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The molecular weight (MW) of regenerated silk fibroin (RSF) decreases during degumming and dissolving processes. Although MW and the MW distribution generally affect polymer material processability and properties, few reports have described studies examining the influences of MW and the distribution on silk fibroin (SF) material. To prepare different MW SF fractions, the appropriate conditions for fractionation of RSF by ammonium sulfate (AS) precipitation process were investigated. The MW and the distribution of each fraction were found using gel permeation chromatography (GPC) and SDS-polyacrylamide electrophoresis (SDS-PAGE). After films of the fractionated SFs formed, the secondary structure, surface properties, and cell proliferation of films were evaluated. Nanofiber nonwoven mats and 3D porous sponges were fabricated using the fractionated SF aqueous solution. Then, their structures and mechanical properties were analyzed. The results showed AS precipitation using a dialysis membrane at low temperature to be a suitable fractionation method for RSF. Moreover, MW affects the nanofiber and sponge morphology and mechanical properties, although no influence of MW was observed on the secondary structure or crystallinity of the fabricated materials.
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We present wetting, hygroscopicity, and interfacial activity of hemicellulose with respect to etherification and contrast it to their potential as interfacial modifiers, which is demonstrated by oil-in-water emulsification containing up to 60 vol% of the oil phase. Tunable amphiphilicity of hardwood and softwood hemicelluloses, xylans, and galactoglucomannans, respectively, was accomplished via controlled etherification. A series of degree of substitution (DS) of hydroxypropylated and 3-butoxy-2-hydroxypropylated ("butylated") grades was synthesized. The hemicellulose ethers were characterized by gel permeation chromatography, spectroscopic techniques, such as NMR, and contact angle measurements. An attenuated total reflectance infrared method was developed for fast identification of the DS. Near infrared analysis was utilized to explore the hygroscopicity of the material and to perform principle component analysis. The modification to butylated grades decreased the hygroscopicity, whereas the hydroxypropylated grades bound moisture. All of the hemicellulose ethers were water-soluble. The interfacial tension of the aqueous hemicellulose solutions was determined by pendant-drop tensiometer, and it was demonstrated to be dependent on the degree of modification.
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Polisacáridos/química , Agua/química , Madera/química , Xilanos/química , Cromatografía en Gel , Tensión Superficial , HumectabilidadRESUMEN
A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC(50) values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.
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Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Bibliotecas de Moléculas Pequeñas , Espectrometría de Fluorescencia , Proteína p53 Supresora de Tumor/metabolismo , Unión Competitiva/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Unión Proteica/efectos de los fármacosRESUMEN
PURPOSE: The simultaneous measurement of PET and magnetic resonance imaging (MRI) is an emerging field for molecular imaging research. Although optical fiber based PET∕MRI systems have advantages on less interference between PET and MRI, there is a drawback in reducing the scintillation light due to the fiber. To reduce the problem, the authors newly developed flexible optical fiber bundle based block detectors and employed them for a high resolution integrated PET∕MRI system. METHODS: The flexible optical fiber bundle used 0.5 mm diameter, 80 cm long double clad fibers which have dual 12 mm × 24 mm rectangular inputs and a single 24 mm × 24 mm rectangular output. In the input surface, LGSO scintillators of 0.025 mol.% (decay time: â¼31 ns: 0.9 mm × 1.3 mm × 5 mm) and 0.75 mol.% (decay time: â¼46 ns: 0.9 mm × 1.3 mm × 6 mm) were optically coupled in depth direction to form depth-of-interaction detector, arranged in 11 × 13 matrix and optically coupled to the fiber bundle. The two inputs of the bundle are bent for 90°, bound to one, and are optically coupled to a Hamamatsu 1-in. square position sensitive photomultiplier tube. RESULTS: Light loss due to the fiber bundle could be reduced and the performance of the block detectors was improved. Eight optical fiber based block detectors (16 LGSO blocks) were arranged in a 56 mm diameter ring to form a PET system. Spatial resolution and sensitivity were 1.2 mm full-width at half-maximum and 1.2% at the central field-of-view, respectively. Sensitivity change was less than 1% for 2 °C temperature changes. This PET system was integrated with a 0.3 T permanent magnet MRI system which has 17 cm diameter hole at the yoke area for insertion of the PET detector ring. There was no observable interference between PET and MRI. Simultaneous imaging of PET and MRI was successfully performed for small animal studies. CONCLUSIONS: The authors confirmed that the developed high resolution PET∕MRI system is promising for molecular imaging research.
