RESUMEN
The distribution of defects and dislocations in graphene layers has become a very important concern with regard to the electrical and electronic transport properties of device applications. Although several experiments have shown the influence of defects on the electrical properties of graphene, these studies were limited to measuring microscopic areas because of their long measurement times. Here, we successfully imaged various local defects in a large area of chemical vapor deposition graphene within a reasonable amount of time by using lock-in thermography (LIT). The differences in electrical resistance caused by the micrometer-scale defects, such as cracks and wrinkles, and atomic-scale domain boundaries were apparent as nonuniform Joule heating on polycrystalline and epitaxially grown graphene. The present results indicate that LIT can serve as a fast and effective method of evaluating the quality and uniformity of large graphene films for device applications.
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Jak1/2 inhibitor ruxolitinib is a promising agent for treating steroid-refractory GvHD after allogeneic hematopoietic stem cell transplantation (SCT) to produce quick and durable responses. However, optimal dose and tapering schedule of ruxolitinib remain to be determined. Discontinuation of ruxolitinib in myelofibrosis often induces 'withdrawal syndrome' characterized by acute relapse of the disease, but this issue is not well addressed in the treatment of GvHD. Four patients with GvHD (one acute and three chronic) after SCT for myelofibrosis were treated with ruxolitinib. Low-dose ruxolitinib at 5 mg/day was safe and effective, but one of two patients treated at 10 mg/day of ruxolitinib was complicated with severe cytopenia. Withdrawal syndrome developed in one patient, who died of recurrence of GvHD shortly after discontinuation of ruxolitinib. Slow tapering or maintenance with low-dose ruxolitinib inhibited GvHD flare. Our experience calls attention that initiation at low-dose of ruxolitinib may be safe and careful tapering schedule is required to avoid withdrawal syndrome in patients with GvHD after SCT for myelofibrosis.
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Enfermedad Injerto contra Huésped/tratamiento farmacológico , Mielofibrosis Primaria/terapia , Pirazoles/administración & dosificación , Adulto , Anciano , Femenino , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Quinasas Janus/antagonistas & inhibidores , Persona de Mediana Edad , Nitrilos , Mielofibrosis Primaria/complicaciones , Pirazoles/efectos adversos , Pirimidinas , Síndrome de Abstinencia a Sustancias/prevención & control , Resultado del TratamientoRESUMEN
The small spin-orbit interaction of carbon atoms in graphene promises a long spin diffusion length and the potential to create a spin field-effect transistor. However, for this reason, graphene was largely overlooked as a possible spin-charge conversion material. We report electric gate tuning of the spin-charge conversion voltage signal in single-layer graphene. Using spin pumping from an yttrium iron garnet ferrimagnetic insulator and ionic liquid top gate, we determined that the inverse spin Hall effect is the dominant spin-charge conversion mechanism in single-layer graphene. From the gate dependence of the electromotive force we showed the dominance of the intrinsic over Rashba spin-orbit interaction, a long-standing question in graphene research.
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Efficacy of reduced-intensity stem-cell transplantation (RIST) for acute lymphoblastic leukemia (ALL) was investigated in 33 patients (median age, 55 years). RIST sources comprised 20 HLA-identical related donors, five HLA-mismatched related, and eight unrelated donors. Six patients had undergone previous transplantation. Disease status at RIST was first remission (n=13), second remission (n=6), and induction failure or relapse (n=14). All patients tolerated preparatory regimens and achieved neutrophil engraftment (median, day 12.5). Acute and chronic graft-versus-host disease (GVHD) developed in 45 and 64%, respectively. Six patients received donor lymphocyte infusion (DLI), for prophylaxis (n=1) or treatment of recurrent ALL (n=5). Nine patients died of transplant-related mortality, with six deaths due to GVHD. The median follow-up of surviving patients was 11.6 months (range, 3.5-37.3 months). The 1-year relapse-free and overall survival rates were 29.8 and 39.6%, respectively. Of the 14 patients transplanted in relapse, five remained relapse free for longer than 6 months. Cumulative rates of progression and progression-free mortality at 3 years were 50.9 and 30.4%, respectively. These findings suggest the presence of a graft-versus-leukemia effect for ALL. RIST for ALL is worth considering for further evaluation.
