RESUMEN
This study aimed to create prognostic signatures to predict AML patients' survival using alternative splicing (AS) events. The AS data, RNA sequencing data, and the survival statistics of 136 AML patients were obtained from The Cancer Genome Atlas (TCGA) and TCGA SpliceSeq databases. Total 34,984 AS events generated from 8,656 genes, 2,583 of which were survival-associated AS events, were identified using univariate Cox regression. The prognostic models constructed using independent survival-associated AS events revealed that low-risk splicing better predicted patients' survival. ROC analysis indicated that the predictive efficacy of the alternate terminator model was best in the area under the curve at 0.781. Enrichment analysis revealed several important genes (TP53, BCL2, AURKB, PPP2R1B, FOS, and BIRC5) and pathways, such as the protein processing pathway in the endoplasmic reticulum, RNA transport pathway, and HTLV-I infection pathway. The splicing network of splicing events and factors revealed interesting interactions, such as the positive correlation between HNRNPH3 and CALHM2-13010-AT, which may indicate the potential splicing regulatory mechanism. Taken together, survival-associated splicing events and the prognostic signatures for predicting survival can help provide an overview of splicing in AML patients and facilitate clinical practice. The splicing regulatory network may improve the understanding of spliceosomes in AML.
Asunto(s)
Empalme Alternativo , Redes Reguladoras de Genes , Leucemia Mieloide Aguda/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Pronóstico , Curva ROC , Análisis de Secuencia de ARNRESUMEN
Paclitaxel (PTX) is a mitotic inhibitor widely used in chemotherapy for many types of cancers, including solid tumors and hematological malignancies. However, the molecular basis of the anti-proliferation activity of PTX is not fully understood. In this paper, we focused on the role of c-Jun N-terminal kinase (JNK) pathways in PTX-induced apoptosis and proliferation inhibition. The effects of PTX were examined in human leukemia cell lines and patients' chronic lymphocytic leukemia (CLL) cells in relation to mitochondrial events, apoptosis, and perturbation of JNK activation using flow cytometry, siRNA, mitochondrial membrane potential determination, and western blotting. Exposure of cells to PTX at concentrations ≥ 10 nM for 18 or 24 h resulted in a significant release of cytochrome c from mitochondria to the cytosol, cleavages of procaspase 3 and poly (ADP-ribose) polymerase (PARP), and JNK activation, leading to apoptosis. The pan-caspase inhibitor BOC-D-FMK blocked the PTX-induced apoptosis but had no effect on cytochrome c release, suggesting that cytochrome c had been released before caspase activation. Moreover, both pharmacological JNK inhibitors SP600125 and JNK siRNA dramatically blocked PTX-induced apoptosis, cytochrome c release, caspase 3, and PARP cleavage. These findings demonstrate that JNK activation plays a critical role in the induction of apoptosis mediated by PTX in human leukemia cell lines and CLL patient-derived primary cancer cells, and this event is upstream of cytochrome c release, caspase 3, and PARP cleavage.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Leucemia/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Paclitaxel/farmacocinética , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Células Jurkat , Leucemia/tratamiento farmacológico , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Paclitaxel/farmacologíaRESUMEN
This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.