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Imagen por Resonancia Magnética/instrumentación , Fibras Ópticas , Tomografía de Emisión de Positrones/instrumentación , Animales , Encéfalo/diagnóstico por imagen , Equipos y Suministros Eléctricos , Corazón/diagnóstico por imagen , Ratones , Ratas , Factores de TiempoRESUMEN
OBJECTIVE: We recently developed an integrated positron emission tomography (PET)/magnetic resonance imaging (MRI) (iPET/MRI) scanner for small animals, which had relatively large field-of-view (FOV) covering up to the size of a rat body. The purpose of this study was to report results of simultaneous PET/MRI of a rat body using this scanner with some radiotracers. METHODS: C-11-methionine (MET), F-18-fluorodeoxyglucose (FDG), or F-18-sodium fluoride (NaF) was injected as a radiotracer for PET portion in addition to gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid, a hepatobiliary contrast agent, for MRI portion. Simultaneous PET/MRI was performed in normal rats. PET, MRI, and co-registered fusion images were evaluated regarding image quality and feasibility for rat imaging studies. RESULTS: MET uptake was clearly shown in the liver and pancreas, which was confirmed with magnetic resonance (MR) and fused PET/MR images. PET/MR images depicted intense FDG uptake in the brain, Harderian glands, and myocardium. NaF uptake was observed in all bones and joints within FOV, except in ribs, which was well recognized with the help of MR and fused PET/MR images. CONCLUSION: This study demonstrated that simultaneous PET/MRI with an integrated dual-modality molecular imaging scanner was a feasible technique for imaging studies targeting on a rat body. However, further developments including attenuation correction methods are required to use this technique routinely in rat imaging studies.
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Imagen por Resonancia Magnética/métodos , Imagen Multimodal/métodos , Tomografía de Emisión de Positrones/métodos , Animales , Fluorodesoxiglucosa F18 , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética/instrumentación , Masculino , Metionina , Imagen Multimodal/instrumentación , Tomografía de Emisión de Positrones/instrumentación , Ratas , Ratas Wistar , Fluoruro de Sodio , Factores de TiempoRESUMEN
The silicon photomultiplier (Si-PM) is a promising photo-detector for PET for use in magnetic resonance imaging (MRI) systems because it has high gain and is insensitive to static magnetic fields. Recently we developed a Si-PM-based depth-of-interaction PET system for small animals and performed simultaneous measurements by combining the Si-PM-based PET and the 0.15 T permanent MRI to test the interferences between the Si-PM-based PET and an MRI. When the Si-PM was inside the MRI and installed around the radio frequency (RF) coil of the MRI, significant noise from the RF sequence of the MRI was observed in the analog signals of the PET detectors. However, we did not observe any artifacts in the PET images; fluctuation increased in the count rate of the Si-PM-based PET system. On the MRI side, there was significant degradation of the signal-to-noise ratio (S/N) in the MRI images compared with those without PET. By applying noise reduction procedures, the degradation of the S/N was reduced. With this condition, simultaneous measurements of a rat brain using a Si-PM-based PET and an MRI were made with some degradation in the MRI images. We conclude that simultaneous measurements are possible using Si-PM-based PET and MRI.