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Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adolescente , Adulto , Anciano , Femenino , Supervivencia de Injerto , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Estudios Retrospectivos , Factores de Riesgo , Análisis de Supervivencia , Acondicionamiento Pretrasplante/métodos , Resultado del TratamientoRESUMEN
This study concerns a patient with minor (m)-BCR/ABL transcript-positive and Philadelphia (Ph) chromosome-negative myelodysplastic syndrome (MDS). The patient was a 78-year-old man whose condition was diagnosed as refractory anemia with excess of blasts in transformation. Molecular genetic studies, using reverse transcriptase polymerase chain reaction analysis detected m-BCR/ABL messenger RNA. We used spectral karyotyping to analyze metaphase cells but could not detect a Ph chromosome. Fluorescence in situ hybridization, however, revealed fusion signals of BCR and ABL probes on an apparently normal chromosome 22.
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Anemia Refractaria con Exceso de Blastos/clasificación , Proteínas de Fusión bcr-abl/genética , Anciano , Anemia Refractaria con Exceso de Blastos/sangre , Anemia Refractaria con Exceso de Blastos/diagnóstico , Anemia Refractaria con Exceso de Blastos/genética , Anemia Refractaria con Exceso de Blastos/patología , Médula Ósea/patología , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 17/ultraestructura , Cromosomas Humanos Par 22/genética , Cromosomas Humanos Par 9/genética , Resultado Fatal , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Cariotipificación , Masculino , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Translocación GenéticaAsunto(s)
Hepacivirus/enzimología , Hepacivirus/genética , ARN Polimerasa Dependiente del ARN , Antivirales , Diseño de Fármacos , Genoma Viral , Hepatitis C/tratamiento farmacológico , Conformación Proteica , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/fisiología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/fisiología , Proteínas ViralesRESUMEN
In order to confirm the effect of interferon-α (IFN-α) in inducing a prolonged duration of the chronic phase (CP) on patients with chronic myelogenous leukemia (CML), we retrospectively compared the duration of CP between patients who continued on IFN-α and the patients in whom IFN-α was discontinued before the blast phase. Of the 32 patients not pretreated for CML in the early CP who received IFN-α therapy, 25 continued on IFN-α while seven discontinued the therapy (side effects, 5; resistance, 1; patient's refusal, 1). Only four of the 25 patients in whom IFN-α was continued (16.0%) progressed to the blast phase or accelerated phase, but six of the seven patients who discontinued IFN-α (85.7%) progressed to the blast phase or accelerated phase. Fourteen of the 25 patients who continued on IFN-α therapy showed cytogenetic response (complete cytogenetic response, 3; minimal cytogenetic response, 11) whereas 11 patients showed no cytogenetic response. However, non-responders showed a longer duration of CP than the patients whom IFN-α was discontinued. Although elderly patients showed a high incidence of side effects, and some patients progressed early after the beginning of IFN-α therapy, our data clearly demonstrated that in accordance with previous large multi-centric randomized studies the continuation of IFN-α, even in low doses, prevents disease progression.
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BACKGROUND: Hepatitis C virus (HCV) is the major etiological agent of hepatocellular carcinoma, and HCV RNA-dependent RNA polymerase (RdRp) is one of the main potential targets for anti-HCV agents. HCV RdRp performs run-off copying replication in an RNA-selective manner for the template-primer duplex and the substrate, but the structural basis of this reaction mechanism has still to be elucidated. RESULTS: The three-dimensional structure of HCV RdRp was determined by X-ray crystallography at 2.5 A resolution. The compact HCV RdRp structure resembles a right hand, but has more complicated fingers and thumb domains than those of the other known polymerases, with a novel alpha-helix-rich subdomain (alpha fingers) as an addition to the fingers domain. The other fingers subdomain (beta fingers) is folded in the same manner as the fingers domain of human immunodeficiency virus (HIV) reverse transcriptase (RT), another RNA-dependent polymerase. The ribose-recognition site of HCV RdRp is constructed of hydrophilic residues, unlike those of DNA polymerases. The C-terminal region of HCV RdRp occupies the putative RNA-duplex-binding cleft. CONCLUSIONS: The structural basis of the RNA selectivity of HCV RdRp was elucidated from its crystal structure. The putative substrate-binding site with a shallow hydrophilic cavity should have ribonucleoside triphosphate (rNTP) as the preferred substrate. We propose that the unique alpha fingers might represent a common structural discriminator of the template-primer duplex that distinguishes between RNA and DNA during the replication of positive single-stranded RNA by viral RdRps. The C-terminal region might exert a regulatory function on the initiation and activity of HCV RdRp.
