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1.
Injury ; 2020 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-32122626

RESUMO

OBJECTIVES: To evaluate the efficacy of conventional microbiological examinations in the diagnostics of septic joint and periprosthetic inflammations. DESIGN AND SETTING: Evidence Level IV, retrospective clinical study of case series. Patients treated with small and large joint septic inflammations or with periprosthetic joint infections (PJI) were entered into the study. Demographics, microbiological cultures and inflammatory mediators were evaluated. PARTICIPANTS: Between 2012 and 2016, total of 1116 hip and 241 knee surgeries were performed at our Department in relation to prostheses; including primary and revision arthroplasties and further surgeries due to PJI. During this period, 72 patients were operated with large joints infections or PJI and another 65 patients were treated due to small joint infections. MAIN OUTCOME MEASURES: The main outcome of interest was to evaluate the sensitivity of conventional microbiological cultures in the primary diagnostics of joint and periprosthetic infections. RESULTS: The most frequent bacteria strains were the Staphylococci: in 43 cases (22.16%) Staphylococcus aureus, in 22 cases (11.34%) coagulase-negative Staphylococcus, in 3 cases (1.54%) Staphylococcus epidermidis and in 4 cases (2.06%) methicillin-resistant S. aureus (MRSA) were detected. In 30 cases (21.88%), the primary microbiological investigation could not reveal the presence of bacteria. CONCLUSION: Based on our data, the efficacy of conventional microbiological testing in the diagnostics of different type of joint infections is questionable. Therefore, further studies are warranted to evaluate the efficacy of novel diagnostic testing tools in prospective randomized controlled trials.

2.
J Photochem Photobiol B ; 164: 276-282, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27718419

RESUMO

The effect of mammalian twinfilin-1 on the structure and dynamics of actin filaments were studied with steady state fluorescence spectroscopy, total internal reflection fluorescence microscopy and differential scanning calorimetry techniques. It was proved before that the eukaryotic budding yeast twinfilin-1 can efficiently bind and severe actin filaments in vitro at low pH values. In the present work steady-state anisotropy measurements revealed that twinfilin can bind efficiently to F-actin. Dilution-induced depolymerization assay proved that mammalian twinfilin-1 has an actin filament severing activity. This severing activity was more pronounced at low pH values. Total internal reflection fluorescence microscopy measurements could support the severing activity of mouse twinfilin-1. The average rate of depolymerization was more apparent at low pH values. The differential scanning calorimetry measurements demonstrated that mammalian twinfilin-1 could reduce the stiffness within the actin filaments before the detachment of the actin protomers. The structural and dynamic reorganization of actin can support the twinfilin-1 induced separation of actin protomers. The measured data indicated that mammalian twinfilin-1 was able to accelerate the monomers dissociation and/or sever the filaments effectively at low pH values. It was concluded that twinfilin-1 can affect the F-actin in biological processes or under stress situations when the pH is markedly under the physiological level.


Assuntos
Concentração de Íons de Hidrogênio , Proteínas dos Microfilamentos/química , Microscopia de Fluorescência/métodos , Animais , Camundongos
3.
FEBS Lett ; 589(20 Pt B): 3085-9, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26348398

RESUMO

The effects of toxofilin (an actin binding protein of Toxoplasma gondii) on G-actin was studied with spectroscopy techniques. Fluorescence anisotropy measurements proved that G-actin and toxofilin interact with 2:1 stoichiometry. The affinity of toxofilin to actin was also determined with a fluorescence anisotropy assay. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of toxofilin. The results can be explained by the shift of the nucleotide binding cleft into a closed conformational state. Differential scanning calorimetry measurements revealed that actin monomers become thermodynamically more stable due to the binding of toxofilin.


