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BACKGROUND AND PURPOSE: Spiral MR imaging may enable improved image quality and higher scan speeds than Cartesian trajectories. We sought to compare a novel spiral 2D T2-weighted TSE sequence with a conventional Cartesian and an artifact-robust, non-Cartesian sequence named MultiVane for routine clinical brain MR imaging. MATERIALS AND METHODS: Thirty-one patients were scanned with all 3 sequences (Cartesian, 4 minutes 14 seconds; MultiVane, 2 minutes 49 seconds; spiral, 2 minutes 12 seconds) on a standard clinical 1.5T MR scanner. Three readers described the presence and location of abnormalities and lesions and graded images qualitatively in terms of overall image quality, the presence of motion and pulsation artifacts, gray-white matter differentiation, lesion conspicuity, and subjective preference. Image quality was objectivized by measuring the SNR and the coefficients of variation for CSF, GM, and WM. RESULTS: Spiral achieved a scan time reduction of 51.9% and 21.9% compared with Cartesian and MultiVane, respectively. The number and location of lesions were identical among all sequences. As for the qualitative analysis, interreader agreement was high (Krippendorff α > .75). Spiral and MultiVane both outperformed the Cartesian sequence in terms of overall image quality, the presence of motion artifacts, and subjective preference (P < .001). In terms of the presence of pulsation artifacts, gray-white matter differentiation, and lesion conspicuity, all 3 sequences performed similarly well (P > .15). Spiral and MultiVane outperformed the Cartesian sequence in coefficient of variation WM and SNR (P < .01). CONCLUSIONS: Spiral 2D T2WI TSE is feasible for routine structural brain MR imaging and offers high-quality, artifact-robust brain imaging in short scan times.
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Imageamento por Ressonância Magnética , Substância Branca , Artefatos , Encéfalo/diagnóstico por imagem , Substância Cinzenta , HumanosAssuntos
Encefalopatias/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/métodos , Neuroimagem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/diagnóstico por imagem , Estudos de Avaliação como Assunto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto JovemRESUMO
BACKGROUND: Effects of beta-amyloid accumulation on neuronal function precede the clinical manifestation of Alzheimer's disease (AD) by years and affect distinct cognitive brain networks. As previous studies suggest a link between beta-amyloid and dysregulation of excitatory and inhibitory neurotransmitters, we aimed to investigate the impact of GABA and glutamate on beta-amyloid related functional connectivity. METHODS: 29 cognitively unimpaired old-aged adults (ageâ¯=â¯70.03⯱â¯5.77â¯years) were administered 11C-Pittsburgh Compound B (PiB) positron-emission tomography (PET), and MRI at 7â¯Tesla (7T) including blood oxygen level dependent (BOLD) functional MRI (fMRI) at rest for measuring static and dynamic functional connectivity. An advanced 7T MR spectroscopic imaging (MRSI) sequence based on the free induction decay acquisition localized by outer volume suppression' (FIDLOVS) technology was used for gray matter specific measures of GABA and glutamate in the posterior cingulate and precuneus (PCP) region. RESULTS: GABA and glutamate MR-spectra indicated significantly higher levels in gray matter than in white matter. A global effect of beta-amyloid on functional connectivity in the frontal, occipital and inferior temporal lobes was observable. Interactive effects of beta-amyloid with gray matter GABA displayed positive PCP connectivity to the frontomedial regions, and the interaction of beta-amyloid with gray matter glutamate indicated positive PCP connectivity to frontal and cerebellar regions. Furthermore, decreased whole-brain but increased fronto-occipital and temporo-parietal dynamic connectivity was found, when GABA interacted with regional beta-amyloid deposits in the amygdala, frontal lobe, hippocampus, insula and striatum. CONCLUSIONS: GABA, and less so glutamate, may moderate beta-amyloid related functional connectivity. Additional research is needed to better characterize their interaction and potential impact on AD.