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Imagen por Resonancia Magnética/instrumentación , Tomografía de Emisión de Positrones/instrumentación , Silicio , Integración de Sistemas , Animales , Encéfalo/diagnóstico por imagen , Fantasmas de Imagen , Ratas , Factores de TiempoRESUMEN
The silicon-photomultiplier (Si-PM) is a promising photodetector, especially for integrated PET/MRI systems, due to its small size, high gain, and low sensitivity to static magnetic fields. The major problem using a Si-PM-based PET system within the MRI system is the interference between the PET and MRI units. We measured the interference by combining a Si-PM-based PET system with a permanent-magnet MRI system. When the RF signal-induced pulse height exceeded the lower energy threshold level of the PET system, interference between the Si-PM-based PET system and MRI system was detected. The prompt as well as the delayed coincidence count rates of the Si-PM-based PET system increased significantly. These noise counts produced severe artifacts on the reconstructed images of the Si-PM-based PET system. In terms of the effect of the Si-PM-based PET system on the MRI system, although no susceptibility artifact was observed on the MR images, electronic noise from the PET detector ring was detected by the RF coil and reduced the signal-to-noise ratio (S/N) of the MR images. The S/N degradation of the MR images was reduced when the distance between the RF coil and the Si-PM-based PET system was increased. We conclude that reducing the interference between the PET and MRI systems is essential for achieving the optimum performance of integrated Si-PM PET/MRI systems.
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Luz , Imagen por Resonancia Magnética/instrumentación , Tomografía de Emisión de Positrones/instrumentación , Silicio , Artefactos , Fantasmas de ImagenRESUMEN
The zinc finger CW (zf-CW) domain is a motif of about 60 residues that is frequently found in proteins involved in epigenetic regulation. Here, we determined the NMR solution structure of the zf-CW domain of the human zf-CW and PWWP domain containing protein 1 (ZCWPW1). The zf-CW domain adopts a new fold in which a zinc ion is coordinated tetrahedrally by four conserved Cys ligand residues. The tertiary structure of the zf-CW domain partially resembles that adopted by the plant homeo domain (PHD) finger bound to the histone tail, suggesting that the zf-CW domain and the PHD finger have similar functions. The solution structure of the complex of the zf-CW domain with the histone H3 tail peptide (1-10) with trimethylated K4 clarified its binding mode. Our structural and biochemical studies have identified the zf-CW domain as a member of the histone modification reader modules for epigenetic regulation.
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Histonas/química , Dedos de Zinc , Secuencia de Aminoácidos , Sitios de Unión , Epigénesis Genética , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de ProteínaRESUMEN
Artifacts in MR(Magnetic Resonance) images of oral cavity produced from non-magnetic metal restorations was verified by measuring the image of index finger and a cylinder of fat test piece with a type 4 gold alloy ring using a compact MRI equipment. In the images of finger, portion around the ring disappeared. However, it was nearly restored with a cut ring. In the cylinder of fat test piece, obvious artifacts appeared when circumferential surface of the ring was placed perpendicular to RF(Radio Frequency) field of MRI equipment's excitation/detection coil. However, in other directions or with a cut ring, artifact disappeared. The cause was simulated with FEM(Finite Element Method) electromagnetic field analysis, and alternating magnetic field was shown to induce surface current on the continuous gold ring. Magnetic field produced by that current interfered with the field from excitation coil. This demonstrated the characteristics and cause of artifacts by non-magnetic dental metals.
