriate Colony Stimulating Factors in the presence of mouse homeobox gene (Hox 2.3) sense or antisense oligodeoxynucleotides and the effects on the haematopoietic colony formation were examined. Treatment of the cells to Hox 2.3 antisense oligodeoxynucleotides led to a selective inhibition of myeloid colony formation, both in size and in numbers, but without significant effect on erythroid and megakaryocytic haematopoiesis. Exposure to Hox 2.3 sense oligodeoxynucleotides (no-oligomers), had no such effect. It was further showed that inhibition of myelopoiesis by Hox 2.3 antisense oligodeoxynucleotides was dependent on the differentiation stage of target cells. These findings demonstrated that Hox 2.3 gene plays a critical role in regulating normal murine myelopoiesis.
Asunto(s)
Genes Homeobox/fisiología , Granulocitos/citología , Hematopoyesis/genética , Macrófagos/citología , Animales , Secuencia de Bases , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , ADN sin Sentido/farmacología , Relación Dosis-Respuesta a Droga , Granulocitos/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Factores de TiempoRESUMEN
To clarify the effects of sex hormones on the expression of atrial natriuretic peptide (ANP), ovariectomized and intact female rats were subcutaneously injected with estradiol, progesterone, a mixture of them or olive oil solvent; castrated and untouched male rats were subcutaneously injected with estradiol, testosterone or olive oil, once a day for 7 days. The relative rANP-mRNA contents of rat atrial were measured by molecular hybridization. rANP-cDNA was labeled with 32P as a probe. The results revealed that estradiol and progesterone increased ANP gene expression. Furthermore their effects were associated with administration dose of these hormones and it was shown that they are probably coordinated. The physiological amounts of estradiol and progesterone may maintain suitable levels of rANP-mRNA and androgen may also increase the ANP gene expression in vivo. These experiments suggested that female sex hormone may have a dual purpose in fluid balance.
Asunto(s)
Factor Natriurético Atrial/genética , Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Testosterona/farmacología , Animales , Factor Natriurético Atrial/efectos de los fármacos , Estradiol/farmacología , Femenino , Masculino , Progesterona/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.
Asunto(s)
Endopeptidasas/aislamiento & purificación , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Ditiotreitol/farmacología , Endopeptidasas/química , Endopeptidasas/metabolismo , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Leupeptinas/farmacología , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Trypanosoma brucei brucei/metabolismoRESUMEN
1. The role played by the modification of protein in determining its fate is reported by us. 2. Post-translational modifications such as acetylation, phosphorylation, sulfation, methylation, hydroxylation, ADP-ribosylation, maturation, amidation, carboxylation, adenylylation, glycosylation, ubiquitination, and prenylation are extensively reviewed. 3. Each post-translational modification's significance and its role played in biological function(s) is summarized in the general discussion and the conclusion's remark is directed at the problems left to solve (e.g. post-translational modification reactions in recombinant protein in modern genetic engineering).
Asunto(s)
Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Ingeniería Genética , Humanos , Datos de Secuencia MolecularRESUMEN
1. The levels of atrial natriuretic peptide(ANP) gene expression in rat atria at 2-3 and 14-18 months of age and the effects of ginsenosides on r-ANP gene expression by determining the concentration of ANP-mRNA were investigated. The male and female rats were abdominally (i.p.) injected with aqueous solution of ginsenosides prepared from ginseng stems and leaves (G-PSL) and ginseng roots (G-PR), 50 mg/kg body wt, once a day for 7 days. Atria total RNA was extracted by the cold phenol method. The ANP-mRNA contents were determined using the Northern blot and dot blot hybridization technique with alpha-32*P-labelled r-prepro-ANP-cDNA probe. 2. The ANP-mRNA contents of 14-18 month rats were remarkably less than that of 2-3 month rats. The levels of male and female rats' atria at 14-18 months were about 15 and 60%, the content of male and female rats at 2-3 months respectively. 3. G-PSL and G-PR increased the ANP-mRNA content of male rats at 14-18 months 1- and 2-fold, respectively, whereas G-PSL and G-PR decreased the ANP-mRNA content in male rats of age 2-3 months. 4. These results revealed that the ANP gene expression declined during ontogenic ageing development and ginsenosides possessed anti-ageing effects in the heart endocrineous function aspect.