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ARN Polimerasas Dirigidas por ADN/química , Hepatitis C/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Moldes GenéticosRESUMEN
Hematopoietic prostaglandin (PG) D synthase is the key enzyme for production of the D and J series of prostanoids in the immune system and mast cells. We isolated a cDNA for the rat enzyme, crystallized the recombinant enzyme, and determined the three-dimensional structure of the enzyme complexed with glutathione at 2.3 A resolution. The enzyme is the first member of the sigma class glutathione S-transferase (GST) from vertebrates and possesses a prominent cleft as the active site, which is never seen among other members of the GST family. The unique 3-D architecture of the cleft leads to the putative substrate binding mode and its catalytic mechanism, responsible for the specific isomerization from PGH2 to PGD2.
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Oxidorreductasas Intramoleculares , Isomerasas/química , Isomerasas/genética , Animales , Sitios de Unión/fisiología , Clonación Molecular , Cristalografía , ADN Complementario , Epoprostenol/metabolismo , Regulación Enzimológica de la Expresión Génica , Hematopoyesis/fisiología , Isomerasas/metabolismo , Isomerismo , Lipocalinas , Datos de Secuencia Molecular , Prostaglandina D2/química , Prostaglandina D2/metabolismo , Prostaglandina H2 , Prostaglandinas H/química , Prostaglandinas H/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tromboxano A2/metabolismoRESUMEN
The crystal structure of recombinant human annexin V complexed with K-201, an inhibitor of the calcium ion channel activity of annexin V, was solved at 3.0 A by molecular replacement including the apo and high-calcium forms. K-201 was bound at the hinge region cavity formed by the N-terminal strand and domains II, III and IV, at the side opposite the calcium and membrane-binding surface, in an L-shaped conformation. Based on the complex and other annexin structures, K-201 is proposed to restrain the hinge movement of annexin V in an allosteric manner, resulting in the inhibition of calcium movement across the annexin V molecule.
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Anexina A5/química , Bloqueadores de los Canales de Calcio/química , Canales de Calcio/efectos de los fármacos , Tiazepinas/química , Anexina A5/genética , Anexina A5/metabolismo , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , Tiazepinas/metabolismoRESUMEN
We have determined the crystal structure of alpha-pokeweed antiviral protein, a member of ribosome-inactivating proteins, at 0.23 nm resolution, by the molecular-replacement method. The crystals belong to the space group P2(1)2(1)2 with unit-cell dimensions a = 4.71, b = 11.63 and c = 4.96 nm, and contain one protein molecule/asymmetric unit based on a crystal volume/unit protein molecular mass of 2.1 x 10(-3) nm3/Da. The crystallographic residual value was reduced to 17.2% (0.6-0.23 nm resolution) with root-mean-square deviations in bond lengths of 1.9 pm and bond angles of 2.2 degrees. The C alpha-C alpha distance map shows that alpha-pokeweed antiviral protein is composed of three modules, the N-terminal (Ala1-Leu76), the central (Tyr77-Lys185) and the C-terminal (Tyr186-Thr266) modules. The substrate-binding site is formed as a cleft between the central and C-terminal modules and all the active residues exist on the central module. The electrostatic potential around the substrate-binding site shows that the central and C-terminal module sides of this cleft have a negatively and a positively charged region, respectively. This charge distribution in the protein seems to provide a suitable interaction with the substrate rRNA.
