RESUMO
CeIrSn with a quasikagome Ce lattice in the hexagonal basal plane is a strongly valence fluctuating compound, as we confirm by hard x-ray photoelectron spectroscopy and inelastic neutron scattering, with a high Kondo temperature of T_{K}â¼480 K. We report a negative in-plane thermal expansion α/T below 2 K, which passes through a broad minimum near 0.75 K. Volume and a-axis magnetostriction for Bâ¥a are markedly negative at low fields and change sign before a sharp metamagnetic anomaly at 6 T. These behaviors are unexpected for Ce-based intermediate valence systems, which should feature positive expansivity. Rather they point towards antiferromagnetic correlations at very low temperatures. This is supported by muon spin relaxation measurements down to 0.1 K, which provide microscopic evidence for a broad distribution of internal magnetic fields. Comparison with isostructural CeRhSn suggests that these antiferromagnetic correlations emerging at TâªT_{K} result from geometrical frustration.
RESUMO
The heavy-fermion compound CeCu_{6-x}Au_{x} has become a model system for unconventional magnetic quantum criticality. For small Au concentrations 0≤x<0.16, the compound undergoes a structural transition from orthorhombic to monoclinic crystal symmetry at a temperature T_{s} with T_{s}â0 for x≈0.15. Antiferromagnetic order sets in close to x≈0.1. To shed light on the interplay between quantum-critical magnetic and structural fluctuations we performed neutron-scattering and thermodynamic measurements on samples with 0≤x≤0.3. The resulting phase diagram shows that the antiferromagnetic and monoclinic phase coexist in a tiny Au concentration range between x≈0.1 and 0.15. The application of hydrostatic and chemical pressure allows us to clearly separate the transitions from each other and to explore a possible effect of the structural transition on the magnetic quantum-critical behavior. Our measurements demonstrate that at low temperatures the unconventional quantum criticality exclusively arises from magnetic fluctuations and is not affected by the monoclinic distortion.
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OBJECTIVE: The aim of this study was to examine the osteoarthritis (OA)-related structural changes associated with histological synovitis in end-stage knee OA patients. METHODS: Forty end-stage knee OA patients (female: 88%, mean age: 71.8 y) were enrolled. All participants underwent 3.0-T MRI. The structural changes, such as cartilage morphology, subchondral bone marrow lesion (BML), subchondral bone cyst (SBC), subchondral bone attrition (SBA), osteophytes, meniscal lesion and synovitis, were scored using the whole-organ MRI scoring (WORMS) method. Synovial samples were obtained from five regions of interest (ROIs) of the knee joint during total joint replacement surgery. The associations between the histological synovitis score (HSS) and WORMS or the synovial expression levels of cyclooxygenase (COX)-2, interleukin (IL)-1ß, IL-6 and transforming growth factor (TGF)-ß were examined using Spearman's correlation coefficient. RESULTS: Among the seven OA-related structural changes, the BML, SBC, SBA and synovitis were significantly associated with the HSS (r = 0.33, 0.35, 0.48 and 0.36, respectively), while other morphological changes were not. Although synovial COX-2, IL-1ß or IL-6 expression levels were not associated with the HSS, the synovial TGF-ß expression levels were associated with the HSS. CONCLUSION: The presence of BML, SBC and SBA was associated with histological synovitis in end-stage knee OA patients.