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Artefactos , Aleaciones Dentales , Imagen por Resonancia Magnética , Tejido Adiposo , Campos Electromagnéticos , Análisis de Elementos Finitos , Aleaciones de Oro , Humanos , Ondas de RadioRESUMEN
OBJECTIVE: Although simultaneous measurements of PET and magnetic resonance imaging (MRI) can provide interesting results in molecular imaging research, most of the combined systems are huge and animal handling in the system is not easy. To minimize these problems, we developed a compact integrated PET/MRI (iPET/MRI) system for small animals. METHODS: For the iPET/MRI system, a new MR-compatible PET and a permanent magnet open MRI were designed. In the MRI, a tunnel is opened at the yoke of the magnet. The position-sensitive photo-multiplier tubes (PSPMTs) of the MR-compatible PET are positioned at the back of the yoke where the magnetic field is sufficiently low. The scintillators for the PET system are positioned at the center of the MRI magnets, and the direction of the scintillation photons is changed by slanted light guides, and they are fed to the PSPMTs by 75 cm long optical fiber bundles. The PET detectors employed two types of LGSO crystals (1.9 mm x 2.2 mm x 6 mm and 7 mm) with different decay times (33 and 43 ns) for depth of interaction detection. Sixteen optical fiber-based block detectors are arranged in a 112 mm diameter ring. RESULTS: The transaxial field-of-view (FOV) of the PET system is ~80 mm, and the axial FOV is 21 mm which can be enlarged by the axial motion of the PET detector ring during MRI acquisition. The transaxial and axial resolutions at the center of the PET system was 2.9 and 2.4 mm FWHM, respectively. The absolute sensitivity was 1.5% at the center of the axial FOV. Phantom images revealed no artifact in either the PET or MRI images. We successfully obtained simultaneously measured small animal images using the iPET/MRI system. CONCLUSION: The open geometry of the developed iPET/ MRI facilitates easy accessibility to the subject. The iPET/ MRI system appears to be a promising tool for molecular imaging research.
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Imagen por Resonancia Magnética/instrumentación , Tomografía de Emisión de Positrones/instrumentación , Integración de Sistemas , Animales , Diseño de Equipo , Masculino , Fantasmas de Imagen , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14h, including 8h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).
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Automatización de Laboratorios/instrumentación , Sistema Libre de Células , Diálisis/instrumentación , Biosíntesis de Proteínas , Proteínas/aislamiento & purificación , Cromatografía de Afinidad/instrumentación , Cromatografía de Afinidad/métodos , Diálisis/métodos , Electroforesis en Gel de Poliacrilamida , Diseño de Equipo , Histidina , Oligopéptidos , Proteínas/genéticaRESUMEN
PURPOSE: We developed a new type of scintillation detector ring for positron emission tomography (PET). In this device the scintillation detectors were connected with 2.6 m length optic fibers to transport scintillation light to a photomultiplier (PMT) located apart from the detector rings. The present study aimed to test the possibility of simultaneous imaging with PET and magnetic resonance imaging (MRI) by means of the present PET device in ex vivo rat brain. MATERIALS AND METHODS: The scintillation detector ring of 4 cm diameter was located in a magnetic field of 0.15 T open MRI. Simultaneous measurements of PET and MRI were performed in ex vivo rat brain after injection of (18)F-fluorodeoxyglucose ((18)F-FDG) 37 MBq and (18)F-NaF 37 MBq. Simultaneous data acquisition was performed for 10 min for PET and 5 min for T1-weighted MRI. RESULTS: Simultaneous imaging of PET and MRI was possible without noticeable image distortion in the PET and MRI scans. In the fusion images, high uptake of (18)F-FDG was identified in the Harderian glands and striatum. High uptake of (18)F-NaF was found in the skull base and vault. CONCLUSIONS: The present study indicated the possibility of simultaneous imaging of PET and MRI by means of optic fiber-based scintillation detectors.
