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1.
BMC Ophthalmol ; 23(1): 469, 2023 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-37981663

RESUMEN

BACKGROUND: Amblyopia treatment by occluding the healthy eye is known to be effective during a sensitive critical period. This study aims to clarify the factors for the total occlusion time (TOT) required for the amblyopic eye to achieve a normal visual acuity (VA) level of 1.0 (0.0 logMAR equivalent). This could contribute to an efficient treatment plan for eyes with hyperopic anisometropic amblyopia. METHODS: Subjects were 58 patients (26 boys and 32 girls; age range, 3.6-9.2, average, 5.8 ± 1.3 years) with hyperopic anisometropic amblyopia. All the subjects had initially visited and completed occlusion therapy with improved VA of 1.0 or better in the amblyopic eye at Kindai University Hospital between January 2007 and March 2017. Using the subjects' medical records, we retrospectively investigated five factors for the TOT: the age at treatment, the initial VA of the amblyopic eye, refraction of the amblyopic eye, anisometropic disparity, and the presence of microstrabismus. Patient's VA improvement at one month after treatment was also evaluated to confirm the effect of the occlusion therapy. RESULTS: The initial VA of the amblyopic eye ranged from 0.1 to 0.9 (median, 0.4). The TOT ranged from 140 to 1795 (median, 598) hours with an average daily occlusion time of 7 hours. The initial VA of the amblyopic eye and presence of microstrabismus were the significant factors for the TOT (p < 0.01). To achieve VA of 1.0 or better, patients with an initial VA of ≤ 0.3 in the amblyopic eye required a longer TOT. Moreover, patients with concomitant microstrabismus required a 1.7-fold longer TOT compared to those without microstrabismus. CONCLUSION: Longer daily occlusion hours and early start of the treatment will be necessary for patients with poor initial VA or microstrabismus to complete occlusion therapy within the sensitive critical period.


Asunto(s)
Ambliopía , Hiperopía , Estrabismo , Masculino , Femenino , Humanos , Preescolar , Niño , Ambliopía/complicaciones , Ambliopía/terapia , Estudios Retrospectivos , Ojo , Hiperopía/complicaciones , Hiperopía/terapia
2.
Clin Ophthalmol ; 17: 1545-1553, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37284056

RESUMEN

Purpose: To investigate the association between stereoacuity and suppression during occlusion therapy for patients with anisometropic amblyopia. Design: Retrospective study. Patients and Methods: This study included 19 patients with hyperopic anisometropic amblyopia who underwent occlusion therapy. The mean age of the patients was 5.5 ± 1.4 years. The participants were evaluated for improvement in stereoacuity and suppression before beginning occlusion therapy, when the highest amblyopic visual acuity (VA) was achieved, during tapering, at the end of occlusion therapy, and at the final visit. Stereoacuity was evaluated using the TNO test or JACO stereo test. The presence of suppression was evaluated using circle No. 1 of the Stereo Fly Test or JACO results as the optotype. Results: Of the 19 patients, 13 (68.4%) had suppression before occlusion, eight (42.1%) when the highest VA was achieved, five (26.3%) during tapering, and none at the final visit. Of the 13 patients with suppression before occlusion, 10 (76.9%) showed further improvement in stereoacuity when suppression disappeared, and nine had foveal stereopsis of 60 arcseconds. A significant association was confirmed between foveal stereopsis and suppression when the highest VA was achieved and during the tapering period (P<0.05, Fisher's exact test). Conclusion: Suppression was observed even when the VA in amblyopic eyes reached the highest score. By gradually decreasing the duration of occlusion, suppression was eliminated, leading to the acquisition of foveal stereopsis.