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Envelhecimento/fisiologia , Peptídeos beta-Amiloides/metabolismo , Cerebelo/fisiologia , Córtex Cerebral/fisiologia , Ácido Glutâmico/metabolismo , Substância Cinzenta/fisiologia , Neuroimagem/métodos , Ácido gama-Aminobutírico/metabolismo , Idoso , Envelhecimento/metabolismo , Compostos de Anilina , Cerebelo/diagnóstico por imagem , Cerebelo/metabolismo , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/metabolismo , Conectoma/métodos , Feminino , Substância Cinzenta/diagnóstico por imagem , Substância Cinzenta/metabolismo , Humanos , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Masculino , Tomografia por Emissão de Pósitrons/métodos , TiazóisRESUMO
Diffusion restriction is the morphologic hallmark of acute ischemic infarcts and excitotoxic brain injury in various cerebral pathologies. Diffusion restriction is visible as hyperintensity on DWI and as hypointensity on ADC maps. Due to the vicinity of multiple anatomic structures in the brain stem and hippocampus, very small lesions with diffusion restriction may result in severe clinical symptomatology, but these small lesions easily go undetected on standard cerebral DWI due to insufficient spatial resolution, T2* blurring, and image artifacts caused by susceptibility-related image distortions. Diffusion-weighted zonal oblique multislice-EPI with reduced FOV acquisition permits a considerable increase in spatial resolution and enhances the visualization of very small pathologic lesions in the brain stem and hippocampus. Improved performance in the depiction of different pathologic lesions with diffusion restriction in the brain stem and hippocampus using this sequence compared with standard DWI in selected cases is presented.
Assuntos
Tronco Encefálico/diagnóstico por imagem , Imagem de Difusão por Ressonância Magnética/métodos , Imagem Ecoplanar/métodos , Hipocampo/diagnóstico por imagem , Neuroimagem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Tronco Encefálico/patologia , Feminino , Hipocampo/patologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We use a scanning nanometer-scale superconducting quantum interference device to map the stray magnetic field produced by individual ferromagnetic nanotubes (FNTs) as a function of applied magnetic field. The images are taken as each FNT is led through magnetic reversal and are compared with micromagnetic simulations, which correspond to specific magnetization configurations. In magnetic fields applied perpendicular to the FNT long axis, their magnetization appears to reverse through vortex states, that is, configurations with vortex end domains or in the case of a sufficiently short FNT with a single global vortex. Geometrical imperfections in the samples and the resulting distortion of idealized magnetization configurations influence the measured stray-field patterns.
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BACKGROUND AND PURPOSE: In postmortem studies, subclinical optic nerve demyelination is very common in patients with MS but radiologic demonstration is difficult and mainly based on STIR T2WI. Our aim was to evaluate 3D double inversion recovery MR imaging for the detection of subclinical demyelinating lesions within optic nerve segments. MATERIALS AND METHODS: The signal intensities in 4 different optic nerve segments (ie, retrobulbar, canalicular, prechiasmatic, and chiasm) were evaluated on 3D double inversion recovery MR imaging in 95 patients with MS without visual symptoms within the past 3 years and in 50 patients without optic nerve pathology. We compared the signal intensities with those of the adjacent lateral rectus muscle. The evaluation was performed by a student group and an expert neuroradiologist. Statistical evaluation (the Cohen κ test) was performed. RESULTS: On the 3D double inversion recovery sequence, optic nerve segments in the comparison group were all hypointense, and an isointense nerve sheath surrounded the retrobulbar nerve segment. At least 1 optic nerve segment was isointense or hyperintense in 68 patients (72%) in the group with MS on the basis of the results of the expert neuroradiologist. Student raters were able to correctly identify optic nerve hypersignal in 97%. CONCLUSIONS: A hypersignal in at least 1 optic nerve segment on the 3D double inversion recovery sequence compared with hyposignal in optic nerve segments in the comparison group was very common in visually asymptomatic patients with MS. The signal-intensity rating of optic nerve segments could also be performed by inexperienced student readers.