Asunto(s)
Leucemia/tratamiento farmacológico , Proteínas Quinasas/genética , Xantonas/administración & dosificación , Proteínas de la Ataxia Telangiectasia Mutada/biosíntesis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Células HL-60 , Humanos , Leucemia/genética , Leucemia/patología , Proteínas de Neoplasias/biosíntesis , Proteínas Quinasas/biosíntesis , ARN Mensajero , Transducción de Señal/efectos de los fármacosRESUMEN
Renal growth, particularly hypertrophy, is a feature of diabetic nephropathy (DN). Adiponectin, an adipocyte-derived hormone, is an important regulator of cell proliferation. Recent studies have suggested that adiponectin has a protective effect in the kidney. The purpose of this study was to investigate the therapeutic effects and the underlying mechanisms of adiponectin in early DN. Mouse mesangial cells (MMCs) were cultured in media containing different concentrations of platelet-derived growth factor-BB (PDGF-BB) with or without adiponectin. MMC proliferation and expression of type IV collagen, laminin, and fibronectin were investigated. Streptozotocin-induced diabetic mice were injected intravenously with recombinant lentivirus encoding the mouse adiponectin gene (Lenti-Acdc-IRES-EGFP). Urinary microalbumin, serum adiponectin level, and expression of proliferating cell nuclear antigen, type IV collagen, laminin, and fibronectin were determined. Adiponectin inhibited the increases in MMC proliferation and expression of type IV collagen, laminin, and fibronectin induced by PDGF-BB. Adiponectin also effectively reduced renal cell proliferation and expression of type IV collagen, laminin, and fibronectin when it was introduced in vivo by lentivirus-mediated gene transfer. These findings suggest that adiponectin exerts renoprotective effects by inhibiting renal cell proliferation and reducing synthesis of extracellular matrix proteins, thus suppressing the development and progression of DN.
Asunto(s)
Adiponectina/metabolismo , Proliferación Celular , Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/metabolismo , Matriz Extracelular/metabolismo , Células Mesangiales/citología , Adiponectina/genética , Animales , Becaplermina , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/fisiopatología , Nefropatías Diabéticas/terapia , Modelos Animales de Enfermedad , Femenino , Terapia Genética , Humanos , Riñón/citología , Riñón/metabolismo , Células Mesangiales/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-sis/metabolismo , EstreptozocinaRESUMEN
OBJECTIVE: The purpose of this study was to assess radio frequency (RF) artefacts in echoplanar imaging (EPI) induced by two 1.5 T MR scanners in close proximity and to find an effective method to correct them. METHODS: Based on the intact shielding of rooms, experiments were performed by two MR scanners with similar centre frequencies. Phantom A (PA) was scanned in one scanner by EPI at different bandwidths (BWs). Simultaneously, phantom B was scanned in a fixed sequence for scanning with the other scanner. RF artefact gaps of PA, scanning time and the image signal-noise ratio (SNR) were measured and recorded. Statistical analysis was performed with the repeated-measures analysis of variance test. Based on findings obtained from PA, three healthy volunteers were studied at a conventional BW and a lower BW to observe the artefact variance. RESULTS: EPI RF artefacts were symmetrically situated in both sides of the image following the phase-encoding direction. The gap size of the artefact became larger and the SNR was significantly improved with a narrower BW. RF artefacts with a lower BW in volunteers presented the same characteristic as PA. CONCLUSION: For EPI RF artefacts produced by two 1.5 T MR scanners with approximately similar centre frequencies, we can reduce BWs in a suitable range to minimize the effect on MRI. ADVANCES IN KNOWLEDGE: MR scanners with the same field strength installed in the same vicinity might produce RF artefacts in the sequence at larger BWs. Reducing BWs properly is effective to control the position of artefacts and improve the image quality.
Asunto(s)
Artefactos , Imagen Eco-Planar/métodos , Adulto , Anciano , Imagen Eco-Planar/instrumentación , Femenino , Arquitectura y Construcción de Hospitales , Humanos , Masculino , Persona de Mediana Edad , Fantasmas de ImagenRESUMEN
BACKGROUND: Association of de novo human leukocyte antigen (HLA) and major histocompatibility complex class I chain-related gene-A (MICA) antibodies and proteinuria with graft survival 5 years after renal transplantation. De novo presence of HLA and MICA antibodies after renal transplantation is associated with poor graft survival. Proteinuria after transplantation is also considered a risk factor for premature graft loss. In this study, we investigated the association of de novo HLA and MICA antibodies on proteinuria after renal transplantation and the association of proteinuria and de novo antibodies with graft survival. METHODS: We enrolled 275 patients without preexisting HLA and MICA antibodies followed for >5 years after renal transplantation. All donor organs were from living-related donors or from an organ donation program. HLA and MICA antibodies were detected by the Luminex method. Patients with proteinuria (>150 mg/d) underwent intermittent 24-hour proteinuria examination. RESULTS: The frequencies of de novo HLA and MICA antibody 5 years after transplantation were 25.8% and 12%, respectively. In total, 26.5% of patients had proteinuria at the 5-year follow-up. De novo HLA antibody was associated with increased proteinuria after transplantation (relative risk, 3.12). HLA antibody and proteinuria were both associated with poor 5-year graft survival (P = .027 and P = .006, respectively). CONCLUSION: De novo HLA and MICA antibodies and proteinuria after renal transplantation are all associated with poor graft survival. De novo HLA antibody is independent risk factor for posttransplant proteinuria, and proteinuria affects the association of de novo antibodies with decreased graft survival after transplantation.