3.
Artículo en Inglés | MEDLINE | ID: mdl-36674179

RESUMEN

AIM: We developed the Training Program on Child Abuse Prevention for Citizens (TCAP-C) and tested its effects and acceptability among citizen leaders (CLs). METHODS: Community-based participatory research using a pretest-posttest follow-up design was conducted in Tokyo, Japan from September 2021 to March 2022. Participants completed questionnaires before, upon completion, and one month and three months after TCAP-C. Recognition, knowledge, and behaviors regarding child abuse and community consciousness were collected and compared before and one and three months after TCAP-C, and the degree of satisfaction, understanding, and meaningfulness were collected upon completion. We analyzed data using repeated-measures ANCOVA. RESULTS: A total of 111, 98, 101, and 94 participants completed the questionnaires before, upon completion, and one and three months after TCAP-C, respectively. Overall, the recognition, knowledge, and community consciousness scores significantly improved from before to one month and three months after TCAP-C. Regarding the behaviors, only the behaviors of learning and watching over were significantly improved from before to one month after TCAP-C; however, those behaviors were not different between before and three months after TCAP-C. Furthermore, 95% participants reported being entirely satisfied with TCAP-C, and 85% and 91% reported good understanding and meaningfulness of the program. CONCLUSIONS: TCAP-C is acceptable and can improve CL recognition, knowledge, and community consciousness.


Asunto(s)
Maltrato a los Niños , Investigación Participativa Basada en la Comunidad , Niño , Humanos , Aprendizaje , Maltrato a los Niños/prevención & control , Encuestas y Cuestionarios
4.
BMC Ophthalmol ; 21(1): 304, 2021 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-34418996

RESUMEN

BACKGROUND: The efficacies of prism adaptation test (PAT) and monocular occlusion (MO) and their optimal test durations to detect the maximum angles of deviation at near and distance in eyes with intermittent exotropia (IXT) were assessed and compared. METHODS: We retrospectively reviewed the medical records of 72 patients with IXT. All the patients had undergone the initial strabismus surgery between April 2015 and October 2018 and had been preoperatively tested by both PAT and MO performed on different days for 30 and 60 min. Near and distance deviations after 30 and 60 min of PAT and MO were compared to their baseline measurements obtained immediately after prism wear and before occlusion by alternate prism cover test. The near/distance measurements and required test duration to reveal the maximum deviation angle were also compared between PAT and MO. RESULTS: Compared with the baseline, the near deviation by PAT significantly increased after 30 (P < 0.05) and 60 (P < 0.01) minutes but not the distance deviation. However, the increase after 30 min was not significant. By MO, neither near nor distance deviation showed a significant difference from the baseline after 30 and 60 min. PAT showed a significantly larger near deviation than MO at 30 and 60 min, but a larger distance deviation by PAT was only observed at 30 min. CONCLUSIONS: In patients with basic and convergence insufficiency types of IXT, a 30-minute PAT appears to be more effective than MO in revealing the maximum angle of deviation before strabismus surgery.


Asunto(s)
Exotropía , Trastornos de la Motilidad Ocular , Enfermedad Crónica , Exotropía/diagnóstico , Exotropía/cirugía , Humanos , Músculos Oculomotores/cirugía , Estudios Retrospectivos
5.
Retina ; 41(6): 1338-1345, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33165297

RESUMEN

PURPOSE: To evaluate the utility of oral fluorescein angiography with ultra-widefield imaging system (oral UWF-FA) predominantly in children. METHODS: We recruited 17 patients aged 2 years to 22 years with retinal disorders. Each patient ingested a dose of fluorescein sodium set by body weight mixed with 100 mL of juice. Images were scored using four parameters as follows: branch retinal vessel identification, retinal vessels visualization, foveal avascular zone visualization, and clinically important findings such as leakage, microaneurysms, neovascularization, or significant nonperfusion area visualization. Based on the aggregate score, we classified the image quality into three grades. RESULTS: Sixteen of 17 patients completely ingested the fluorescein sodium, and ultra-widefield fluorescein angiography was performed. Images were classified as high quality in nine cases, moderate quality in four, and poor quality in three. In 13 cases (81.3%), images had adequate quality to evaluate retinal conditions. Of three patients with poor-quality images, 2 took 10 minutes to ingest fluorescein sodium and the other ingested only half the dose. The adverse event of a mild skin rash was noted in one patient. CONCLUSION: Oral ultra-widefield fluorescein angiography is effective in evaluating retinal pathology and is a useful alternative especially for pediatric patients who cannot tolerate intravenous line placement.