Assuntos
Cistos Ósseos/patologia , Doenças da Medula Óssea/patologia , Medula Óssea/patologia , Cartilagem Articular/patologia , Imageamento por Ressonância Magnética/métodos , Osteoartrite do Joelho/patologia , Sinovite/patologia , Idoso , Cistos Ósseos/complicações , Cistos Ósseos/metabolismo , Doenças da Medula Óssea/complicações , Doenças da Medula Óssea/metabolismo , Estudos Transversais , Citocinas/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Masculino , Osteoartrite do Joelho/complicações , Osteoartrite do Joelho/metabolismo , Líquido Sinovial/metabolismo , Sinovite/etiologia , Sinovite/metabolismoRESUMO
Magnetic field (B) variation of the electrical polarization P(c) (â¥c) of the perfect triangular lattice antiferromagnet RbFe(MoO(4))(2) is examined up to the saturation point of the magnetization for Bâ¥c. P(c) is observed only in phases for which chirality is predicted in the in-plane magnetic structures. No strong anomaly is observed in P(c) at the field at which the spin modulation along the c axis, and hence the spin helicity, exhibits a discontinuity to the commensurate state. These results indicate that the ferroelectricity in this compound originates predominantly from the spin chirality, the explanation of which would require a new mechanism for magnetoferroelectricity. The obtained field-temperature phase diagram of ferroelectricity agree well with those theoretically predicted for the spin chirality of a Heisenberg spin triangular lattice antiferromagnet.
RESUMO
OBJECTIVES: Knee osteoarthritis (OA) pain is suggested to be associated with inflammation and detrimental mechanical loading across the joint. In this cross-sectional study, we simultaneously examined the inflammation and alignment of the lower limb and examined how the pain components varied depending on the disease progression. DESIGN: One-hundred sixty female medial type of early- [n = 74 in Kellgren-Lawrence (K/L) 2] to advanced-stage (n = 96 in K/L >2) knee OA subjects (70.5 years on average) were enrolled. Knee pain was evaluated using a pain visual analog scale (VAS) and the pain-related subcategory of the Japanese Knee Osteoarthritis Measure (JKOM-pain). The serum interleukin (sIL)-6 level reflecting synovitis, and the high sensitivity C-reactive protein (hs-CRP) level were measured to evaluate the severity of inflammation. The anatomical axis angle (AAA) was measured as an alignment index. The ß-coefficient was estimated after adjusting for age and the body mass index (BMI) using a multiple linear regression analysis. RESULTS: Multiple linear regression analyses showed that the sIL-6 levels, but not AAA, associated with the pain VAS [ß = 10.77 (95% confidence interval (CI): 4.14-17.40), P < 0.01] and JKOM-pain scores [ß = 3.19 (95% CI: 1.93-4.44), P < 0.001] in the early stage. Conversely, AAA, but not the sIL-6 levels, was found to be associated with the pain VAS [ß = -1.29 (95% CI: -2.51 to -0.08), P < 0.05] and JKOM-pain scores [ß = -0.49 (95% CI: -0.82 to -0.16), P < 0.01] in the advanced stage. CONCLUSIONS: The presence of a higher level of sIL-6 and the varus alignment of the joint is associated with pain in early- and advanced-stage knee OA patients, respectively.
Assuntos
Artralgia/diagnóstico por imagem , Artralgia/epidemiologia , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/epidemiologia , Índice de Gravidade de Doença , Idoso , Artralgia/fisiopatologia , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Estudos Transversais , Progressão da Doença , Feminino , Humanos , Interleucina-6/sangue , Pessoa de Meia-Idade , Osteoartrite do Joelho/fisiopatologia , Medição da Dor , Radiografia , Fatores de Risco , Sinovite/diagnóstico por imagem , Sinovite/epidemiologia , Sinovite/fisiopatologia , Suporte de Carga/fisiologiaRESUMO
We have succeeded in synthesizing single crystals of a new organic radical 3-Cl-4-F-V [3-(3-chloro-4-fluorophenyl)-1,5-diphenylverdazyl]. Through the ab initio molecular orbital calculation and the analysis of the magnetic properties, this compound was confirmed to be the first experimental realization of an S=1/2 spin-ladder system with ferromagnetic leg interactions. The field-temperature phase diagram indicated that the ground state is situated very close to the quantum critical point. Furthermore, we found an unexpected field-induced successive phase transition, which possibly originates from the interplay of low dimensionality and frustration.