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Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética/métodos , Esclerose Múltipla/diagnóstico por imagem , Neuroimagem/métodos , Nervo Óptico/diagnóstico por imagem , Adolescente , Adulto , Idoso , Doenças Desmielinizantes/diagnóstico por imagem , Doenças Desmielinizantes/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Nervo Óptico/patologia , Adulto JovemRESUMO
Quantitative Susceptibility Mapping (QSM) MRI at 7 Tesla and 11-Carbon Pittsburgh-Compound-B PET were used for investigating the relationship between brain iron and Amyloid beta (Aß) plaque-load in a context of increased risk for Alzheimer's disease (AD), as reflected by the Apolipoprotein E ε4 (APOE-e4) allele and mild cognitive impairment (MCI) in elderly subjects. Carriers of APOE-e4 with normal cognition had higher cortical Aß-plaque-load than non-carriers. In MCI an association between APOE-e4 and higher Aß-plaque-load was observable both for cortical and subcortical brain-regions. APOE-e4 and MCI was also associated with higher cortical iron. Moreover, cerebral iron significantly affected functional coupling, and was furthermore associated with increased Aß-plaque-load (R2-adjusted = 0.80, p < 0.001) and APOE-e4 carrier status (p < 0.001) in MCI. This study confirms earlier reports on an association between increased brain iron-burden and risk for neurocognitive dysfunction due to AD, and indicates that disease-progression is conferred by spatial colocalization of brain iron deposits with Aß-plaques.
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Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Disfunção Cognitiva/metabolismo , Ferro/metabolismo , Idoso , Idoso de 80 Anos ou mais , Apolipoproteína E4/genética , Encéfalo/patologia , Estudos de Casos e Controles , Disfunção Cognitiva/diagnóstico por imagem , Demografia , Feminino , Heterozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Tomografia por Emissão de Pósitrons , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologiaAssuntos
Lentes de Contato , Distrofias Hereditárias da Córnea/etiologia , Distrofias Hereditárias da Córnea/terapia , Epidermólise Bolhosa Distrófica/complicações , Epidermólise Bolhosa Distrófica/terapia , Epitélio Corneano/patologia , Idoso , Distrofias Hereditárias da Córnea/diagnóstico , Epidermólise Bolhosa Distrófica/diagnóstico , Feminino , Humanos , Pessoa de Meia-Idade , Recidiva , Resultado do TratamentoRESUMO
PURPOSE: The aim of this work is to evaluate the geometric accuracy of a prerelease version of a new six degrees of freedom (6DoF) couch. Additionally, a quality assurance method for 6DoF couches is proposed. METHODS: The main principle of the performance tests was to request a known shift for the 6DoF couch and to compare this requested shift with the actually applied shift by independently measuring the applied shift using different methods (graph paper, laser, inclinometer, and imaging system). The performance of each of the six axes was tested separately as well as in combination with the other axes. Functional cases as well as realistic clinical cases were analyzed. The tests were performed without a couch load and with a couch load of up to 200 kg and shifts in the range between -4 and +4 cm for the translational axes and between -3° and +3° for the rotational axes were applied. The quality assurance method of the new 6DoF couch was performed using a simple cube phantom and the imaging system. RESULTS: The deviations (mean ± one standard deviation) accumulated over all performance tests between the requested shifts and the measurements of the applied shifts were -0.01 ± 0.02, 0.01 ± 0.02, and 0.01 ± 0.02 cm for the longitudinal, lateral, and vertical axes, respectively. The corresponding values for the three rotational axes couch rotation, pitch, and roll were 0.03° ± 0.06°, -0.04° ± 0.12°, and -0.01° ± 0.08°, respectively. There was no difference found between the tests with and without a couch load of up to 200 kg. CONCLUSIONS: The new 6DoF couch is able to apply requested shifts with high accuracy. It has the potential to be used for treatment techniques with the highest demands in patient setup accuracy such as those needed in stereotactic treatments. Shifts can be applied efficiently and automatically. Daily quality assurance of the 6DoF couch can be performed in an easy and efficient way. Long-term stability has to be evaluated in further tests.