Asunto(s)
Autoanticuerpos/inmunología , Supervivencia de Injerto , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Trasplante de Riñón , Proteinuria/diagnóstico , Adulto , Femenino , Humanos , MasculinoRESUMEN
An aberrant proliferation of mesangial cells (MCs) is one of the more important features of diabetic nephropathy (DN). Adiponectin, an adipocyte-derived hormone, has been associated with type 2 diabetes, a known cause of DN. Recent studies have suggested that adiponectin has a protective effect on the kidney. To elucidate the potential protective mechanism of adiponectin on kidney, we investigated the effects of adiponectin on platelet-derived growth factor (PDGF)-induced cell proliferation and intracellular signaling pathways in cultured Human MCs (HMCs). PDGF-induced HMC proliferation was significantly inhibited by the co-treatment of adiponectin. Adiponectin alone had no effect on HMC proliferation. The mammalian target of rapamycin (mTOR) and 40 S ribosomal S6 kinase 1 (S6K1) were activated by PDGF stimulation in HMCs. PDGF-induced mTOR and S6K1 phosphorylations were significantly attenuated by the co-treatment of adiponectin in HMC. Adiponectin alone had no effects on PDGF-receptor autophosphorylation by PDGF. We also confirmed that the inhibitory effect of adiponectin on PDGF-induced HMC proliferation was significantly suppressed by compound C, an adenosine 5'-monophosphate-activated protein kinase (AMPK) inhibitor. From these findings, it is implied that adiponectin could attenuate renal dysfunction associated with MC disorders through AMPK-mTOR signal pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adiponectina/farmacología , Células Mesangiales/citología , Células Mesangiales/enzimología , Proteínas Proto-Oncogénicas c-sis/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Becaplermina , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Células Mesangiales/efectos de los fármacos , Fosforilación/efectos de los fármacos , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Rejection in renal transplantation is the most frequent event causing transplant failure. It is important to identify parameters to predict rejection, which are helpful in a timely fashion. METHODS: Fifty-nine renal transplant recipients were divided into two groups: group 1 (stable renal function) and group 2 (acute rejection episodes). The levels of HLA-A mRNA in peripheral blood lymphocytes (both pre- and posttransplantation) were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) with glucose-6-phosphate dehydrogenase (G6PDH) as an internal reference. The TEST software was used to analyze the relative expressions of HLA-A mRNA. RESULTS: There was no statistical significance between features of the two groups pretransplant versus normal controls. Posttransplant, the HLA-A mRNA levels decreased significantly compared to those of pretransplant and normal control individuals. The levels of HLA-A mRNA among the 10 patients with acute rejection episodes were significantly increased. There was no significant change in the lymphocyte populations in the early stage of an acute rejection episode compared with the prerejection value. CONCLUSION: HLA-A mRNA expression was strongly correlated with immune status. The HLA-A mRNA levels may provide an effective and reliable indicator to predict acute rejection episodes in renal transplantation.