RESUMO
Treatment outcomes for chronic myelogenous leukemia (CML) have shown major improvements as a result of the development of the tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib for the disease-specific molecular target BCR-ABL1 tyrosine kinase (TK), but a cure of CML by BCR-ABL1 TKIs has been rarely achieved. CML cells are protected from cytotoxic insults, including those by TKIs, through various collaborative BCR-ABL1- mediated and -independent mechanisms, as well as cell-intrinsic and -extrinsic molecular mechanisms. These protective mechanisms include overlapping cell signaling pathways for normal hematopoietic proliferation, modulation of molecules associated with the BCL2 family protein-regulated programmed cell death pathway, autophagic cell protection capability, bone marrow environment-mediated cell protective signaling, abnormally upregulated genetic instability and other BCR-ABL1- independent kinase activities. To develop a more effective treatment strategy for a cure by means of total leukemic cell killing, a thorough understanding of how CML cells survive and resist cytotoxic insults is essential. In this article, we review current knowledge about multifaceted BCR-ABL1-related and -unrelated mechanisms for survival and death of CML cells and present suggestions for the development of new therapeutic strategies for complete elimination of residual CML cells during TKI treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Terapia de Alvo Molecular , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/agonistas , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Instabilidade Cromossômica/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Fatores de Crescimento de Células Hematopoéticas/antagonistas & inibidores , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
A surface-illuminated photoconductive detector based on Ge0.91Sn0.09 quantum wells with Ge barriers grown on a silicon substrate is demonstrated. Photodetection up to 2.2µm is achieved with a responsivity of 0.1 A/W for 5V bias. The spectral absorption characteristics are analyzed as a function of the GeSn/Ge heterostructure parameters. This work demonstrates that GeSn/Ge heterostructures can be used to developed SOI waveguide integrated photodetectors for short-wave infrared applications.
Assuntos
Eletrônica/instrumentação , Germânio/química , Silício/química , Espectrofotometria Infravermelho/instrumentação , Estanho/química , Desenho de Equipamento , Raios Infravermelhos , Pontos QuânticosRESUMO
How ground states of quantum matter transform between one another reveals deep insights into the mechanisms stabilizing them. Correspondingly, quantum phase transitions are explored in numerous materials classes, with heavy-fermion compounds being among the most prominent ones. Recent studies in an anisotropic heavy-fermion compound have shown that different types of transitions are induced by variations of chemical or external pressure, raising the question of the extent to which heavy-fermion quantum criticality is universal. To make progress, it is essential to broaden both the materials basis and the microscopic parameter variety. Here, we identify a cubic heavy-fermion material as exhibiting a field-induced quantum phase transition, and show how the material can be used to explore one extreme of the dimensionality axis. The transition between two different ordered phases is accompanied by an abrupt change of Fermi surface, reminiscent of what happens across the field-induced antiferromagnetic to paramagnetic transition in the anisotropic YbRh2Si2. This finding leads to a materials-based global phase diagram--a precondition for a unified theoretical description.
Assuntos
Cério/química , Paládio/química , Anisotropia , Metais/química , Transição de Fase , Dióxido de Silício/química , Itérbio/químicaRESUMO
The transition of the bacterial community structure and predominant bacteria in the ceca of chicks from hatching to 2 wk of age was investigated using denaturing gradient gel electrophoresis with the 16S ribosomal RNA gene, followed by phylogenetic analysis. The results demonstrated that most of the cecal bacterial flora from hatching to a few days old consisted of Escherichia coli (sequence similarity: 100%), and the floral diversity was still low 2 wk posthatch. These findings will help contribute to the development of a novel competitive exclusion product.
