RESUMEN
Objective. To investigate the effects of different moving speeds on isokinetic dynamometry of knee flexors and extensors, and to provide normative data for comparative purposes in strength evaluations. Method. Thirty healthy male were tested at 60, 180, and 240 deg/sec angular velocities using a REV9000 testing and rehabilitation system. Result. Both PT (peak torque) and PT/BW (peak torque/body weights) showed significant differences (P< 0.01, P< 0.05) between flexors and extensors, and declined significantly with increased speed (P< 0.001). Peak torque differences between dominant and nondominant knee (Di%) was 10%. TPT (time to peak torque) tended to decrease as moving speeds increased (P< 0.001). APT (angle of peak torque) were 50%-70% for quadriceps, and 32%-45% for hamstrings; H/Q (hamstrings peak torque/quadriceps peak torque) was 50%-60%; WF (work fatigue) was 65%. Conclusion. The results provided a reference for strength evaluations in space medicine and space physiology, especially under simulated weightlessness and microgravity.
Asunto(s)
Articulación de la Rodilla/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Esfuerzo Físico/fisiología , Medicina Aeroespacial , Ergometría , Humanos , Masculino , Fatiga Muscular/fisiología , TorqueRESUMEN
Tumor necrosis factor receptors (TNFR) are single transmembrane-spanning glycoproteins that bind cytokines and trigger multiple signal transduction pathways. Many of these TNFRs rely on interactions with TRAF proteins that bind to the intracellular domain of the receptors. CD40 is a member of the TNFR family that binds to several different TRAF proteins. We have determined the crystal structure of a 20-residue fragment from the cytoplasmic domain of CD40 in complex with the TRAF domain of TRAF3. The CD40 fragment binds as a hairpin loop across the surface of the TRAF domain. Residues shown by mutagenesis and deletion analysis to be critical for TRAF3 binding are involved either in direct contact with TRAF3 or in intramolecular interactions that stabilize the hairpin. Comparison of the interactions of CD40 with TRAF3 vs. TRAF2 suggests that CD40 may assume different conformations when bound to different TRAF family members. This molecular adaptation may influence binding affinity and specific cellular triggers.
Asunto(s)
Antígenos CD40/metabolismo , Proteínas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Antígenos CD40/química , Cristalización , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas/antagonistas & inhibidores , Factor 3 Asociado a Receptor de TNFRESUMEN
Objective. To probe into the optimum exercise load in sports training. Method. Myosin heavy chain degradation fragments produced by different-intensity electric stimulation were identified in toad gastrocnemius muscle. The fragments were identified by electrophoresis of unfractionated extracts of toad gastrocnemius muscle on sodium dodecyl sulfate/polyacrylamide gels followed by immunoblotting on nitrocellulose sheets. Polyclonal antibody directed against the entire myosin were used to characterize the fragments. Result. Toad gastrocnemius muscle protein degradation increased under high-intensity electric stimulation; two kinds of Myosin degradation fragments whose molecular weight were 43KD,38KD respectively were produced by double high-intensity electric stimulation; the molecular weight 43KD immunoreactive myosin fragments produced by high-intensity electric stimulation disappeared under low-intensity electric stimulation. Conclusion. Continuous high-intensity exercise load would make muscle contractile protein further degradate while low-intensity exercise load would enhance the recover of contraction and function of the muscle.
Asunto(s)
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Subfragmentos de Miosina/metabolismo , Animales , Bufonidae , Proteínas Contráctiles/metabolismo , Estimulación Eléctrica , Immunoblotting , Peso Molecular , Músculo Esquelético/patología , Condicionamiento Físico Animal/fisiologíaRESUMEN
Antibodies are important tools to explore receptor-ligand interactions. The anti-integrin antibody OPG2 binds in an RGD-related manner to the alphaIIb beta3 integrin as a molecular mimic of fibrinogen. The Fab fragment from OPG2 was cocrystallized with a peptide from the beta3 subunit of the integrin representing a site that binds RGD. The crystal structure of the complex was determined at 2.2-A resolution and compared with the unbound Fab. On binding the integrin peptide there were conformational changes in CDR3 of the heavy chain. Also, a significant shift across the intermolecular interface between the CH1-CL domains was observed so that the angle of rotation relating the two domains was reduced by 15 degrees. This unusual conformational adjustment represents the first example of ligand-induced conformational changes in the carboxyl domains of a Fab fragment.