Asunto(s)
Rechazo de Injerto/epidemiología , Antígenos HLA-A/genética , Trasplante de Riñón/efectos adversos , Linfocitos/inmunología , ARN Mensajero/genética , Adolescente , Adulto , Biopsia , Femenino , Glucosafosfato Deshidrogenasa/genética , Rechazo de Injerto/genética , Rechazo de Injerto/patología , Humanos , Trasplante de Riñón/patología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Angiogenesis and hematopoiesis are closely linked and interactive with each other, but few studies were given to identify possible links between angiogenesis-promoting proteins and hematopoiesis-related transcription factors. Here we investigated the potential relationship of oxygen-sensitive alpha-subunit of angiogenesis-related hypoxia-inducible factor-1alpha (HIF-1alpha) with Runt-related protein 1 (Runx1, also known as acute myeloid leukemia-1, AML-1), an important hematopoietic transcription factor. The results demonstrated that Runx1 and HIF-1alpha proteins directly interacted with each other to a degree, in which Runt homology domain of Runx1 was mainly involved. Leukemia-related abnormal Runx1 fusion protein AML1-ETO, which fuses the N-terminal 177 amino acid residues of the Runx1 protein in frame to ETO (eight-twenty-one) protein, also interacted with HIF-1alpha protein with greater ability than Runx1 itself. More intriguingly, Runx1 overexpression inhibited DNA-binding and transcriptional activity of HIF-1 protein with reduced expression of HIF-1-targeted genes such as vascular endothelial growth factor, while silence of Runx1 expression by specific small interfering RNA significantly increased transcriptional activity of HIF-1 protein, suggesting that Runx1 inhibited transcription-dependent function of HIF-1. Vice versa, HIF-1alpha increased DNA-binding ability and transcriptional activity of Runx1 protein. All these data would shed new insight to understanding Runx1 and HIF-1alpha-related hematopoietic cell differentiation and angiogenesis.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Transcripción Genética , Línea Celular Tumoral , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , ADN/metabolismo , Hematopoyesis/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neovascularización Fisiológica/genética , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/farmacologíaRESUMEN
Leukemia-associated fusion protein AML1-ETO is a product of the chromosome translocation (8;21) frequently occurred in acute myeloid leukemia (AML). The fusion oncoprotein blocks leukemic cell differentiation, and it also induces growth arrest with increased sensitivity to apoptosis induction. Such dichotomous functions make it difficult to clarify the role of AML1-ETO in leukemogenesis. Here, we systematically showed that constitutively and overexpressed AML1-ETO protein was cleaved to four fragments of 70, 49, 40 and 25 kDa by activated caspase-3 during apoptosis induction by extrinsic mitochondrial and death receptor signaling pathways. The in vitro proteolytic system combined with MALDI-TOF/TOF mass spectrometer confirmed that AML1-ETO and wild-type ETO but not RUNX1 (AML1) proteins were direct substrates of apoptosis executioner caspase-3. Site-directed mutagenesis analyses identified two nonclassical aspartates (TMPD188 and LLLD368) as caspase-3-targeted sites in the AML1-ETO sequence. When these two aspartates were mutated into alanines, more intriguingly, the apoptosis-amplified action of AML1-ETO induction completely disappeared, while inducible expression of the caspase-3-cleaved 70 kDa fragment of AML1-ETO after tetracycline removal is sufficient to enhance apoptotic sensitivity. Further investigations on the potential in vivo effects of such a cleavage and its possible role in leukemogenesis would provide new insights for understanding the biology and treatment of AML1-ETO-associated leukemia.
Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Leucemia/patología , Proteínas de Fusión Oncogénica/metabolismo , Fragmentos de Péptidos/análisis , Sustitución de Aminoácidos , Ácido Aspártico , Sitios de Unión , Línea Celular Tumoral , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Hidrólisis , Leucemia Mieloide Aguda/patología , Espectrometría de Masas , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 Compañera de Translocación de RUNX1 , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
We reported recently that cobalt chloride-simulated hypoxia and mild hypoxia modified the differentiation of human acute myeloid leukemic (AML) cells, probably acting via a hypoxia-inducible factor-1 alpha (HIF-1 alpha)-dependent mechanism. In this study, we investigated the effect of desferrioxamine (DFO), an iron chelator with 'hypoxia-mimetic' activity, on the differentiation of AML cells. The results showed that DFO at nontoxic concentrations induced the differentiation of AML cell lines NB4 and U937, as assessed by morphological criteria and differentiation-associated antigens. DFO-induced differentiation parallel to the rapid accumulation of HIF-1 alpha protein in these two cell lines. Of importance, the transient transfection of HIF-1 alpha cDNA induced U937 cells to develop the differentiation-related alterations such as growth arrest and increased CD11b expression. Furthermore, the inducible expression of chromosome translocation t(8;21)-generated leukemogenic AML1-ETO fusion gene attenuated DFO-induced differentiation of U937 cells with the decrease of CCAAT/enhancer-binding protein alpha (C/EBP alpha), a critical factor for granulocytic differentiation. Using immunoprecipitation and luciferase reporter assay, HIF-1 alpha was also shown to interact physically with and to increase the transcriptional activity of C/EBP alpha. Taken together, these results provided novel evidence for a role of HIF-1 alpha in AML cell differentiation, and suggested that C/EBP alpha might be a downstream effector for HIF-1 alpha-mediated differentiation.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Deferoxamina/farmacología , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Enfermedad Aguda , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Regulación Leucémica de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína 1 Compañera de Translocación de RUNX1 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Magnetic particles about 10 nm in size were prepared by chemical precipitation under nitrogen and used for the selective and sequential adsorption of bovine serum albumin (BSA) (pI = 4.7) and lysozyme (LSZ) (pI = 1.1) under different conditions, such as pH and initial protein concentration. The separation ratio of BSA over LSZ at pH 4.6 is about 5, which is about 1.5 times the separation ratio of LSZ over BSA at pH 11.0. Only 10% of the preadsorbed BSA could be displaced by the sequential adsorption of LSZ at pH 11.0. On the other hand, 60% of the preadsorbed LSZ was desorbed due to the sequential adsorption of BSA at pH 4.6. Over 50% desorption of BSA or LSZ could be achieved either by 0.5 M Na(2)HPO(4) or 0.5 M NaH(2)PO(4) after 2 h. Over 80% of the enzymatic activity of LSZ was preserved when it was desorbed from magnetic particles.
Asunto(s)
Magnetismo , Muramidasa/aislamiento & purificación , Nanotecnología , Albúmina Sérica Bovina/aislamiento & purificación , Adsorción , Animales , Bovinos , Concentración de Iones de Hidrógeno , Cinética , Muramidasa/química , Nitrógeno/química , Tamaño de la Partícula , Albúmina Sérica Bovina/químicaRESUMEN
Adsorption and desorption of lysozyme on nano-sized magnetic particles and its conformational change were studied in this work. Adsorption of lysozyme on nano-sized magnetic particles (Fe(3)O(4)) was carried out at different pH. Maximum adsorption of lysozyme (4.65 mg/m2) occurred at its isoelectric point (pI = 11.1). Differential scanning calorimetry (DSC) results show that the lysozyme adsorbed on magnetic particles did not show any thermal transition over the range 20-100 degrees C. High desorption of lysozyme from magnetic particles was achieved using NaH(2)PO(4) (pH 4.0) (90%) and NaSCN (pH 6.0) (97%) as desorbents. The conformational change of the lysozyme desorbed by NaH(2)PO(4) was small, while the lysozyme desorbed by NaSCN underwent a significant conformational change as measured by the intrinsic fluorescence. Eighty-eight and 82% activity was retained in the desorbed enzyme for desorption by NaH(2)PO(4) and NaSCN, respectively.