Assuntos
Envelhecimento/fisiologia , Bactérias/classificação , Ceco/microbiologia , Galinhas , Animais , Bactérias/genética , DNA Bacteriano/genética , Masculino , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Molecularly imprinted cyclodextrins (MI-CDs) are prepared by cross-linking CDs in the presence of a template molecule. The binding ability of MI-CDs to the template molecule is specific; therefore, MI-CDs will prove to be useful materials. In this study, we prepared microspheres of MI-CDs (MSs-MI-CD) in a dimethylsulfoxide/poly(dimethylsiloxane) (PDMS) emulsion, using cholesterol as the template molecule. MSs-MI-CD were prepared under various conditions and were evaluated with respect to their morphology, size, and binding ability. MSs-MI-CD prepared at 65 degrees C were in an aggregated form; however, we could prepare separated and uniform MSs-MI-CD at 95 degrees C. The viscosity of PDMS influenced the size of MSs-MI-CD. The mean particle diameters of MSs-MI-CD prepared with 50 and 1000 mm(2)/s PDMS were 146 and 43 microm, respectively. The binding ability of MSs-MI-CD to cholesterol was higher than that of non-imprinted microspheres. Cholesterol imprinting also promoted the binding ability to other steroids; however, the increase in binding ability was most remarkable in the case of cholesterol, suggesting that we successfully introduced the cholesterol-specific binding ability into MSs-MI-CD. The novel MSs-MI-CD preparation method is useful and simple, and it will provide opportunities for further studies on the specific binding ability of MI-CDs.
Assuntos
Ciclodextrinas/síntese química , Microesferas , Colesterol/metabolismo , Ciclodextrinas/metabolismoRESUMO
Phe286 located in the center of the active site of alpha-amylase 2 from Thermoactinomyces vulgaris R-47 (TVAII) plays an important role in the substrate recognition for cyclomaltooligosaccharides (cyclodextrins). The X-ray structures of mutant TVAIIs with the replacement of Phe286 by Ala (F286A) and Tyr (F286Y) were determined at 3.2 A resolution. Their structures have no significant differences from that of the wild-type enzyme. The kinetic analyses of Phe286-replaced variants showed that the variants with non-aromatic residues, Ala (F286A) and Leu (F286L), have lower enzymatic activities than those with aromatic residues, Tyr (F286Y) and Trp (F286W), and the replacement of Phe286 affects enzymatic activities for CDs more than those for starch.
Assuntos
Substituição de Aminoácidos/genética , Ciclodextrinas/química , Micromonosporaceae/enzimologia , Micromonosporaceae/genética , Fenilalanina/química , alfa-Amilases/química , alfa-Amilases/genética , Alanina/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Cinética , Mutagênese Sítio-Dirigida , Fenilalanina/genética , Tirosina/genéticaRESUMO
Matrix metalloproteinases (MMPs) are important hydrolytic enzymes with profound physiological and pathological functions in living organisms. MMPs are produced in their inactive zymogenic forms, which are subsequently proteolytically activated in an elaborate set of events. The propeptide in the zymogen blocks the active site, with a cysteine side-chain thiolate from this propeptide achieving coordination with the catalytically important zinc ion in the active site. Molecular dynamics simulations, ab initio calculations, and wet chemistry experiments presented herein argue for the critical importance of a protonation event at the coordinated thiolate as a prerequisite for the departure of the propeptide from the active site. Furthermore, a catalytically important glutamate is shown to coordinate transiently to the active-site zinc ion to "mask" the positive potential of the zinc ion and lower the energy barrier for dissociation of the protonated cysteine side chain from the zinc ion. In addition, a subtle conformational change by the propeptide is needed in the course of zymogen activation. These elaborate processes take place in concert in the activation process of MMPs, and the insight into these processes presented herein sheds light on a highly regulated physiological process with profound consequences for eukaryotic organisms.
