RESUMO
A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (delta23KGF-R144Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a beta-trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti-parallel beta-strands in which three pairs of the strands form a six-stranded beta-barrel structure and the other three pairs of beta-strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well-defined beta strands, which form five double-stranded anti-parallel beta-sheets. A sixth poorly defined beta-strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24-33) compared to the other FGF structures, which are important for biological activity. Based on mutagenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His127 predominantly confer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of delta23KGF-R144Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main-chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic.
Assuntos
Cristalografia por Raios X , Análise Mutacional de DNA , Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/química , Sequência de Aminoácidos , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Heparina/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
A precise knowledge of the myocardial fiber architecture is essential to accurately understand and interpret cardiac electrical and mechanical functions. Diffusion tensor imaging has been used to noninvasively and quantitatively characterize myocardial fiber orientations. However, because the approach necessitates diffusion to be measured in multiple encoding directions and frequently at multiple weighting levels, the required data set size may present a limitation on its acquisition time efficiency. Applying the principles of reduced encoding imaging (REI), four basic reconstruction schemes, keyhole using direct substitution, keyhole with baseline correction, symmetrically encoded REI with generalized-series reconstruction (RIGR), and asymmetrically encoded RIGR, are evaluated in terms of their accuracy in diffusion tensorfiber orientation mapping of excised myocardial samples. Results show that the performances of all REI schemes, at approximately 50% reduced encoding, are at least comparable with that of a control experiment consisting of proportionally reduced number of full k-space images. Moreover, although performances of the symmetrically and asymmetrically encoded RIGR schemes are similar, both methods provide significant improvements over the control experiment and the direct-substitution keyhole technique. These findings demonstrate the potential of the general REI methodology for diffusion tensor imaging and pave the way for modified schemes involving rapid imaging sequences or alternative k-space sampling strategies to achieve even better data acquisition time efficiency and performance.
Assuntos
Coração/anatomia & histologia , Imageamento por Ressonância Magnética/métodos , Fibras Musculares Esqueléticas/citologia , Miocárdio/citologia , Análise de Variância , Animais , Anisotropia , Cães , Aumento da Imagem , Modelos BiológicosRESUMO
NMR measurements of anisotropic diffusion were studied using a three-dimensional random-walk model. It was found that the apparent diffusion coefficient can be expressed in a canonical form as the product of a diagonal matrix, an orthonormal rotation matrix, and a vector representing the encoding magnetic field gradient. The diffusion coefficient can be interpreted as the sum of the corresponding coefficients measured along the principal diffusion axes, weighted by the squares of the directional cosines of the encoding direction with respect to the principal axes. The analysis revealed that determining the orientation of anisotropy, in a cylindrically symmetric system, requires a minimum of four diffusion measurements. A special pulse sequence which minimized gradient cross-terms and possible restricted diffusion effects was used to characterize diffusion anisotropy in cut chicken gizzards. Diffusion coefficients parallel to the muscle fibers were found to be approximately two to three times larger than those in the transverse direction. Furthermore, the method was successful in detecting the angular change when the sample was rotated by 30 degrees. Results indicate that the proposed approach to measure fiber orientation is valid and may be used to improve the time efficiency of diffusion anisotropy measurements.
Assuntos
Espectroscopia de Ressonância Magnética , Fibras Musculares Esqueléticas/ultraestrutura , Algoritmos , Animais , Galinhas , Difusão , Mucosa Gástrica/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Modelos Teóricos , Fibras Musculares Esqueléticas/metabolismo , Reprodutibilidade dos Testes , Rotação , Estômago/ultraestrutura , Água/metabolismoRESUMO
Morphologically accurate biomechanical models of the intervertebral disc anulus fibrosus (AF) require precise knowledge of its lamellar architecture; however, available methods of assessment are limited by poor spatial resolution or the destructive nature of the technique. In a novel approach, diffusion tensor microscopy was used in this study to characterize the microstructure of excised porcine AF samples. Results show diffusion in the AF to be anisotropic. The orientations of anisotropy exhibit a layered morphology that agrees with light micrographs of the corresponding samples, and the behavior of the orientation angles is consistent with the known AF collagen fiber architecture. A static magnetic field-dependent relaxation anisotropy was observed in the AF, which has methodological implications for magnetic resonance (MR) imaging of ordered collagenous tissues. These findings present MR diffusion tensor microscopy as a potentially valuable tool to assess quantitatively and nondestructively water diffusion anisotropy and lamellar structure of the intervertebral disc AF.