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Mapeo Encefálico/métodos , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Imagen por Resonancia Magnética/métodos , Fibras Ópticas , Tomografía de Emisión de Positrones/métodos , Animales , Fluorodesoxiglucosa F18/administración & dosificación , Procesamiento de Imagen Asistido por Computador/métodos , Radiofármacos/administración & dosificación , RatasRESUMEN
A compact magnetic resonance imaging (MRI) system for a cold (-5 degrees C) room has been developed to acquire MR images below the freezing point of water. The MRI system consists of a 1.0 T permanent magnet, a higher-order shim coil set, and a gradient coil probe, installed in the cold room, and a compact MRI console installed in a room at normal temperature (20-25 degrees C). The most difficult problem for the installation of the MRI system in the cold room was the degradation of the field homogeneity of the permanent magnet shimmed at 25 degrees C. To overcome this problem, higher-order shim coils were developed and the temperature variation of the magnetic field distribution was measured using a standard phantom with and without shim coil currents. As a result, it was confirmed that the homogeneity (the difference between the minimum and maximum values) of the magnetic field in the 17x17x19 mm(3) rectangular parallelepiped region was improved from 117 to 59 ppm using an appropriate combination of shim coil currents. A snowpack immersed in dodecane (C(12)H(26)) was imaged using a driven-equilibrium three-dimensional (3D) spin-echo sequence at -5 degrees C. The visualized 3D structure of the snowpack demonstrated the effectiveness of our approach.
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The muscleblind-like (MBNL) proteins 1, 2, and 3, which contain four CCCH zinc finger motifs (ZF1-4), are involved in the differentiation of muscle inclusion by controlling the splicing patterns of several pre-mRNAs. Especially, MBNL1 plays a crucial role in myotonic dystrophy. The CCCH zinc finger is a sequence motif found in many RNA binding proteins and is suggested to play an important role in the recognition of RNA molecules. Here, we solved the solution structures of both tandem zinc finger (TZF) motifs, TZF12 (comprising ZF1 and ZF2) and TZF34 (ZF3 and ZF4), in MBNL2 from Homo sapiens. In TZF12 of MBNL2, ZF1 and ZF2 adopt a similar fold, as reported previously for the CCCH-type zinc fingers in the TIS11d protein. The linker between ZF1 and ZF2 in MBNL2 forms an antiparallel beta-sheet with the N-terminal extension of ZF1. Furthermore, ZF1 and ZF2 in MBNL2 interact with each other through hydrophobic interactions. Consequently, TZF12 forms a single, compact global fold, where ZF1 and ZF2 are approximately symmetrical about the C2 axis. The structure of the second tandem zinc finger (TZF34) in MBNL2 is similar to that of TZF12. This novel three-dimensional structure of the TZF domains in MBNL2 provides a basis for functional studies of the CCCH-type zinc finger motifs in the MBNL protein family.
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ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Análisis de Secuencia de ProteínaRESUMEN
Ferrimagnetic materials can be expected to be useful as thermo seeds for hyperthermic treatment of cancer, especially where the cancer is located in deep parts of body, as they can generate heat by magnetic hysteretic loss when they are placed in an alternating magnetic field. Recently, it was reported that ferrimagnetic maghemite (gamma-Fe2O3) microspheres 20-30 microm in diameter prepared in aqueous solution can show excellent heat generating ability. However, these microspheres have many cracks on their surfaces. In this study, the preparation conditions for the microspheres was further optimized in order to obtain crack-free ferrimagnetic microspheres, and the in vitro heat generation of the obtained microspheres was measured in an agar phantom under an alternating magnetic field. Crack-free gamma-Fe2O3 microspheres 20-30 microm in diameter were obtained successfully. Their saturation magnetization and coercive force were 68 emu g(-1) and 198 Oe, respectively. Their heat generation under an alternating magnetic field of 300 Oe at 100 kHz was estimated to be 42 W g(-1). The microspheres showed in vitro heat generation when they were dispersed in an agar phantom and placed under an alternating magnetic field. It is believed that these microspheres may be useful for the in situ hyperthermic treatment of cancer.
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Compuestos Férricos/química , Hipertermia Inducida , Magnetismo , Microesferas , Neoplasias/terapia , Agar , Campos Electromagnéticos , Diseño de Equipo , Calor , Humanos , Ensayo de Materiales , Modelos Estadísticos , Fantasmas de Imagen , Factores de Tiempo , Agua/químicaRESUMEN
The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 A for the structured residues 12-48. The fold consists of two consecutive antiparallel beta-sheets and two short C-terminal helices packed against the second beta-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain.