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Antivirales/química , N-Glicosil Hidrolasas , Proteínas de Plantas/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1 , Homología de Secuencia de AminoácidoRESUMEN
Mirabilis anti-viral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F which exhibits a lower (1/100) activity against E. coli ribosomes while retaining almost full activity against mammalian cells [Habuka, Miyano, Kataoka, Tsuge & Noma (1992). J. Biol. Chem. 267, 7758-7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F MAP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the native MAP crystals [Miyano et al. (1992). J. Mol. Biol. 226, 281-283]: 50% ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3(1)21 (or P3(2)21) with a = b = 104.1 and c = 134.3 A. The crystals are isomorphous with the wild-type crystals but diffract to higher resolution. Imaging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.
RESUMEN
Recombinant human leukotriene A4 hydrolase complexed with bestatin, an inhibitor of metalloprotease, has been crystallized by the hanging drop vapor diffusion method using 0.1 M phosphate buffer (pH 6.5) and 50 to 54% saturated ammonium sulfate. The orthorhombic crystals belong to the space group I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 273.6 A, b = 261.3 A and c = 52.9 A. They diffract beyond 2.5 A resolution and a native data set up to 3 A resolution has been collected on an imaging plate Weissenberg camera using synchrotron radiation.
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Epóxido Hidrolasas/química , Leucina/análogos & derivados , Inhibidores de Proteasas/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Humanos , Leucina/química , Datos de Secuencia Molecular , Estructura Molecular , Proteínas Recombinantes/químicaRESUMEN
A genomic gene of tritin, a ribosome-inactivating protein (RIP) from Triticum aestivum, was cloned using a barley RIP gene as a probe. The 5'-non-coding region has potential TATA boxes and three sequences homologous to the binding sequence of the transcriptional activator protein Opaque-2 which activates maize RIP gene expression. The cloned DNA encoded tritin consists of 275 amino acids with no secretion signal sequence. The coding region of tritin was expressed in Escherichia coli using lac promoter and yielded a protein similar to the native one, as determined by SDS-polyacrylamide gel electrophoresis and immunological analysis.
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Genes de Plantas/genética , Proteínas de Plantas/genética , Inhibidores de la Síntesis de la Proteína , Triticum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Inactivadoras de Ribosomas Tipo 1 , Homología de Secuencia de AminoácidoRESUMEN
We examined 60 patients with various chronic liver diseases for cellular sensitivity to native human type I and IV collagens using an in vitro leucocyte migration inhibition test. Mononuclear cells from 7 (33%) of 21 patients with chronic active hepatitis, 14 (52%) of 27 patients with liver cirrhosis and 11 (92%) of 12 patients with primary biliary cirrhosis exhibited cellular sensitivity to type IV collagen, although cells from almost all patients responded to type I collagen. None except one for type I collagen of 25 normal controls showed positive response to both collagens. In chronic active hepatitis and liver cirrhosis, cellular sensitivity to type IV collagen was significantly lower than to type I collagen (p < 0.01). Patients with primary biliary cirrhosis showed significantly higher cellular sensitivity to type IV collagen when compared to patients with other chronic liver diseases (p < 0.01). Cellular sensitivity to type IV collagen was significantly correlated with serum levels of the 7S domain of type IV collagen in all 85 subjects (r = +0.462, p < 0.001). These findings suggest that cellular sensitivity to type IV collagen as well as to type I collagen exists in chronic liver disease, especially in primary biliary cirrhosis, and may reflect the accelerated metabolism of the perisinusoidal and peribiliary basement membranes.