Asunto(s)
Diagnóstico por Imagen/instrumentación , Angiografía con Fluoresceína/estadística & datos numéricos , Mácula Lútea/diagnóstico por imagen , Enfermedades de la Retina/diagnóstico , Vasos Retinianos/diagnóstico por imagen , Tomografía de Coherencia Óptica/normas , Agudeza Visual , Adolescente , Niño , Preescolar , Femenino , Angiografía con Fluoresceína/métodos , Fondo de Ojo , Humanos , Masculino , Enfermedades de la Retina/fisiopatología , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodos , Adulto Joven
6.
Ophthalmic Res ; 60(4): 231-237, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30326478

RESUMEN

PURPOSE: This study aimed to evaluate the utility of optical coherence tomography (OCT) in the quantification of eccentric fixation in amblyopic patients. MATERIAL AND METHODS: In this study, 14 amblyopic patients and 10 healthy volunteers were enrolled. Under non-mydriatic conditions, fixation tests were performed directly using a fixation ophthalmoscope and indirectly using spectral-domain OCT. For evaluations using OCT, the distance between the fovea and the fixation point, which was determined by a cross-sectional image, was measured. RESULTS: On evaluations of healthy volunteers by OCT, the mean distance between the fixation point and the fovea was 80.4 ± 37.7 µm for the dominant eyes and 63.7 ± 36.4 µm for non-dominant eyes (p = 0.41). In amblyopic patients, on evaluation by OCT, the mean distance between the fixation point and the fovea was 193.8 ± 188.3 µm in amblyopic eyes and 83.5 ± 39.3 µm in paired fellow eyes (p = 0.02). Although OCT could detect eccentric fixation points in all the affected eyes of amblyopic patients, fixation ophthalmoscope was unable to quantify them in 2 of 14 affected eyes. CONCLUSIONS: Compared with a fixation ophthalmoscope, our method using OCT seems to be superior both in quantification and detection of eccentric fixation in amblyopic patients, without the need for mydriasis.


Asunto(s)
Ambliopía/diagnóstico , Fijación Ocular/fisiología , Fóvea Central/patología , Tomografía de Coherencia Óptica/métodos , Agudeza Visual , Adolescente , Adulto , Ambliopía/fisiopatología , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , Masculino , Reproducibilidad de los Resultados , Adulto Joven
7.
Case Rep Ophthalmol ; 9(2): 304-309, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29928227

RESUMEN

Binocular vision may be compromised in children after unilateral cataract surgery because the distances at which clear vision is present are different for the two eyes. We believe that wearing progressive additional lenses can be effective in improving the binocular vision in children with pseudophakia.

8.
Jpn J Ophthalmol ; 62(3): 307-314, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29556848

RESUMEN

PURPOSE: To examine the role of fusional convergence amplitude in postoperative phoria maintenance in childhood intermittent exotropia [X(T)]. METHODS: The medical records of 29 children aged 15 years or younger (mean age, 10.8 ± 2.4 years) and treated with monocular recession-resection for X(T) were reviewed retrospectively. The patients' fusional convergence amplitude (break point/total amplitudes), physiologic diplopia, and phoria maintenance (presence/absence of phoria maintenance and ability to maintain phoria) were assessed. The presence of phoria maintenance was confirmed by a cover test, and the ability to maintain phoria was quantified using the Bagolini red filter bar. Correlations of the amplitude size with the presence and ability of phoria maintenance were investigated. RESULTS: A significant correlation was seen between fusional amplitude (break point/total) and ability to maintain phoria at near and at far (break point: P < .05 at near/P < .01 at far; total: P < .05 at near/far). Neither the break point amplitude nor the total amplitude significantly differed between the patients with phoria maintenance and those without it (break point: P = .71 at near, P = .29 at far; total: P = .98 at near, P = .85 at far). Phoria maintenance correlated with the suppression of physiologic diplopia during phoria (P < .01). The deviation angle did not significantly correlate with fusional amplitude either at near (P = .58) or at far (P = .27). CONCLUSIONS: In childhood X(T), fusional amplitude plays a role in enforcing the patient's ability to maintain phoria. However, sufficient fusional amplitude does not guarantee fully functioning fusion if suppression is present during phoria.