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Fosfoproteínas/química , Dedos de Zinc , Secuencia de Aminoácidos , Secuencia de Consenso , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones/química , Propiedades de Superficie , Zinc/química , Zinc/metabolismoRESUMEN
The general transcription factor TFII-I, with the corresponding gene name GTF2I, is an unusual transcriptional regulator that associates with both basal and signal-induced transcription factors. TFII-I consists of six GTF2I repeat domains, called I-repeats R1-R6. The structure and function of the GTF2I domain are not clearly understood, even though it contains a helix-loop-helix motif, which is considered to be the protein-protein interaction area, based on biochemical analyses. Here, we report the solution structure of the fifth repeat of the six GTF2I repeat domains from murine TFII-I, which was determined by heteronuclear multidimensional NMR spectroscopy (PDB code 1Q60). The three-dimensional structure of the GTF2I domain is classified as a new fold, consisting of four helices (residues 8-24, 34-39, 63-71, and 83-91), two antiparallel beta strands (residues 44-47 and 77-80), and a well-defined loop containing two beta-turns between sheet 1 and helix 3. All of the repeats probably have similar folds to that of repeat 5, because the conserved residues in the GTF2I repeat domains are assembled on the hydrophobic core, turns, and secondary structure elements, as revealed by a comparison of the sequences of the first through the sixth GTF2I repeats in TFII-I.
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Factores de Transcripción TFII/química , Secuencia de Aminoácidos , Animales , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones , Factores de Transcripción TFII/genéticaRESUMEN
SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.
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Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , ADN/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones , Relación Estructura-Actividad , Transactivadores , Proteasas Ubiquitina-EspecíficasRESUMEN
Cell-free protein synthesis has become one of the standard methods for protein expression. The cell-free method is suitable for the synthesis of a protein that requires a ligand for its enzymatic activity and/or structure formation and stabilization, since it is an open system, which allows us to add the proper ligand to the reaction mixture. A large number of proteins that require zinc for their function are involved in diverse cellular processes, including transcription, DNA replication, metabolism, and cell signaling. In this study, we analyzed the effects of zinc on the cell-free synthesis of plant-specific zinc-binding transcription factors. The solubility and/or stability of the proteins were significantly increased in the presence of the proper concentration of zinc during the cell-free reaction. NMR analyses confirmed that correctly folded proteins were synthesized by the cell-free method. These results indicate that the cell-free method can be used to synthesize correctly folded and functional zinc-binding proteins.
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Proteínas de Arabidopsis/biosíntesis , Genómica/métodos , Proteínas de Plantas/biosíntesis , Zinc/farmacología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sistema Libre de Células , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/química , Cloranfenicol O-Acetiltransferasa/genética , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Isótopos de Nitrógeno/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Represoras/biosíntesis , Proteínas Represoras/química , Proteínas Represoras/genética , Solubilidad , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
UNC5Hs (UNC5H1-4) are netrin 1 receptors that are involved in axonal guidance and neuronal migration. They are dependence receptors that mediate apoptosis in the absence of netrin 1. UNC5H2-induced apoptosis depends on the interaction of the death domain at the C-terminus with the DAP-kinase death domain and caspase cleavage near the transmembrane region. Here, the crystal structure of the mouse UNC5H2 death domain has been determined at 2.1 A resolution. The domain adopts a six-helix bundle fold, which is similar to those of the other members of the death-domain superfamily. The UNC5H2 death domain is a dimer in the crystal and in solution. This homodimerized structure may represent the structure of the death domain when netrin 1 binds to the UNC5H2 receptor. Homodimerization of UNC5H2 may block the access of caspase to the cleavage site. In the death-domain dimer, residues in alpha3 and the 3(10)-helix preceding alpha3 and the residues in alpha4 make significant contacts, mainly by hydrophobic and van der Waals interactions.