Asunto(s)
Sitios de Unión de Anticuerpos , Fragmentos Fab de Inmunoglobulinas/química , Oligopéptidos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Conformación Proteica , Secuencia de Aminoácidos , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Cristalización , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , SolucionesRESUMEN
There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.
Asunto(s)
Bacteriófagos/química , Cápside/química , Modelos Moleculares , Secuencia de Aminoácidos , Cápside/genética , Clonación Molecular , Cristalización , Datos de Secuencia Molecular , Conformación Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Alineación de SecuenciaRESUMEN
Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.
Asunto(s)
Fibronectinas/química , Conformación Proteica , Sulfato de Amonio , Sitios de Unión , Precipitación Química , Cristalización , Humanos , Concentración de Iones de Hidrógeno , Integrinas/metabolismo , Sulfato de Magnesio , Fragmentos de Péptidos/química , Polietilenglicoles , Proteínas Recombinantes/química , Difracción de Rayos XRESUMEN
Transcription factors belonging to the ets family regulate gene expression and share a conserved ETS DNA-binding domain that binds to the core sequence 5'-(C/A)GGA(A/T)-3'. The domain is similar to alpha+beta ("winged") helix-turn-helix DNA-binding proteins. The crystal structure of the PU.1 ETS domain complexed to a 16-base pair oligonucleotide revealed a pattern for DNA recognition from a novel loop-helix-loop architecture (Kodandapani, R., Pio, F., Ni. C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460). Correlation of this model with mutational analyses and chemical shift data on other ets proteins confirms this complex as a paradigm for ets DNA recognition. The second helix in the helix-turn-helix motif lies deep in the major groove with specific contacts with bases in both strands in the core sequence made by conserved residues in alpha3. On either side of this helix, two loops contact the phosphate backbone. The DNA is bent (8 degrees) but uniformly curved without distinct kinks. ETS domains bind DNA as a monomer yet make extensive DNA contacts over 30 A. DNA bending likely results from phosphate neutralization of the phosphate backbone in the minor groove by both loops in the loop-helix-loop motif. Contacts from these loops stabilize DNA bending and may mediate specific base interactions by inducing a bend toward the protein.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Secuencias Hélice-Giro-Hélice , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Análisis Mutacional de ADN , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas c-ets , Homología de Secuencia de Aminoácido , Transactivadores/química , Factores de Transcripción/químicaRESUMEN
The Ets family of transcription factors, of which there are now about 35 members regulate gene expression during growth and development. They share a conserved domain of around 85 amino acids which binds as a monomer to the DNA sequence 5'-C/AGGAA/T-3'. We have determined the crystal structure of an ETS domain complexed with DNA, at 2.3-A resolution. The domain is similar to alpha + beta (winged) 'helix-turn-helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8 degrees. The domain contacts the DNA by a novel loop-helix-loop architecture. Four of amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix-turn-helix' motif, which binds downstream and on the opposite strand.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Secuencias Hélice-Giro-Hélice , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Proteínas Oncogénicas de Retroviridae , Homología de Secuencia de AminoácidoRESUMEN
The PU.1 transcription factor is a member of the ets gene family of regulatory proteins. These molecules play a role in normal development and also have been implicated in malignant processes such as the development of erythroid leukemia. The Ets proteins share a conserved DNA-binding domain (the ETS domain) that recognizes a purine-rich sequence with the core sequence: 5'-C/AGGAA/T-3'. This domain binds to DNA as a monomer, unlike many other DNA-binding proteins. The ETS domain of the PU.1 transcription factor has been crystallized in complex with a 16-base pair oligonucleotide that contains the recognition sequence. The crystals formed in the space group C2 with a = 89.1, b = 101.9, c = 55.6 A, and beta = 111.2 degrees and diffract to at least 2.3 A. There are two complexes in the asymmetric unit. Production of large usable crystals was dependent on the length of both protein and DNA components, the use of oligonucleotides with unpaired A and T bases at the termini, and the presence of polyethylene glycol and zinc acetate in the crystallization solutions. This is the first ETS domain to be crystallized, and the strategy used to crystallize this complex may be useful for other members of the ets family.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Oligodesoxirribonucleótidos/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , ADN/síntesis química , ADN/aislamiento & purificación , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/aislamiento & purificación , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/aislamiento & purificación , Unión Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Oncogénicas de Retroviridae , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Factores de Transcripción/aislamiento & purificaciónRESUMEN
The coat protein from the MS2 bacteriophage plays a dual role by encapsidating viral RNA and also by binding RNA as a translational repressor. In order to study the isolated dimer in a conformation not influenced by capsid interactions, a mutant molecule was crystallized that is defective in capsid assembly but is an active repressor. The unassembled dimer crystallized in the space group P21212 with a = 76.2, b = 55.7, and c = 28.4 A. In these crystals, monomers were related by twofold symmetry. When this dimer was co-crystallized with 5-bromouridine, crystals formed in space group R3 with a = b = 155.9 A, c = 29.9 A, gamma = 120 degrees; the dimer was the asymmetric unit.