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Colágeno/farmacología , Cirrosis Hepática Biliar/tratamiento farmacológico , Adulto , Anciano , Inhibición de Migración Celular , Femenino , Hepatitis Crónica/sangre , Hepatitis Crónica/tratamiento farmacológico , Humanos , Factores Inhibidores de la Migración de Leucocitos/farmacología , Cirrosis Hepática/sangre , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/patología , Masculino , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
Two expression vectors were constructed to produce a putative mature alpha-pokeweed antiviral protein (alpha-PAP) in Escherichia coli with its NH2- and COOH-terminal extrapeptides excised. One was for its intracellular expression with a methionine at its NH2-terminal. The other was for its secretion using an ompA signal peptide. The former product was purified from the total soluble proteins of the transformant with a yield of 1.74 mg/liter and the latter had a yield of 5.55 mg/liter. Both products exhibited RNA N-glycosidase activity on wheat ribosomes and inhibitory activity to protein synthesis in a rabbit reticulocyte system.
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Antivirales , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Antivirales/química , Antivirales/metabolismo , Secuencia de Bases , Cromatografía en Gel , Clonación Molecular , ADN de Cadena Simple , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Inactivadoras de Ribosomas , Proteínas Inactivadoras de Ribosomas Tipo 1RESUMEN
Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of crystallographic quality were grown in 20-microliters droplets of an equi-volume mixture of 1.5% (w/v) protein solution and a reservoir solution containing 49 to 50% (w/v) ammonium sulfate and 50 mM-ammonium citrate (pH 5.4) at room temperature. Addition of 2 mM-adenine sulfate reduced twinning and "crystal shower". The resulting trigonal crystals diffract beyond 2.5 A resolution using a rotating anode X-ray generator. The space group was determined to be P3(1)21 or P3(2)21 (a = b = 103.9.A, c = 134.6 A, alpha = beta = 90 degrees, gamma = 120 degrees) based on their precession photography of h0l and hk0 zones. There seems to be three monomers in an asymmetric unit for VM = 2.51 A3/Da.
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Antivirales/química , N-Glicosil Hidrolasas , Proteínas de Plantas/química , Plantas/química , Cristalización , Sustancias Macromoleculares , Proteínas Inactivadoras de Ribosomas Tipo 1 , Difracción de Rayos XRESUMEN
The crystal structure of a calcium binding equine lysozyme has been determined at 2.5 A resolution by means of molecular replacement. The energy minimized equine lysozyme as the starting model, was refined with the molecular dynamics program, X-PLOR, and the R factor of the current model was found to be 24% without any water molecules. The conformation of the calcium binding loop is similar to that of alpha-lactalbumin. The profiles of backbone atomic displacements throughout the lysozyme and alpha-lactalbumin superfamilies are comparable as well as their homologous tertiary structures.
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Proteínas de Unión al Calcio/análisis , Caballos/metabolismo , Leche/enzimología , Muramidasa/análisis , Secuencia de Aminoácidos , Animales , Bovinos , Cristalografía , Humanos , Datos de Secuencia MolecularRESUMEN
Mirabilis antiviral protein (MAP) is a rigid, heat-stable protein composed of 250 amino acids with an intramolecular disulfide bond. MAP inhibits the in vitro protein synthesis of rabbit reticulocyte with approximately one-thirtieth the activity of the ricin A chain, a homologous protein with no such bond (Habuka, N., Murakami, Y., Noma, M., Kudo, T., and Horikoshi, K. (1989) J. Biol. Chem. 264, 6629-6637; Habuka, N., Akiyama, K., Tsuge, H., Miyano, M., Matsumoto, T., and Noma, M. (1990) J. Biol. Chem. 265, 10988-10992). The bond is presumed to induce some structural perturbation that alters the mode of interaction with the substrate ribosome and thus lowers the activity. To confirm this hypothesis, a mutant MAP gene in which the codons of both cysteines were replaced by those of serines was constructed and expressed in Escherichia coli, and its product (C36/22OS) was purified. In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, C36/220S showed the same mobility as that of MAP reduced by 2-mercaptoethanol, whereas nonreduced MAP showed faster migration. The inhibitory activity of C36/220S was approximately 22 times higher than that of native MAP, that is the mutant had an IC50 of 0.16 nM for the protein synthesis of the rabbit reticulocyte system, whereas the native MAP had an IC50 of 3.5 nM. The results indicate that the activity of MAP is increased by the elimination of the disulfide bond, and this supports the hypothesis.