Assuntos
Disco Intervertebral/anatomia & histologia , Imageamento por Ressonância Magnética/métodos , Microscopia/métodos , Algoritmos , Animais , Fenômenos Biomecânicos , Água Corporal/química , Colágeno/ultraestrutura , Difusão , Processamento de Imagem Assistida por Computador/métodos , Disco Intervertebral/metabolismo , Disco Intervertebral/ultraestrutura , Microscopia Eletrônica , Modelos Biológicos , Reprodutibilidade dos Testes , SuínosRESUMO
The apparent diffusion coefficient (ADC) of water was measured in single Aplysia californica neurons by using NMR microscopy encoded in each of two perpendicular gradient directions. Comparisons of the mean ADCs of the gross nuclear and cytoplasmic compartments in five cells, and 50 subregions within these cells, showed no significant difference between the diffusion measurements in the majority of cases. Since anisotropic diffusion would make the ADC dependent on the encoding direction, the results indicate that the ADC in these single neurons is isotropic at the spatial and temporal resolutions used in these studies. Consequently, a single scalar ADC measurement is sufficient for characterizing the ADC in these cells, hence reducing the acquisition time and measurement complexity that would have been required had the ADC been anisotropic.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Microscopia/métodos , Neurônios/metabolismo , Água/metabolismo , Animais , Anisotropia , Aplysia , Difusão , Neurônios/citologiaRESUMO
Nuclear magnetic resonance (NMR) characteristics of water in perfused single neurons undergoing a 20% hypotonic perturbation were examined quantitatively using NMR microscopy. The transverse relaxation times (T2) in the cytoplasm and nucleus increased by 24.0 +/- 8.5% (average +/- SE, n = 8) and 29.7 +/- 5.3% (n = 6), respectively, whereas the apparent diffusion coefficients (ADC) showed no significant change. These findings are consistent with the behaviors of a perfect osmometer and with accepted molecular relaxation and diffusion models and have significant impacts on current views of properties of cellular water. Furthermore, the results suggest that the increase of tissue intracellular-to-extracellular volume ratio during cell swelling is the predominant mechanism underlying the ADC reduction in acute brain ischemia. These data are the first direct quantitative measurements of the NMR characteristics of water in the cytoplasm and nucleus of single cells undergoing physiological perturbations and may lead to an improved diagnostic capability for NMR imaging in a variety of disease states.
Assuntos
Neurônios/fisiologia , Água/fisiologia , Animais , Aplysia , Soluções Hipotônicas , Espectroscopia de Ressonância Magnética , Concentração OsmolarRESUMO
Adiabatic pulses, although useful in generating uniform spin nutation in the presence of inhomogeneous B1 fields, are limited for NMR imaging applications due to the lack of slice-selective excitation capability. Selective excitation techniques using gradient modulation have been introduced; however, present methods require either a minimum of two excitations or eight adiabatic segments. Here, a scheme is presented that allows single-shot, arbitrary flip-angle, and slice-selective excitation with only four adiabatic half-passage segments. The technique is demonstrated via computer simulation and experimental tests on a phantom. Furthermore, issues associated with the implementation of these gradient-modulated adiabatic pulses are discussed.