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Radioterapia/instrumentação , Radioterapia/métodos , Tomografia Computadorizada de Feixe Cônico , Desenho de Equipamento , Cabeça/diagnóstico por imagem , Humanos , Imageamento Tridimensional , Posicionamento do Paciente , Imagens de Fantasmas , Controle de Qualidade , Radiocirurgia , Planejamento da Radioterapia Assistida por Computador , Radioterapia Guiada por Imagem , Reprodutibilidade dos Testes , RotaçãoRESUMO
We study the thermal relaxation of artificial spin ice with photoemission electron microscopy, and are able to directly observe how such a system finds its way from an energetically excited state to the ground state. On plotting vertex-type populations as a function of time, we can characterize the relaxation, which occurs in two stages, namely a string and a domain regime. Kinetic Monte Carlo simulations agree well with the temporal evolution of the magnetic state when including disorder, and the experimental results can be explained by considering the effective interaction energy associated with the separation of pairs of vertex excitations.
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Nanopartículas de Magnetita/química , Imãs/química , Modelos Teóricos , Cinética , Magnetismo , Microscopia Eletrônica/métodos , Método de Monte CarloRESUMO
The human insula has been the focus of great attention in the last decade due to substantial progress in neuroimaging methodology and applications. Anatomical support for functional localization and interpretations, however, is still fragmented. The aim of the present study was to re-examine the microanatomical organization of the insula and relate cytoarchitectonic maps to major sulcal/gyral patterns by registration to high-resolution MR images of the same brains. The insula was divided into seven architectonic subdivisions (G, Ig, Id1-3, Ia1-2) that were charted on unfolded maps of the insula following a method used previously in monkeys. The results reveal overall similar patterns of Nissl, and to some extent also, myelin and parvalbumin (PV), as in monkeys, with a postero-dorsal to antero-ventral gradient of hypergranular to granular, dysgranular and agranular fields. Reversals occur ventrally along the inferior peri-insular sulcus (IPS), at the margin with the temporal operculum, and anteriorly at the limit with orbitofrontal cortex (OFC). A large portion of agranular cortex is characterized by a dense accumulation of the spindle-shaped von Economo neurons (VENs) in layer V. The distribution of VENs is not restricted to agranular insula but also extends into the anterior part of dysgranular fields. The patterns of intracortical myelin and of PV neuropil in the middle layers follow decreasing gradients from postero-dorsal granular to antero-ventral agranular insula, with particularly strong staining in posterior and dorsal insula. A separate PV enhanced area in the middle-dorsal insula corresponds in location to the presumed human gustatory area. Projections of the cytoarchitectonic maps onto high-resolution stereotactic MRI reveal a near concentric organization around the limen insula, with each cytoarchitectonic subdivision encompassing several major insular gyri/sulci. The dysgranular domain is the largest, taking up about half of the insula. The present study of the human insula provides a new anatomical basis for MR imaging and clinical applications.
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Córtex Cerebral/citologia , Imageamento por Ressonância Magnética , Autopsia , Humanos , Processamento de Imagem Assistida por ComputadorRESUMO
Laser speckle imaging (LSI) based on the speckle contrast analysis is a simple and robust technique for imaging of heterogeneous dynamics. LSI finds frequent application for dynamical mapping of cerebral blood flow, as it features high spatial and temporal resolution. However, the quantitative interpretation of the acquired data is not straightforward for the common case of a speckle field formed by both by moving and localized scatterers such as blood cells and bone or tissue. Here we present a novel processing scheme, we call dynamic laser speckle imaging (dLSI), that can be used to correctly extract the temporal correlation parameters from the speckle contrast measured in the presence of a static or slow-evolving background. The static light contribution is derived from the measurements by cross-correlating sequential speckle images. In-vivo speckle imaging experiments performed in the rodent brain demonstrate that dLSI leads to improved results. The cerebral hemodynamic response observed through the thinned and intact skull are more pronounced in the dLSI images as compared to the standard speckle contrast analysis. The proposed method also yields benefits with respect to the quality of the speckle images by suppressing contributions of non-uniformly distributed specular reflections.