Asunto(s)
Bromodesoxiuridina/metabolismo , Proteínas de la Cápside , Cápside/química , Levivirus/química , Proteínas de Unión al ARN , Cápside/genética , Cápside/aislamiento & purificación , Cápside/metabolismo , Cristalización , Cristalografía por Rayos X , Levivirus/genética , Mutación Puntual , Conformación Proteica , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
BACKGROUND: The coat protein in RNA bacteriophages binds and encapsidates viral RNA, and also acts as translational repressor of viral replicase by binding to an RNA hairpin in the RNA genome. Because of its dual function, the MS2 coat protein is an interesting candidate for structural studies of protein-RNA interactions and protein-protein interactions. In this study, unassembled MS2 coat protein dimers were selected to analyze repressor activity and virus assembly. RESULTS: The crystal structure of a mutant MS2 coat protein that is defective in viral assembly yet retains repressor activity has been determined at 2.0 A resolution. The unassembled dimer is stabilized by interdigitation of alpha-helices, and the formation of a 10-stranded antiparallel beta-sheet across the interface between monomers. The substitution of arginine for tryptophan at residue 82 results in the formation of two new inter-subunit hydrogen bonds that further stabilize the dimer. Residues that influence RNA recognition, identified by molecular genetics, were located across the beta-sheet. Two of these residues (Tyr85 and Asn87) are displaced in the unliganded dimer and are located in the same beta-strand as the Trp-->Arg mutation. CONCLUSIONS: When compared with the structure of the coat protein in the assembled virus, differences in orientation of residues 85 and 87 suggest conformational adjustment on binding RNA in the first step of viral assembly. The substitution at residue 82 may affect virus assembly by imposing conformational restriction on the loop that makes critical inter-subunit contacts in the capsid.
Asunto(s)
Proteínas de la Cápside , Cápside/química , Cápside/metabolismo , Cristalización , Conformación Proteica , ARN Viral/metabolismo , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Pliegue de Proteína , Fagos ARN/química , Programas Informáticos , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The CHCl3 extract of the whole plant of Arundo donax yielded N-(4'-bromophenyl)-2,2-diphenylacetanilide [1]. The structure was determined on the basis of chemical, spectroscopic, and X-ray crystallographic data. Compound 1 has not been previously reported as a natural product and showed inhibition of feeding for boll weevils.
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Acetanilidas/química , Compuestos de Bencidrilo/química , Insectos , Insecticidas/química , Plantas/química , Acetanilidas/farmacología , Animales , Compuestos de Bencidrilo/farmacología , Cristalografía por Rayos X , Conducta Alimentaria/efectos de los fármacos , Insecticidas/farmacología , Espectroscopía de Resonancia Magnética , TailandiaRESUMEN
The alkaloid extract of the whole plant of Gelsemium elegans has afforded four new alkaloids: N-desmethoxyrankinidine [1], 11-hydroxrankinidine [3], 11-hydroxyhuman-humantenine [4] and humantenirine [6]. The structures of 5 was established through X-ray crystallographic analysis, and the structures of the other three new alkaloids were deduced by spectral analysis (1H, 13C, APT, 2D-COSY and 2D-HETCOR).