Assuntos
Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/metabolismo , Ativação Enzimática/genética , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz/genéticaRESUMO
The structures of Thermoactinomyces vulgaris R-47 alpha-amylase II mutant (d325nTVA II) complexed with substrate analogues, methyl beta-cyclodextrin (m beta-CD) and maltohexaose (G6), were solved by X-ray diffraction at 3.2 A and 3.3 A resolution, respectively. In d325nTVA II-m beta-CD complex, the orientation and binding-position of beta-CD in TVA II were identical to those in cyclodextin glucanotransferase (CGTase). The active site residues were essentialy conserved, while there are no residues corresponding to Tyr89, Phe183, and His233 of CGTase in TVA II. In d325nTVA II-G6 complex, the electron density maps of two glucosyl units at the non-reducing end were disordered and invisible. The four glucosyl units of G6 were bound to TVA II as in CGTase, while the others were not stacked and were probably flexible. The residues of TVA II corresponding to Tyr89, Lys232, and His233 of CGTase were completely lacking. These results suggest that the lack of the residues related to alpha-glucan and CD-stacking causes the functional distinctions between CGTase and TVA II.
Assuntos
Ciclodextrinas/química , Oligossacarídeos/química , alfa-Amilases/química , beta-Ciclodextrinas , Glucosiltransferases/química , Micromonosporaceae/enzimologia , Modelos Moleculares , Estrutura Terciária de Proteína , Especificidade por Substrato , alfa-Amilases/fisiologiaRESUMO
Several physicochemical experiments were done to obtain further information on the conformational changes occurring in beta-conglycinin in acidic-ethanol solution, using a single molecular species of this protein, beta3. By far-UV circular dichroism (CD), a transition from beta-sheet to alpha-helical structure was observed upon addition of acidic-ethanol, and the alpha-helix content was found to reach 76% in 70% ethanol (pH 2). From analyses of near-UV CD and difference absorption spectra, it was found that the tertiary structure of the beta3 species was significantly altered at ethanol concentrations between 10 and 20%. The profiles of binding of 1-anilinonaphthalene-8-sulfonic acid to the beta3 species during acidic-ethanol denaturation were indicative of the existence of intermediate conformers in the molten globule-like denaturation state. By measuring Fourier transform infrared spectra and estimating the Stokes radius by dynamic light scattering, the beta3 molecules were found to aggregate with an increase in ethanol concentration.
Assuntos
Globulinas/química , Glycine max/química , Proteínas de Plantas/química , Proteínas de Soja , Naftalenossulfonato de Anilina , Antígenos de Plantas , Dicroísmo Circular , Etanol , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Luz , Conformação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Espalhamento de Radiação , Proteínas de Armazenamento de Sementes , Soluções , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The tissue inhibitors of metalloproteinases (TIMPs) are specific inhibitors of MMP enzymatic activity. However, TIMP-2 can promote the activation of pro-MMP-2 by MT1-MMP. This process is mediated by the formation of a complex between MT1-MMP, TIMP-2, and pro-MMP-2. Binding of TIMP-2 to active MT1-MMP also inhibits the autocatalytic turnover of MT1-MMP on the cell surface. Thus, under certain conditions, TIMP-2 is a positive regulator of MMP activity. TIMP-4, a close homologue of TIMP-2 also binds to pro-MMP-2 and can potentially participate in pro-MMP-2 activation. We coexpressed MT1-MMP with TIMP-4 and investigated its ability to support pro-MMP-2 activation. TIMP-4, unlike TIMP-2, does not promote pro-MMP-2 activation by MT1-MMP. However, TIMP-4 binds to MT1-MMP inhibiting its autocatalytic processing. When coexpressed with TIMP-2, TIMP-4 competitively reduced pro-MMP-2 activation by MT1-MMP. A balance between TIMP-2 and TIMP-4 may be a critical factor in determining the degradative potential of cells in normal and pathological conditions.