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Algoritmos , Velocidade do Fluxo Sanguíneo/fisiologia , Circulação Cerebrovascular/fisiologia , Interpretação de Imagem Assistida por Computador/métodos , Fluxometria por Laser-Doppler/métodos , HumanosRESUMO
Two-photon microscopy is a powerful method in biomedical research that allows functional and anatomical imaging at a subcellular resolution in vivo. The technique is seriously hampered by absorption and scattering of light by blood, which prevents imaging through large vessels. Here, we demonstrate in the rat cerebral cortex that blood replacement by perfluorocarbon emulsion, a compound also used in human critical care medicine, yields superior image quality, while preserving neuronal integrity. Shadows of large superficial vessels disappear completely and cells can be imaged underneath them. For the first time, it is possible to image complete populations of neurons and astrocytes in the upper layers of neocortex in vivo.
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Substitutos Sanguíneos , Fluorocarbonos , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Transfusão de Sangue , Sinalização do Cálcio , Humanos , Masculino , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Córtex Somatossensorial/irrigação sanguínea , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismoRESUMO
Dental identification is the most valuable method to identify human remains in single cases with major postmortem alterations as well as in mass casualties because of its practicability and demanding reliability. Computed tomography (CT) has been investigated as a supportive tool for forensic identification and has proven to be valuable. It can also scan the dentition of a deceased within minutes. In the present study, we investigated currently used restorative materials using ultra-high-resolution dual-source CT and the extended CT scale for the purpose of a color-encoded, in scale, and artifact-free visualization in 3D volume rendering. In 122 human molars, 220 cavities with 2-, 3-, 4- and 5-mm diameter were prepared. With presently used filling materials (different composites, temporary filling materials, ceramic, and liner), these cavities were restored in six teeth for each material and cavity size (exception amalgam n = 1). The teeth were CT scanned and images reconstructed using an extended CT scale. Filling materials were analyzed in terms of resulting Hounsfield units (HU) and filling size representation within the images. Varying restorative materials showed distinctively differing radiopacities allowing for CT-data-based discrimination. Particularly, ceramic and composite fillings could be differentiated. The HU values were used to generate an updated volume-rendering preset for postmortem extended CT scale data of the dentition to easily visualize the position of restorations, the shape (in scale), and the material used which is color encoded in 3D. The results provide the scientific background for the application of 3D volume rendering to visualize the human dentition for forensic identification purposes.
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Restauração Dentária Permanente , Restauração Dentária Temporária , Odontologia Legal/métodos , Radiografia Dentária/métodos , Tomografia Computadorizada por Raios X/métodos , Cerâmica , Compômeros , Resinas Compostas , Porcelana Dentária , Adesivos Dentinários , Cimentos de Ionômeros de Vidro , Humanos , Maleatos , Metilmetacrilatos , Dente Molar/diagnóstico por imagem , Cimentos de Resina , Cimento de Óxido de Zinco e EugenolRESUMO
Abstract The aim of this study was to evaluate if hypoxia in feline fibrosarcomas can be detected. This was done using positron emission tomography (PET), two hypoxia tracers and polarographic pO(2) measurements. Of the seven cats included, five received [(18)F]-fluoromisonidazole and two 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide. Perfusion was evaluated with [(15)O]-H(2)O (n = 4) and with contrast-enhanced power Doppler ultrasonography (n = 5). Hypoxia was detected in three cats. Polarographic pO(2) measurements did not confirm PET results. In the ultrasonographic evaluation, low vascularity and low perfusion were seen with a peripheral vascular pattern and no perfusion in the centre of the tumour. This was in contrast to the [(15)O]-H(2)O scans, where central perfusion of the tumour was also found. In conclusion, it appears that hypoxia exists in this tumour type. The presence of tumour necrosis and heterogeneous hypoxia patterns in these tumours may explain the found discrepancies between the applied techniques.