Asunto(s)
Convergencia Ocular/fisiología , Exotropía/cirugía , Músculos Oculomotores/cirugía , Procedimientos Quirúrgicos Oftalmológicos/métodos , Estudios Retrospectivos , Visión Binocular/fisiología , Adolescente , Niño , Enfermedad Crónica , Exotropía/fisiopatología , Femenino , Humanos , Masculino , Músculos Oculomotores/fisiopatología , Periodo Posoperatorio , Agudeza Visual
9.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 3): 541-54, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25760604

RESUMEN

Environmentally friendly absorbents are needed for Sr(2+) and Cs(+), as the removal of the radioactive Sr(2+) and Cs(+) that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs(+) or Sr(2+). The crystal structure of a halophilic ß-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Šin space group P31 using X-ray crystallography. Moreover, the locations of bound Sr(2+) and Cs(+) ions were identified by anomalous X-ray diffraction. The location of one Cs(+)-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na(+) (90 mM Na(+)/10 mM Cs(+)). From an activity assay using isothermal titration calorimetry, the bound Sr(2+) and Cs(+) ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs(+)-binding site provides important information that is useful for the design of artificial Cs(+)-binding sites that may be useful in the bioremediation of radioactive isotopes.


Asunto(s)
Cesio/química , Chromohalobacter/enzimología , beta-Lactamasas/química , Sitios de Unión , Cristalografía por Rayos X , Unión Proteica , Estroncio/química
10.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 3): 811-20, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24598750

RESUMEN

Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1-4 M NaCl). In order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.1 Šresolution. The unit cell of HaAP contained one dimer unit corresponding to the biological unit. The monomer structure of HaAP contains a domain comprised of an 11-stranded ß-sheet core with 19 surrounding α-helices similar to those of APs from other species, and a unique `crown' domain containing an extended `arm' structure that participates in formation of a hydrophobic cluster at the entrance to the substrate-binding site. The HaAP structure also displays a unique distribution of negatively charged residues and hydrophobic residues in comparison to other known AP structures. AP from Vibrio sp. G15-21 (VAP; a slight halophile) has the highest similarity in sequence (70.0% identity) and structure (C(α) r.m.s.d. of 0.82 Šfor the monomer) to HaAP. The surface of the HaAP dimer is substantially more acidic than that of the VAP dimer (144 exposed Asp/Glu residues versus 114, respectively), and thus may enable the solubility of HaAP under high-salt conditions. Conversely, the monomer unit of HaAP formed a substantially larger hydrophobic interior comprising 329 C atoms from completely buried residues, whereas that of VAP comprised 264 C atoms, which may maintain the stability of HaAP under low-salt conditions. These characteristics of HaAP may be responsible for its unique functional adaptation permitting activity over a wide range of salt concentrations.


Asunto(s)
Fosfatasa Alcalina/química , Halomonas/enzimología , Potenciales de Acción , Proteínas Bacterianas/química , Cristalización , Cristalografía por Rayos X , Multimerización de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Electricidad Estática
11.
J Mol Biol ; 425(22): 4295-306, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-23831226

RESUMEN

NADH-Cytochrome b5 reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5 (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD(+). Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.


Asunto(s)
Citocromo-B(5) Reductasa/química , Animales , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Citocromo-B(5) Reductasa/metabolismo , Transporte de Electrón , Flavina-Adenina Dinucleótido/química , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes , Porcinos
12.
Protein Sci ; 21(4): 498-510, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22275000

RESUMEN

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography. The molecular weight of HaNDK is 33,660, and the X-ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1-65.3 kDa, and the oligomerization state was investigated by X-ray crystallography to 2.3 and 2.5 Å resolution with space groups of P2(1) and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild-type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.