Asunto(s)
Muramidasa/química , Muramidasa/fisiología , Adsorción , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Concentración de Iones de Hidrógeno , Magnetismo , Muramidasa/efectos de los fármacos , Tamaño de la Partícula , Fosfatos/farmacología , Conformación Proteica/efectos de los fármacos , Espectrometría de Fluorescencia , Tiocianatos/farmacologíaRESUMEN
Adsorption of bovine serum albumin (BSA) on nanosized magnetic particles (Fe(3)O(4)) was carried out in the presence of carbodiimide. The equilibrium and kinetics of the adsorption process were studied. Nanosized magnetic particles (Fe(3)O(4)) were prepared by the chemical precipitation method using Fe2+, Fe3+ salts, and ammonium hydroxide under a nitrogen atmosphere. Characterizations of magnetic particles were carried out using transmission electron microscopy (TEM) and a vibrating sample magnetometer (VSM). Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to confirm the attachment of BSA on magnetic particles. Effects of pH and salt concentrations were investigated on the adsorption process. The experimental results show that the adsorption of BSA on magnetic particles was affected greatly by the pH, while the effect of salt concentrations was insignificant at a low concentration range. The adsorption equilibrium isotherm was fitted well by the Langmuir model. The maximum adsorption of BSA on magnetic particles occurred at the isoelectric point of BSA. Adsorption kinetics was analyzed by a linear driving force mass-transfer model. BSA was desorbed from magnetic particles under alkaline conditions, which was confirmed by SDS-PAGE electrophoresis and FTIR results.
Asunto(s)
Hierro/química , Óxidos/química , Albúmina Sérica Bovina/química , Adsorción , Algoritmos , Animales , Bovinos , Microanálisis por Sonda Electrónica , Electroforesis en Gel de Poliacrilamida , Etildimetilaminopropil Carbodiimida/química , Óxido Ferrosoférrico , Concentración de Iones de Hidrógeno , Cinética , Magnetismo , Microscopía Electrónica , Óxidos/síntesis química , Tamaño de la Partícula , Cloruro de Sodio/química , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad Estática , Propiedades de Superficie , TermodinámicaRESUMEN
The ADP-ribosylation factor (ARF) is the small (21 kb) GTP-binding protein required for the efficient cholera toxin-catalyzed ADP-ribosylation of purified Gs, the stimulating regulatory component of adenylate cyclase. Human ARF cDNA clones were obtained from a human cDNA library by cross-species hybridization with bovine ARF1, and the nucleotide and deduced amino acid sequences were determined. Comparison of the sequences of human and bovine ARF1 showed 90% identity at the nucleotide level and 100% identity at the amino acid level, demonstrating the highly conserved nature of the ARF protein. Using human ARF cDNA as the probe, we have detected ARF messenger RNA (approximately 2.2-2.3 kb) in a wide variety of human tissues and tumor cell lines.
Asunto(s)
Clonación Molecular , ADN/genética , Proteínas de Unión al GTP/genética , Genes , ARN Mensajero/genética , Transcripción Genética , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Animales , Ganglios Basales/metabolismo , Secuencia de Bases , Northern Blotting , Bovinos , Línea Celular , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
Two distinct types of protein kinase C cDNA clones were isolated from a Xenopus laevis oocyte cDNA library, and the complete nucleotide sequences were determined. The sequences encode a single open reading frame with a domain structure that consists of four constant (designated C1-C4) and five variable (designated V1-V5) regions. Comparison of the two sequences shows good homology at the nucleotide and deduced amino acid level. The differences reside primarily in the variable regions. Each clone encodes 671 and 676 amino acids, respectively, having extensive homology with published sequences of human, rat, and bovine protein kinase C. These results provide evidence that these two distinct types of protein kinase C are members of a multigene family in amphibian and mammalian species.
Asunto(s)
Isoenzimas/genética , Proteína Quinasa C/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Ácido Nucleico , Xenopus laevisRESUMEN
An improved rapid method for sequencing a target DNA is described. A new plasmid, pAA-PZ1, which contains the origin of replication from phage M13 and a portion of the Tn9 transposon was constructed. A long fragment of target DNA cloned into this vector is progressively shortened in vivo from one end by transposon-mediated deletions. The plasmids carrying different lengths of target DNA are then made into single-stranded DNA in the same host upon infection with an M13 phage and their sequence is determined using the dideoxynucleotide chain-termination method. This method bypasses the in vitro enzymatic manipulations for progressive deletions and requires no subcloning. Using this strategy, we sequenced 1.3 kb of rice DNA containing a histone 3 gene within three weeks.