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1.
J Dermatol ; 46(10): 914-916, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31389046

RESUMEN

A 75-year-old man presented with a 1-cm large elastic soft subcutaneous nodule on the left side of the umbilicus, which when excised showed presence of a helminthic form within the granulomatous lesions. Morphologically, the helminth was considered to be of the genus Dirofilaria, and the patient showed increased serum antibody titer against canine filaria. The partial DNA sequence of the mitochondrial 12S rRNA gene locus of this clinical isolate showed the highest nucleotide identity (89.6%) with Dirofilaria repens; however, the phylogenetic analysis addressed the haplotype and Dirofilaria ursi as outgroups of the clusters of D. repens and Dirofilaria immitis, which are the causal agents of most human dirofilariasis. As like bear filaria D. ursi, a wide variety of other carnivore-parasitizing filaria species have rarely been reported in humans. The newly detected genetic haplotype in this case may correspond to one of these species of Dirofilaria, though the genetic references are not available thus far.


Asunto(s)
ADN de Helmintos/genética , Dirofilaria/genética , Dirofilariasis/parasitología , Tejido Subcutáneo/parasitología , Anciano , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , ADN de Helmintos/aislamiento & purificación , ADN Mitocondrial/genética , ADN Mitocondrial/aislamiento & purificación , Dirofilaria/inmunología , Dirofilaria/aislamiento & purificación , Dirofilariasis/sangre , Dirofilariasis/diagnóstico , Técnicas de Genotipaje , Haplotipos , Humanos , Masculino , Filogenia , ARN Ribosómico/genética , Tejido Subcutáneo/patología , Ombligo
2.
J Mol Biol ; 341(2): 589-604, 2004 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-15276846

RESUMEN

To investigate whether the structure partially formed in the molten globule folding intermediate of goat alpha-lactalbumin is further organized in the transition state of folding, we constructed a number of mutant proteins and performed Phi-value analysis on them. For this purpose, we measured the equilibrium unfolding transitions and kinetic refolding and unfolding reactions of the mutants using equilibrium and stopped-flow kinetic circular dichroism techniques. The results show that the mutants with mutations located in the A-helix (V8A, L12A), the B-helix (V27A), the beta-domain (L52A, W60A), the C-helix (K93A, L96A), the C-D loop (Y103F), the D-helix (L105A, L110A), and the C-terminal 3(10)-helix (W118F), have low Phi-values, less than 0.2. On the other hand, D87N, which is located on the Ca(2+)-binding site, has a high Phi-value, 0.91, indicating that tight packing of the side-chain around Asp87 occurs in the transition state. One beta-domain mutant (I55V) and three C-helix mutants (I89V, V90A, and I95V) demonstrated intermediate Phi-values, between 0.4 and 0.7. These results indicate that the folding nucleus in the transition state of goat alpha-LA is not extensively distributed over the alpha-domain of the protein, but very localized in a region that contains the Ca(2+)-binding site and the interface between the C-helix and the beta-domain. This is apparently in contrast with the fact that the molten globule state of alpha-lactalbumin has a partially formed structure inside the alpha-domain. It is concluded that the specific docking of the alpha and beta-domains at a domain interface is necessary for this protein to organize its native structure from the molten globule intermediate.


Asunto(s)
Lactalbúmina/química , Pliegue de Proteína , Animales , Dicroismo Circular , Cabras , Modelos Moleculares , Desnaturalización Proteica
3.
J Mol Biol ; 327(1): 183-91, 2003 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-12614617

RESUMEN

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.


Asunto(s)
Chaperonina 60/química , Espectrometría de Fluorescencia/métodos , Difracción de Rayos X/métodos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Regulación Alostérica , Chaperonina 60/metabolismo , Escherichia coli , Cinética , Conformación Proteica/efectos de los fármacos , Termodinámica
4.
J Mol Biol ; 321(1): 121-32, 2002 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-12139938

RESUMEN

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.


Asunto(s)
Lactalbúmina/química , Lactalbúmina/metabolismo , Pliegue de Proteína , Animales , Bovinos , Dicroismo Circular , Cinética , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , Renaturación de Proteína , Dispersión de Radiación , Soluciones , Termodinámica , Agua/metabolismo , Rayos X
5.
J Biosci Bioeng ; 100(5): 524-30, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16384791

RESUMEN

The effect of conditioning for a variety of inoculums on fermentative hydrogen production was investigated. In addition, the effects of pH condition on hydrogen fermentation and bacterial community were investigated. The effect of conditioning on hydrogen production was different depending on the inoculum types. An appreciable hydrogen production was shown with anaerobic digested sludge and lake sediment without conditioning, however, no hydrogen was produced when refuse compost and kiwi grove soil were used as inoculums without conditioning. The highest hydrogen production was obtained with heat-conditioned anaerobic digested sludge, almost the same production was also obtained with unconditioned digested sludge. The pH condition considerably affected hydrogen fermentation, hydrogen gas was efficiently produced with unconditioned anaerobic sludge when the pH was controlled at 6.0 throughout the culture period and not when only the initial pH was adjusted to 6.0 and 7.0. Hydrogen production decreased when the culture pH was only adjusted at the beginning of each batch in continuous batch culture, and additionally, bacterial community varied with the change in hydrogen production. It was suggested that Clostridium and Coprothermobacter species played important role in hydrogen fermentation, and Lactobacillus species had an adverse effect on hydrogen production.


Asunto(s)
Reactores Biológicos/microbiología , Bacterias Grampositivas/crecimiento & desarrollo , Hidrógeno/metabolismo , Aguas del Alcantarillado/microbiología , Eliminación de Residuos Líquidos , Anaerobiosis/fisiología , Calor , Concentración de Iones de Hidrógeno , Eliminación de Residuos Líquidos/métodos
8.
Biochemistry ; 44(17): 6685-92, 2005 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-15850402

RESUMEN

The intermediate in the equilibrium unfolding of canine milk lysozyme induced by a denaturant is known to be very stable with characteristics of the molten globule state. Furthermore, there are at least two kinetic intermediates during refolding of this protein: a burst-phase (first) intermediate formed within the dead time of stopped-flow measurements and a second intermediate that accumulates with a rate constant of 22 s(-)(1). To clarify the relationships of these intermediates with the equilibrium intermediate, and also to characterize the structural changes of the protein during refolding, here we studied the kinetic refolding reactions using stopped-flow circular dichroism at 10 different wavelengths and obtained the circular dichroism spectra of the intermediates. Comparison of the circular dichroism spectra of the intermediates, as well as the absence of observed kinetics in the refolding from the fully unfolded state to the equilibrium intermediate, has demonstrated that the burst-phase intermediate is equivalent to the equilibrium intermediate. The difference circular dichroism spectrum that represented changes from the kinetic intermediate to the native state had characteristics of an exciton coupling band, indicating that specific packing of tryptophan residues in this protein occurred in this phase. From these findings, we propose a schematic model of the refolding of canine milk lysozyme that is consistent with the hierarchical mechanism of protein folding.


Asunto(s)
Dicroismo Circular/métodos , Proteínas de la Leche/química , Muramidasa/química , Pliegue de Proteína , Animales , Perros , Guanidina , Calor , Cinética , Modelos Químicos , Modelos Moleculares , Desnaturalización Proteica , Proteínas Recombinantes/química , Triptófano/química
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