Asunto(s)
Proteínas Bacterianas/química , Halomonas/enzimología , Nucleósido-Difosfato Quinasa/química , Multimerización de Proteína , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Dominio Catalítico , Cromatografía en Gel/métodos , Cristalografía por Rayos X , Activación Enzimática , Pruebas de Enzimas , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Halomonas/química , Halomonas/genética , Enlace de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Nucleósido-Difosfato Quinasa/genética , Mutación Puntual , Conformación Proteica , Estabilidad Proteica , Alineación de Secuencia , Electricidad Estática , Especificidad por Sustrato
13.
Eur Biophys J ; 40(5): 661-71, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21249494

RESUMEN

Quasielastic neutron scattering (QENS) experiments were carried out on powders of F-actin and G-actin hydrated with D(2)O to characterize the internal dynamics on the picosecond time scale and the Ångstrom length scale. To investigate the effects of hydration, the measurements were done on samples at hydration ratio (h) of 0.4 (mg D(2)O/mg protein), containing only the first layer of hydration water, and at h = 1.0, containing more layers of water. The QENS spectra, obtained from the measurements at two energy resolutions of 110 and 15 µeV, indicated that the internal motions of both F-actin and G-actin have distributions of motions with distinct correlation times and amplitudes. Increasing hydration changes relative populations of these distinct motions. The effects of hydration were shown to be different between F-actin and G-actin. Elastic incoherent neutron scattering measurements provided the concerted results. The observed effects were interpreted in terms of the dynamical heterogeneity of the actin molecule: in G-actin, more surface loops become flexible and undergo diffusive motions of large amplitudes, whereas in F-actin the molecular interactions that keep the polymerized state suppress the large motions of the surface loops involved with polymerization so that the population of atoms undergoing large motions can increase only to a lesser degree.


Asunto(s)
Actinas/química , Actinas/metabolismo , Movimiento , Difracción de Neutrones/métodos , Animales , Óxido de Deuterio/química , Elasticidad
14.
Anal Biochem ; 399(2): 299-301, 2010 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-20064482

RESUMEN

We have developed a technique by which muscle thin filaments are reconstituted from the recombinant troponin components and the native thin filaments. By this technique, the reconstituted troponin complex is exchanged into the native thin filaments in the presence of 20% glycerol and 0.3M KCl at pH 6.2. More than 90% of endogenous troponin complex was replaced with the recombinant troponin complex. Structural integrity and Ca(2+) sensitivity of the reconstituted thin filament prepared by this technique was confirmed by X-ray fiber diffraction measurements and the thin filament-activated myosin subfragment 1 ATPase measurements, respectively.


Asunto(s)
Citoesqueleto de Actina/fisiología , Troponina/metabolismo , Animales , Calcio/metabolismo , Bovinos , Contracción Muscular/fisiología , Músculo Esquelético , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Troponina/química , Troponina/genética
15.
Biochem Biophys Res Commun ; 382(1): 205-9, 2009 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-19275886

RESUMEN

Two cardiomyopathy-causing mutations, E244D and K247R, in human cardiac troponin T (TnT) are located in the coiled-coil region of the Tn-core domain. To elucidate effects of mutations in this region on the regulatory function of Tn, we measured Ca(2+)-dependent ATPase activity of myofibrils containing various mutants of TnT at these residues. The results confirmed that the mutant E244D increases the maximum ATPase activity without changing the Ca(2+)-sensitivity. The mutant K247R was shown for the first time to have the effect similar to the mutant E244D. Furthermore, various TnT mutants (E244D, E244M, E244A, E244K, K247R, K247E, and K247A) showed various effects on the maximum ATPase activity while the Ca(2+)-sensitivity was unchanged. Molecular dynamics simulations of the Tn-core containing these TnT mutants suggested that the hydrogen-bond network formed by the side chains of neighboring residues around residues 244 and 247 is important for Tn to function properly.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Calcio/metabolismo , Cardiomiopatías/enzimología , Miofibrillas/enzimología , Troponina T/metabolismo , Cardiomiopatías/genética , Humanos , Enlace de Hidrógeno , Mutación , Estructura Terciaria de Proteína/genética , Troponina T/química , Troponina T/genética
16.
Biophys J ; 94(12): 4880-9, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18326640