Assuntos
Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Inibidores Teciduais de Metaloproteinases/fisiologia , Animais , Catálise , Linhagem Celular , Membrana Celular/metabolismo , Ativação Enzimática , Vetores Genéticos/metabolismo , Haplorrinos , Humanos , Immunoblotting , Metaloproteinases da Matriz Associadas à Membrana , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Transfecção , Vaccinia virus/metabolismo , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
The FILAMENTOUS FLOWER gene from Arabidopsis thaliana is a member of a gene family whose role is to specify abaxial cell fate in lateral organs. Analysis of the amino-terminal region of the FILAMENTOUS FLOWER protein suggests that seven cysteine residues at positions 14, 26, 30, 33, 54, 56, and 57, and two histidine residues at positions 18 and 24 contribute to a putative zinc finger motif, Cys-X(3)-His-X(5)-His-X-Cys-X(3)-Cys-X(2)-Cys-X(20)-Cys-X-Cys-Cys. Zinc determination experiments revealed that the FILAMENTOUS FLOWER protein binds two zinc ions per molecule. Chemical modification was required to release one zinc ion, whereas the other was released spontaneously or more rapidly in the presence of metallochromic indicator. The loss of a zinc ion and the subsequent structural change of the zinc finger domain were correlated with the multimerization of the FILAMENTOUS FLOWER protein. A cysteine residue at position 56 in the FILAMENTOUS FLOWER protein potentially interferes with zinc ligation within the zinc finger and causes this zinc release. In support of this, substitution of the Cys(56) by alanine suppressed both the zinc release and the multimerization of the FILAMENTOUS FLOWER protein. Deletion analysis showed that the region between positions 45 and 107 functions in the intermolecular contacts between FILAMENTOUS FLOWER proteins. This region corresponds to the carboxyl-terminal half of the zinc finger domain and the following hydrophobic region containing two putative alpha-helices. Our results suggest that the FILAMENTOUS FLOWER protein forms a range of different conformers. This attribute may lead to a greater degree of functional flexibility that is central to its role as an abaxial cell fate regulator.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Proteínas de Plantas/química , Dedos de Zinco , Zinco/metabolismo , Alanina/química , Sequência de Aminoácidos , Quelantes/farmacologia , Cromatografia em Gel , Dicroísmo Circular , Cisteína/química , Ácido Edético/farmacologia , Deleção de Genes , Vetores Genéticos , Glutationa Transferase/metabolismo , Histidina/química , Leucina/química , Ligantes , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Compostos de Sulfidrila/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sx1) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5-2H]uridine phosphoramidite, and synthesized a series of 2H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-2H]uridine in the target sequence, GU8C. By observing the H5-H6 TOCSY cross peaks of the series of 2H-labeled RNAs complexed with the Sx1 RBDI-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU2GU8, AU8, and UAU8, were assigned by comparison with those of GU8C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Nucleares/genética , Fragmentos de Peptídeos/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Deutério/química , Deutério/metabolismo , Drosophila/metabolismo , Hormônios de Inseto/genética , Hormônios de Inseto/metabolismo , Substâncias Macromoleculares , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Nucleotídeos de Purina/metabolismo , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Uridina/química , Uridina/metabolismoRESUMO
To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006% of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05% of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.
Assuntos
Domínio Catalítico , Micromonosporaceae/enzimologia , alfa-Amilases/genética , alfa-Amilases/metabolismo , Glucose/metabolismo , Glicosilação , Hidrólise , Maltose/metabolismo , Mutagênese Sítio-Dirigida , Oligossacarídeos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
To clarify the effect of GH on the development of seminiferous tubules in premature male rats, we investigated whether GH accelerates spermatogenesis under the condition of gonadotropin deprivation. Male Wistar rats aged three weeks were divided into three groups and subjected to administration of either long-acting GnRH agonist (GnRHa) or a combination of GnRHa and rat GH, with normal saline solution as control. After the 4-week treatment, sperm density and motility in the right epididymis were measured and seminiferous tubules of right testes were histologically examined. Sperm density and motility were significantly higher in GnRHa+GH-treated rats than in GnRHa-treated rats. In histological examination, the numbers of germ cells in various stages were increased in GnRHa+GH-treated rats compared with GnRHa-treated rats, with the number of mature spermatid being noticeably higher in GnRHa+GH-treated rats. These results suggest that administration of GH decreases loss of germ cells at various stages of spermatogenesis under the condition of gonadotropin withdrawal.