Asunto(s)
Envejecimiento/genética , Factor Natriurético Atrial/genética , Regulación de la Expresión Génica/efectos de los fármacos , Saponinas/farmacología , Animales , Factor Natriurético Atrial/metabolismo , Northern Blotting , Sondas de ADN , Femenino , Ginsenósidos , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas EndogámicasRESUMEN
1. Adult male Wistar rats were injected with streptozotocin (STZ: 55 mg/kg) for inducing diabetes. Then blood and atria for RNA extraction were withdrawn from rats treated 3 and 11 weeks previously with STZ respectively. Atrial total RNA were extracted with cold phenol method. The ANP mRNA contents were determined using Dot blot hybridization technique with alpha-32-P-labelled r-prepro ANP cDNA probe. 2. Plasma glucose was increased and plasma immunoreactive insulin was lowered in rats at 3 and 11 weeks after injection of STZ. ANP gene expression in diabetic rats was depressed. ANP mRNA contents in rats treated 3 and 11 weeks with STZ were 86.4% and 31.7% of that of control rats. 3. Three weeks after treatment of STZ, the rats were gastrically perfused with FOC (Fish Oil Compound) (0.355 ml/kg) once a day successively until 11 weeks. This treatment induces lower blood pressure in rats. ANP gene expression in FOC group was apparently recovered which had been decreased because of the effect of diabetes mellitus.
Asunto(s)
Factor Natriurético Atrial/genética , Diabetes Mellitus Experimental/genética , Aceites de Pescado/farmacología , Animales , Glucemia/análisis , ADN/genética , Sondas de ADN , Electroforesis en Gel de Agar , Expresión Génica/efectos de los fármacos , Insulina/sangre , Masculino , Hibridación de Ácido Nucleico , ARN/análisis , Ratas , Ratas Endogámicas , Estreptozocina/farmacologíaRESUMEN
Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is known as an inducer of proliferation and functional activation of myeloid cells. This study was carried out to characterize the effect of purified recombinant human GM-CSF (rhGM-CSF) on induction of TGF-alpha in macrophages. Using Northern blot analysis and immunoassays, we show here that rhGM-CSF markedly stimulates production of TGF-alpha messenger RNA and protein in normal tonsil macrophages. The findings are consistent with macrophages being a normal inducible source of TGF-alpha which may be an important mediator of various activities of GM-CSF both in hematopoietic and non-hematopoietic cells.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Macrófagos/metabolismo , Factor de Crecimiento Transformador alfa/biosíntesis , Animales , Northern Blotting , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Cinética , Ratas , Proteínas Recombinantes/farmacologíaRESUMEN
Human promyelocytic leukemia HL-60 cells were induced to differentiate into macrophages by PMA (phorbol 12-myristate-13-acetate), 1-alpha-25-(OH)2D3(1-alpha-25-dihydroxyvitamin D3, hrGM-CSF (human recombinant granulocyte-macrophage colony-stimulating factor) and into granulocytes by DMSO (dimethylsulfoxide). We found that the differentiation of HL-60 cells into macrophages was accompanied by transcription of the c-fms oncogene, which was assessed by a modified PCR (polymerase-chain reaction) method. After treatment with a c-fms anti-sense oligomer, the PMA and hrGM-CSF induced macrophage differentiation of HL-60 cells was significantly inhibited, whereas either 1-alpha-25-(OH)2D3 induced macrophage or DMSO and hrGM-CSF induced granulocytic differentiation was not inhibited. Furthermore, we treated the HL-60 cells with M-CSF (macrophage-colony stimulating factor or CSF-1) anti-sense N degrees 2 (see Figure 1) in the presence of PMA, hrGM-CSF, 1-alpha-25-(OH)2D3 and DMSO. The results showed that this treatment leads to a significant inhibition of PMA and hrGM-CSF-induced macrophage differentiation, but has no influence on the 1-alpha-25-(OH)2D3-induced macrophage differentiation and DMSO-induced granulocytic differentiation. It was further demonstrated that the M-CSF (or CSF-1) and c-fms antisense oligomers acted synergistically on inhibition of macrophage formation induced by PMA and hrGM-CSF, but had no inhibitory effect on the macrophage formation induced by 1-alpha-25-(OH)2D3. Thus we concluded firstly, that HL-60 cells differentiate into macrophages along two different pathways: one is involved in the action of the c-fms oncogene and the other is not. Secondly, an autocrine circuit of M-CSF (or CSF-1) action may exist in the macrophage formation induced by PMA and hrGM-CSF.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Leucemia Promielocítica Aguda/patología , Proteínas Proto-Oncogénicas/fisiología , Secuencia de Bases , Calcitriol/farmacología , Transformación Celular Neoplásica/patología , Factores Estimulantes de Colonias/farmacología , Dimetilsulfóxido/farmacología , Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Sustancias de Crecimiento/farmacología , Humanos , Factor Estimulante de Colonias de Macrófagos , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Datos de Secuencia Molecular , Oligonucleótidos/farmacología , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Receptor de Factor Estimulante de Colonias de Macrófagos , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patologíaRESUMEN
1. Effects of sodium loading and dehydration on ANP gene expression were investigated in rats. 2. ANP-mRNA was determined using Northern blot and dot blot hybridization technique with alpha-32P-labeled r-preproANP-cDNA probe. Salt loading increased the ANP-mRNA content in atria. Correlation with ANP-mRNA content and the plasma sodium concentration was established. 3. Deprivation of water for 2 and 4 days increased ANP-mRNA 2.1- and 1.6-fold, respectively. 4. These results demonstrated that water-salt balance affects the ANP-gene expression.