Assuntos
Proteínas de Capeamento de Actina/química , Actinas/química , Proteínas de Protozoários/química , Termodinâmica , Proteínas de Capeamento de Actina/genética , Proteínas de Capeamento de Actina/metabolismo , Actinas/metabolismo , Algoritmos , Animais , Sítios de Ligação/genética , Ligação Competitiva , Varredura Diferencial de Calorimetria , Polarização de Fluorescência , Temperatura Alta , Cinética , Modelos Químicos , Músculo Esquelético/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo , Ligação Proteica , Desnaturação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura de Transição
4.
Biochemistry ; 47(15): 4530-4, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18361506

RESUMO

Actin plays important roles in eukaryotic cell motility. During actin polymerization, the actin-bound ATP is hydrolyzed to ADP and P i. We carried out differential scanning calorimetry experiments to characterize the cooperativity of the stabilizing effect of phalloidin on actin filaments in their ADP.P i state. The ADP.P i state was mimicked by using ADP.BeF x or ADP.AlF 4. The results showed that the binding of the nucleotide analogues or phalloidin stabilized the actin filaments to a similar extent when added separately. Phalloidin binding to ADP.BeF x- or ADP.AlF 4-actin filaments further stabilized them, indicating that the mechanism by which phalloidin and the nucleotide analogues affect the filament structure was different. The results also showed that the stabilization effect of phalloidin binding to ADP.BeF x or ADP.AlF 4-bound actin filaments was not cooperative. Since the effect of phalloidin binding was cooperative in the absence of these nucleotide analogues, these results suggest that the binding of ADP.BeF x or ADP.AlF 4 to the actin modified the protomer-protomer interactions along the actin filaments.


Assuntos
Citoesqueleto de Actina/química , Difosfato de Adenosina/análogos & derivados , Faloidina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Difosfato de Adenosina/química , Berílio/química , Varredura Diferencial de Calorimetria , Fluoretos/química , Compostos Organometálicos/química
5.
Biochem Biophys Res Commun ; 368(3): 696-702, 2008 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-18261974

RESUMO

The thermodynamic properties of the actin filaments prepared from cardiomyocytes were investigated with differential scanning calorimetry. This method could distinguish between the alpha-cardiac and alpha-skeletal components of the actin filaments polymerised from ADP-actin monomers by their different melting temperatures (T(m)). Similar separation was not possible with filaments polymerised from ATP-actin monomers. Further analyses revealed that the activation energy (E(act)) was greater for filaments of alpha-skeletal actin than for alpha-cardiac actin monomers when the filaments were polymerised from ADP-actin monomers. These results showed that the alpha-cardiac actin filaments were thermodynamically less stable than the filaments of alpha-skeletal actin and their difference was nucleotide dependent. Based on these results and considering previous observations it was concluded that the existence of two actin isoforms and their nucleotide dependent conformational differences are part of the tuning regulatory mechanism by which the cardiac muscle cells can maintain their biological function under pathological conditions.


Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Actinas/química , Actinas/ultraestrutura , Fibras Musculares Esqueléticas/metabolismo , Miócitos Cardíacos/metabolismo , Nucleotídeos/química , Animais , Bovinos , Células Cultivadas , Simulação por Computador , Modelos Químicos , Modelos Moleculares , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/ultraestrutura , Relação Estrutura-Atividade
6.
BMC Struct Biol ; 7: 41, 2007 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-17588264

RESUMO

BACKGROUND: Thermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC). RESULTS: SETARAM Micro DSC-II was used to monitor the thermal denaturation of the fibre system in rigor and in the presence of nucleotide and nucleotide analogues. The AM.ADP.Pi state of the ATP hydrolysis cycle has a very short lifetime therefore, we mimicked the different intermediate states with AMP.PNP and/or inorganic phosphate analogues Vi and AlF4 or BeFx. Studying glycerol-extracted muscle fibres from the rabbit psoas muscle by DSC, three characteristic thermal transitions were detected in rigor. The thermal transitions can be assigned to myosin heads, myosin rods and actin with transition temperatures (Tm) of 52.9 +/- 0.7 degrees C, 57.9 +/- 0.7 degrees C, 63.7 +/- 1.0 degrees C. In different intermediate states of the ATP hydrolysis mimicked by nucleotide analogues a fourth thermal transition was also detected which is very likely connected with nucleotide binding domain of myosin and/or actin filaments. This transition temperature Tm4 depended on the mimicked intermediate states, and varied in the range of 66-77 degrees C. CONCLUSION: According to DSC measurements, strongly and weakly binding states of myosin to actin were significantly different. In the presence of ADP only a moderate change of the DSC pattern was detected in comparison with rigor, whereas in ADP.Pi state trapped by Vi, AlF4 or BeFx a remarkable stabilization was detected on the myosin head and actin filament which is reflected in a 3.0-10.0 degrees C shift in Tm to higher temperature. A similar effect was observed in the case of the nonhydrolyzable AMP.PNP analogue. Differential DSC measurements suggest that stabilization actin structure in the intermediate states of ATP hydrolysis may play an additional role in actin-myosin interaction.