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Oxidative metabolism and cerebral blood flow (CBF) are two of the most important measures in neuroimaging. However, results from concurrent imaging of the two with high spatial and temporal resolution have never been published. We used flavoprotein autofluorescence (AF) and laser speckle imaging (LSI) in the anaesthetized rat to map oxidative metabolism and CBF in response to single vibrissa stimulation. Autofluorescence responses reflecting oxidative metabolism demonstrated a fast increase with a delay of 0.1 s. The sign-reversed speckle contrast reflecting CBF started to rise with a delay of 0.6 s and reached its maximum 1.4 s after the stimulation offset. The fractional signal changes were 2.0% in AF and 9.7% in LSI. Pixelwise modelling revealed that CBF maps spread over an area up to 2.5-times larger than metabolic maps. The results provide evidence that the increase in cerebral oxidative metabolism in response to sensory stimulation is considerably faster and more localized than the CBF response. This suggests that future developments in functional imaging concentrating on the metabolic response promise an increased spatial resolution.
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Córtex Cerebral/fisiologia , Circulação Cerebrovascular/fisiologia , Vibrissas/fisiologia , Animais , Córtex Cerebral/irrigação sanguínea , Flavoproteínas/metabolismo , Processamento de Imagem Assistida por Computador , Fluxometria por Laser-Doppler , Lasers , Imageamento por Ressonância Magnética/métodos , Masculino , Oxigênio/sangue , Estimulação Física/métodos , Ratos , Ratos Sprague-Dawley , Tempo de Reação , Fluxo Sanguíneo Regional/fisiologia , Vibrissas/inervaçãoRESUMO
In human medicine positron emission tomography (PET) is a modern diagnostic imaging method. In the present paper we outline the physical principles of PET and give an overview over the main clinic fields where PET is being used, such as neurology, cardiology and oncology. Moreover, we present a current project in veterinary medicine (in collaboration with the Paul Scherrer Institute and the University Hospital Zurich), where a hypoxia tracer is applied to dogs and cats suffering from spontaneous tumors. Finally new developments in the field of PET were discussed.
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Tomografia Computadorizada de Emissão/veterinária , Medicina Veterinária/instrumentação , Medicina Veterinária/métodos , Animais , Técnicas de Diagnóstico Neurológico/veterinária , Cardiopatias/diagnóstico , Cardiopatias/diagnóstico por imagem , Cardiopatias/veterinária , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias/diagnóstico , Neoplasias/diagnóstico por imagem , Neoplasias/veterinária , Tomografia Computadorizada de Emissão/instrumentação , Tomografia Computadorizada de Emissão/métodosRESUMO
Aspergillus fumigatus phytase has previously been identified as a phytase with a series of favourable properties that may be relevant in animal and human nutrition, both for maximising phytic acid degradation and for increasing mineral and amino acid availability. To study the natural variability in amino acid sequence and its impact on the catalytic properties of the enzyme, we cloned and overexpressed the phytase genes and proteins from six new purported A. fumigatus isolates. Five of these phytases displayed < or= 2 amino acid substitutions and had virtually identical stability and catalytic properties when compared with the previously described A. fumigatus ATCC 13073 phytase. In contrast, the phytase from isolate ATCC 32239 ( Sartorya fumigata, the anamorph of which was identified as A. fumigatus) was more divergent (only 86% amino acid sequence identity), had a higher specific activity with phytic acid, and displayed distinct differences in substrate specificity and pH-activity profile. Finally, comparative experiments confirmed the favourable stability and catalytic properties of A. fumigatus phytase.
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6-Fitase/genética , 6-Fitase/metabolismo , Aspergillus fumigatus/enzimologia , 6-Fitase/química , Sequência de Aminoácidos , Substituição de Aminoácidos/fisiologia , Catálise , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/fisiologia , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Ácido Fítico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.
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Evolução Molecular Direcionada/métodos , Desenho de Fármacos , Estabilidade de Medicamentos , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Alinhamento de Sequência/métodos , Temperatura Alta , Engenharia de Proteínas/métodosRESUMO
Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60 degrees C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U. mg, of protein(-1) and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, (1)H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26).