RESUMEN

F-actin, a helical polymer formed by polymerization of the monomers (G-actin), plays crucial roles in various aspects of cell motility. Flexibility of F-actin has been suggested to be important for such a variety of functions. Understanding the flexibility of F-actin requires characterization of a hierarchy of dynamical properties, from internal dynamics of the actin monomers through domain motions within the monomers and relative motions between the monomers within F-actin to large-scale motions of F-actin as a whole. As a first step toward this ultimate purpose, we carried out elastic incoherent neutron scattering experiments on powders of F-actin and G-actin hydrated with D(2)O and characterized the internal dynamics of F-actin and G-actin. Well established techniques and analysis enabled the extraction of mean-square displacements and their temperature dependence in F-actin and in G-actin. An effective force constant analysis with a model consisting of three energy states showed that two dynamical transitions occur at approximately 150 K and approximately 245 K, the former of which corresponds to the onset of anharmonic motions and the latter of which couples with the transition of hydration water. It is shown that behavior of the mean-square displacements is different between G-actin and F-actin, such that G-actin is "softer" than F-actin. The differences in the internal dynamics are detected for the first time between the different structural states (the monomeric state and the polymerized state). The different behavior observed is ascribed to the differences in dynamical heterogeneity between F-actin and G-actin. Based on structural data, the assignment of the differences observed in the two samples to dynamics of specific loop regions involved in the polymerization of G-actin into F-actin is proposed.


Asunto(s)
Actinas/química , Actinas/ultraestructura , Modelos Químicos , Modelos Moleculares , Difracción de Neutrones/métodos , Simulación por Computador , Cinética , Movimiento (Física) , Conformación Proteica
17.
J Mol Biol ; 367(1): 16-24, 2007 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-17254604

RESUMEN

In striated muscles contraction is regulated by the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, and TnT), and tropomyosin. Knowledge of in situ structures of these proteins is indispensable for elucidating this Ca(2+)-sensitive regulatory mechanism. We employed neutron scattering to investigate the structure of TnC within the thin filament, and found that TnC assumes extended dumbbell-like structures and moves toward the filament axis by binding of Ca(2+). Here, in order to obtain more detailed in situ structural information of TnC, neutron fiber diffraction measurements were performed. Sols of native thin filaments and the thin filaments containing deuterated TnC were prepared in (2)H(2)O. The oriented samples were obtained by placing these sols sealed in quartz capillaries with a diameter of 3 mm in a magnetic field of 18 Tesla. Neutron fiber diffraction patterns were obtained from these oriented samples in the absence and presence of Ca(2+). The patterns obtained showed strong equatorial diffraction due to the thin filaments, 59 A and 51 A layer-lines due to actin, and meridional reflections due to Tn-complex. Analysis of the meridional reflections due to Tn-complex with aid of model calculation showed that the angle between the thin filament axis and the long axis of TnC was estimated to be 67(+/-7) degrees and 49(+/-17) degrees , in the absence and presence of Ca(2+), respectively, suggesting that TnC, which assumes orientations rather perpendicular to the filament axis in the absence of Ca(2+), tilts toward the filament axis and the orientational and positional disorder increases by binding Ca(2+). It also showed that the relative position of the TnC moved by about 22 A by binding Ca(2+), and this apparent movement was concomitant with the movements of other Tn-subunits. This implies that by binding Ca(2+), significant structural rearrangements of Tn-subunits occur.