Assuntos
Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miosinas/metabolismo , Músculos Psoas/metabolismo , Actinas/fisiologia , Animais , Varredura Diferencial de Calorimetria , Glicerol , Hidrólise , Miosinas/fisiologia , Nucleotídeos/metabolismo , Desnaturação Proteica/fisiologia , Coelhos , Temperatura Ambiente
7.
Thermochim Acta ; 463(1-2): 77-80, 2007 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-20543906

RESUMO

The effect of BeF(x) and a natural toxin (jasplakinolide) was examined on the thermal stability of actin filaments by using differential scanning calorimetry. The phosphate analogue beryllium fluoride shifted the melting temperature of actin filaments (67.4 degrees C) to 83.7 degrees C indicating that the filaments were thermodynamically more stable in their complex with ADP.BeF(x). A similar tendency was observed when the jasplakinolide was used in the absence of BeF(x). When both the ADP.BeF(x) and the jasplakinolide bound to the actin filaments their collective effect was similar to that observed with ADP.BeF(x) or jasplakinolide alone. These results suggested that ADP.BeF(x) and jasplakinolide probably stabilize the actin filaments by similar molecular mechanisms.

8.
J Biol Chem ; 281(16): 10727-36, 2006 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-16490788

RESUMO

The members of the formin family nucleate actin polymerization and play essential roles in the regulation of the actin cytoskeleton during a wide range of cellular and developmental processes. In the present work, we describe the effects of mDia1-FH2 on the conformation of actin filaments by using a temperature-dependent fluorescence resonance energy transfer method. Our results revealed that actin filaments were more flexible in the presence than in the absence of formin. The effect strongly depends on the mDia1-FH2 concentration in a way that indicates that more than one mechanism is responsible for the formin effect. In accordance with the more flexible filament structure, the thermal stability of actin decreased and the rate of phosphate dissociation from actin filaments increased in the presence of formin. The interpretation of the results supports a model in which formin binding to barbed ends makes filaments more flexible through long range allosteric interactions, whereas binding of formin to the sides of the filaments stabilizes the protomer-protomer interactions. These results suggest that formins can regulate the conformation of actin filaments and may thus also modulate the affinity of actin-binding proteins to filaments nucleated/capped by formins.


Assuntos
Citoesqueleto de Actina/química , Proteínas de Transporte/fisiologia , Proteínas Fetais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Actinas/química , Sítio Alostérico , Animais , Varredura Diferencial de Calorimetria , Proteínas de Transporte/química , Citoesqueleto/metabolismo , Dimerização , Proteínas Fetais/química , Transferência Ressonante de Energia de Fluorescência , Temperatura Alta , Camundongos , Proteínas dos Microfilamentos/química , Microscopia de Fluorescência , Modelos Biológicos , Modelos Químicos , Conformação Molecular , Proteínas Nucleares/química , Fosfatos/química , Ligação Proteica , Subunidades Proteicas/química , Proteínas/química , Temperatura Ambiente , Termodinâmica
9.
FEBS Lett ; 579(1): 6-10, 2005 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-15620683

RESUMO

The stabilisation of magnesium actin filaments by phalloidin and jasplakinolide was studied using the method of differential scanning calorimetry. The results showed that actin could adapt three conformations in the presence of drugs. One conformation was adapted in direct interaction with the drug, while another conformation was identical to that observed in the absence of drugs. A third conformation was induced through allosteric inter-protomer interactions. The effect of both drugs propagated cooperatively along the actin filaments. The number of the cooperative units determined by using a quantitative model was larger for jasplakinolide (15 actin protomers) than for phalloidin (7 protomers).


Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/efeitos dos fármacos , Depsipeptídeos/farmacologia , Modelos Químicos , Faloidina/farmacologia , Citoesqueleto de Actina/metabolismo , Animais , Varredura Diferencial de Calorimetria , Magnésio/metabolismo , Conformação Molecular , Coelhos , Temperatura Ambiente
10.
FEBS Lett ; 565(1-3): 163-6, 2004 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-15135072

RESUMO

In this work the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments was studied. Temperature dependent fluorescence resonance energy transfer measurements showed that filaments of Ca-actin became more rigid in the presence of phalloidin or jasplakinolide. Differential scanning calorimetric data implied that the stiffer filaments also had greater thermal stability in the presence of phalloidin or jasplakinolide. The fluorescence and calorimetric measurements provided evidences that the extent of stabilization by jasplakinolide was greater than that by phalloidin.


Assuntos
Actinas/química , Depsipeptídeos , Peptídeos Cíclicos/farmacologia , Faloidina/farmacologia , Animais , Antineoplásicos/farmacologia , Calorimetria , Varredura Diferencial de Calorimetria , Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência , Músculo Esquelético/metabolismo , Conformação Proteica/efeitos dos fármacos , Coelhos , Temperatura Ambiente , Termodinâmica
11.
Environ Toxicol Pharmacol ; 16(3): 163-8, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21782703

RESUMO

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on human erythrocyte membranes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model systems in order to study the effect the polycyclic aromatic hydrocarbons on lipid structure and dynamics. Eight different compounds among others naphthalene and pyrene were compared, which occur in significant concentrations in dust collected from the air in large cities. Experiments using spin label technique showed that the compounds induced mobility changes in the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes. The effects depended on the structure and concentration of the different compounds. Similarly to EPR observations, DSC measurements reported decrease of transition temperature in comparison to control DPPC vesicles. These results suggest that polycyclic aromatic hydrocarbons were able to modify the internal dynamics of erythrocyte membranes which might lead to damage of the biological functions.

12.
J Biochem Biophys Methods ; 53(1-3): 67-74, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12406588

RESUMO

The domain movement in myosin head plays a decisive role in the energy transduction process of the muscle contraction. During hydrolysis of ATP, the specific formation of strong binding of myosin head for actin causes conformational changes. As a consequence, the light chain-binding motif generates the powerstroke. In our work maleimide spin labels were covalently attached to Cys-177 residue of ELC in subfragment-1 (S1). Our goal was to study the orientation dependence and the motion of S1, which were incorporated into glycerinated skeletal muscle fibres. The electron paramagnetic resonance spectroscopy (EPR) spectra of the probes depended strongly on the orientation of the fibre axis relative to the magnetic field, indicating that the essential light chain (ELC) and the neck were ordered. The probes were undergoing rapid motion within a cone. The half-width of the cone was estimated to be 65+/-5 degrees (SD, n=8). Addition of ADP affected little the hyperfine splitting and the angular spread of the probe distribution. In the presence of ADP and orthovanadate the intensity of the spectra decreased, which showed the dissociation of S1 and this was accompanied with the disappearance of the orientation dependence.


Assuntos
Difosfato de Adenosina/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas Motores Moleculares/química , Fibras Musculares Esqueléticas/química , Miosinas/química , Difosfato de Adenosina/metabolismo , Animais , Simulação por Computador , Técnicas In Vitro , Maleimidas , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Técnicas de Sonda Molecular , Movimento (Física) , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Miosinas/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo , Estrutura Terciária de Proteína , Músculos Psoas , Coelhos , Marcadores de Spin
13.
Eur J Biochem ; 269(8): 2168-77, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11985595

RESUMO

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) was used to study the orientation of probe molecules in muscle fibers in different intermediate states of the ATP hydrolysis cycle. A separate procedure was used to obtain ST EPR spectra with precise phase settings even in the case of samples with low spectral intensity. Fibers prepared from rabbit psoas muscle were labeled with isothiocyanate spin labels at the reactive thiol sites of the catalytic domain of myosin. In comparison with rigor, a significant difference was detected in the orientation-dependence of spin labels in the ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[CH2]P) states, indicating changes in the internal dynamics and domain orientation of myosin. In the AdoPP[CH2]P state, approximately half of the myosin heads reflected the motional state of ADP-myosin, and the other half showed a different dynamic state with greater mobility.


Assuntos
Adenilil Imidodifosfato/química , Miosinas/química , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Motores Moleculares , Fibras Musculares Esqueléticas/química , Estrutura Terciária de Proteína , Coelhos
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