Asunto(s)
Factor Natriurético Atrial/genética , Regulación de la Expresión Génica , Sodio/farmacología , Animales , Northern Blotting , Deshidratación , Femenino , ARN/análisis , Ratas , Ratas EndogámicasRESUMEN
In human brain extracts, most proteins of pathological interest in Alzheimer's disease are insoluble and their analysis is often performed on denatured and reduced samples by immunoblotting after electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulfate. Because we needed to accurately compare the concentration of several proteins in brain extracts to investigate the etiology of the disease, the quantitative aspect of immunoblotting was assessed and the results compared for a soluble component with those obtained by electroimmunoassay. Glial fibrillary acidic protein (GFAP) and Tau proteins were analysed by immunoblotting in brain homogenates treated with the Laemmli sample buffer from 10 control and 25 Alzheimer's disease brains. The linearity of densitometric measures of dilutions for one given sample was demonstrated. A 8 to 16-fold GFAP increase in Alzheimer brain was established. With regard to Tau proteins it was possible to show the presence of two pathological Tau variants (Tau 64 and 69) in all the Alzheimer brain homogenates, furthermore, the amount of Tau 64 and 69 was proportional to the presence of neurofibrillary degeneration. As far as alpha 1-antichymotrypsin is concerned, we showed, in a second set of brain samples (14 control and 12 Alzheimer brains), discrepancies between the results obtained by immunoblotting and by electroimmunoassay while for a given sample linearity of immunoblotting measures of dilutions of this sample was demonstrated. Quantitation by immunoblotting of such components which can be quantified using other procedures is uncertain whereas the interest of immunoblotting is undoubted for the insoluble proteins in the brain extracts.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Química Encefálica , Proteína Ácida Fibrilar de la Glía/análisis , Proteínas Asociadas a Microtúbulos/análisis , Proteínas del Tejido Nervioso/análisis , alfa 1-Antiquimotripsina/análisis , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoensayo/métodos , Immunoblotting , Proteínas tauRESUMEN
Conditions for unblocking reversible chemical modifications such as maleylation or citraconylation 'in situ' at the N-terminus of proteins after transfer of proteins to immobilon membranes from SDS-PAGE are described. Demaleylation or decitraconylation occurred at 55 degrees C in 70% formic acid (pH 1.50) during 60 min. During the unblocking reaction, Coomassie blue dye was completely removed, resulting in superior high performance liquid chromatographic separation of phenylthiohydantoin-amino acid (PTH-AA) after Edman degradation (automatic gas phase sequencer). The protein fixed on the matrix after demaleylation and removal of Coomassie blue was not degraded. The possible cleavage at the aspartyl-prolyl peptide bonds was considered, but no side reaction was observed. Furthermore, the incubation time in 70% formic acid at 55 degrees C could be reduced to 10 min in the absence of maleylation of the starting material, and this was suitable for the removal of Coomassie blue and the quantification of phenylthiolhydantoin-amino acids (PTH-AAs) by HPLC. The yield from the starting protein through SDS-PAGE, blotting, and Edman degradation to quantitative analysis of PTH-aminoacid(s) by HPLC was established.
Asunto(s)
Proteínas/análisis , Aminoácidos/análisis , Western Blotting , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Indicadores y Reactivos , Membranas Artificiales , Polivinilos , Colorantes de Rosanilina , Dodecil Sulfato de Sodio , Coloración y EtiquetadoRESUMEN
Human trypsin inhibitor (home prepared), lactalbumin, trypsinogen, carbonic anhydrase, and bovine serum albumin were submitted to succinylation and their molecular masses were determined by SDS-PAGE according to the method of Weber and Osborn (1969 J. Biol. Chem. 244, 4406) before and after chemical modification. High estimates of their molecular masses were obtained. The monomer and dimer of arrowhead inhibitor proteinase-B (Chinese vegetable legume) obtained after chemical crosslink(s) were also submitted to SDS-PAGE and their apparent molecular masses were also determined and compared to the native arrowhead inhibitor proteinase-B. Abnormally high estimates of their molecular masses were obtained. Our results agree with those in the literature.