Asunto(s)
Difracción de Neutrones/métodos , Troponina C/química , Citoesqueleto de Actina/química , Animales , Contracción Muscular/fisiología , Conformación Proteica
18.
J Mol Biol ; 342(4): 1209-21, 2004 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-15351646

RESUMEN

Regulation of skeletal and cardiac muscle contraction is associated with structural changes of the thin filament-based proteins, troponin consisting of three subunits (TnC, TnI, and TnT), tropomyosin, and actin, triggered by Ca2+-binding to TnC. Knowledge of in situ structures of these proteins is indispensable for elucidating the molecular mechanism of this Ca2+-sensitive regulation. Here, the in situ structure of TnC within the thin filaments was investigated with neutron scattering, combined with selective deuteration and the contrast matching technique. Deuterated TnC (dTnC) was first prepared, this dTnC was then reconstituted into the native thin filaments, and finally neutron scattering patterns of these reconstituted thin filaments containing dTnC were measured under the condition where non-deuterated components were rendered "invisible" to neutrons. The obtained scattering curves arising only from dTnC showed distinct difference in the absence and presence of Ca2+. These curves were analyzed by model calculations using the Monte Carlo method, in which inter-dTnC interference was explicitly taken into consideration. The model calculation showed that in situ radius of gyration of TnC was 23 A (99% confidence limits between 22 A and 23 A) and 24 A (99% confidence limits between 23 A and 25 A) in the absence and presence of Ca2+, respectively, indicating that TnC within the thin filaments assumes a conformation consistent with the extended dumbbell structure, which is different from the structures found in the crystals of various Tn complexes. Elongation of TnC by binding of Ca2+ was also suggested. Furthermore, the radial position of TnC within the thin filament was estimated to be 53 A (99% confidence limits between 49 A and 57 A) and 49 A (99% confidence limits between 44 A and 53 A) in the absence and presence of Ca2+, respectively, suggesting that this radial movement of TnC by 4A is associated with large conformational changes of the entire Tn molecule by binding of Ca2+.


Asunto(s)
Troponina C/química , Animales , Bovinos , Modelos Moleculares , Neutrones , Conformación Proteica , Dispersión de Radiación , Difracción de Rayos X
19.
J Mol Biol ; 331(1): 21-8, 2003 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-12875832

RESUMEN

Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.


Asunto(s)
Amiloide/química , Muramidasa/química , Difracción de Neutrones , Cloruro de Sodio/farmacología , Amiloide/ultraestructura , Animales , Pollos , Dimerización , Relación Dosis-Respuesta a Droga , Proteínas del Huevo/química , Proteínas del Huevo/ultraestructura , Cinética , Muramidasa/ultraestructura , Conformación Proteica/efectos de los fármacos , Electricidad Estática
20.
Graefes Arch Clin Exp Ophthalmol ; 240(9): 743-7, 2002 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12271372

RESUMEN

BACKGROUND: Visual information projected onto corresponding points on the right and left retinas converges on the binocular cells in the visual cortex. The aim of this study is to investigative the characteristics of the receptive field for binocular stimulation in the central visual field of normal-sighted human subjects. METHODS: We investigated the receptive field for binocular stimulation under fusion conditions by combining the Octopus 201 with the space synoptophore. We measured binocular and monocular sensitivities while the fusion patterns were projected onto the Octopus 201 cupola, using the space synoptophore. We designed a new program to test 37 points in the central 6 degrees visual field. Six target sizes were tested: the white-spot targets of 0.054 degrees, 0.108 degrees, 0.216 degrees, 0.431 degrees, 0.862 degrees and 1.724 degrees projected diameters. RESULTS: The threshold energy necessary for binocular stimulation was lower than that for the monocular stimulation in all subjects. This difference was more obvious on the test points that were more distant from the fovea when target sizes of 0.054 degrees and 0.108 degrees were used. The amount of binocular summation ratio was highest for target size 0.054 degrees in each stimulus area in the central 6 degrees of the visual field. When we measured binocular summation using target sizes larger than 0.108 degrees, the result was the constant summation. CONCLUSIONS: The size of the receptive field for binocular stimulation is smaller than monocular stimulation under the same fusion condition. The amount of binocular summation varies as a function of target size.


Asunto(s)
Visión Binocular/fisiología , Campos Visuales/fisiología , Percepción Visual/fisiología , Adulto , Humanos , Umbral Sensorial , Pruebas de Visión
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