Assuntos
Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Simulação por Computador , Análise de Fourier , Humanos , Imagens de Fantasmas , Sensibilidade e EspecificidadeRESUMO
Functional mapping of the rat somatosensory cortex was performed with T2*-sensitized MRI using a forepaw electrical stimulation model in alpha-chloralose-anesthetized rats at 7 T under both normocapnia and mild hyperventilation-induced hypocapnia. A highly localized activation area, consistent with the known somatosensory cortical region, was detected in all seven animals studied during hypocapnia and in five of the same animals during normocapnia. Quantitatively, hypocapnia was found to significantly increase both the size of the fMRI activation area (3.4 +/- 0.6 mm2 versus 1.5 +/- 0.6 mm2 in normocapnia, mean +/- standard error, n = 7, P < 0.03) and the average fMRI signal intensity increase (3.4 +/- 0.6% versus 2.7 +/- 0.4%, n = 5, P < 0.05). The increased sensitivity of fMRI to functional activation may reflect a widened arterial-venous oxygenation difference resulting from an increased effective oxygen extraction during hyperventilation. The dependence of the fMRI response on the ventilation state underscores the need to control for physiological parameters in animal fMRI studies.
Assuntos
Dióxido de Carbono/sangue , Imageamento por Ressonância Magnética/métodos , Oxigênio/sangue , Córtex Somatossensorial/irrigação sanguínea , Vias Aferentes/fisiologia , Animais , Mapeamento Encefálico , Estimulação Elétrica , Membro Anterior/inervação , Ratos , Ratos Sprague-Dawley , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
We have used an isolated neuronal preparation of single Aplysia californica L7 cells to study the effects of development on the nuclear magnetic resonance (NMR) characteristics, T2 spin-spin relaxation rate (RT2) and apparent diffusion coefficient (D), of intracellular water within the nuclear and cytoplasmic compartments. These studies demonstrate a significant correlation of animal weight, but not age, with RT2. On the other hand, D was not significantly different as a function of either age or weight in single L7 neurons. Demonstration of maturation dependence in single cells is important in understanding the cellular origins of developmental effects on NMR characteristics in cell assemblies such as brain.
Assuntos
Aplysia/crescimento & desenvolvimento , Neurônios/fisiologia , Fatores Etários , Animais , Aplysia/anatomia & histologia , Peso Corporal , Compartimento Celular , Núcleo Celular/fisiologia , Tamanho Celular , Citoplasma/fisiologia , Difusão , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica , Água/fisiologiaRESUMO
Damage to the meniscus after trauma or injury is associated with detrimental changes in joint function that can lead to pain, disability, and degenerative joint changes. Recently, tissue engineering strategies for meniscal repair have been suggested including using biocompatible grafts as a substrate for regeneration, and cellular supplementation to promote remodeling and healing. Little is known, however, about the contributions of these novel repair strategies to restoration of normal meniscal function. Biomechanical factors play a role in the design and synthesis of tissue engineered biomaterials and bioreactors, and also are important for evaluating the efficacy of these new strategies for restoring normal meniscal function. In this report, an overview is presented of biomechanical factors that are critical to meniscal function followed by a review of biomechanical considerations for the design and evaluation of tissue engineered strategies for meniscal repair. Recommendations for future study of biomechanical factors in tissue engineered meniscal repair also are provided.
Assuntos
Materiais Biocompatíveis , Biotecnologia , Técnicas de Cultura de Células , Meniscos Tibiais/citologia , Meniscos Tibiais/fisiologia , Regeneração , Animais , Fenômenos Biomecânicos , Transplante de Células , Meniscos Tibiais/cirurgiaRESUMO
The apparent diffusion coefficient (ADC) of water after regional myocardial ischemia was measured in isolated, perfused rabbit hearts by using magnetic resonance imaging (MRI) techniques. After ligation of the left anterior descending coronary artery, the ADC of the nonperfused region showed a gradual but significant decreasing trend over time, whereas that of the normally perfused myocardium remained constant. Morphological analysis revealed that the ADC decrease reflected the expansion of a subregion of reduced ADC within the nonperfused myocardium. The dynamics of the diffusion change and the morphological progression of the affected tissue suggest that the ADC decrease may be linked to the onset of myocardial infarction, which is known to involve myocyte swelling. The ADC reduction provides a potentially valuable MRI tissue-contrast mechanism for noninvasively determining the viability of the ischemic myocardium and assessing the dynamics of acute myocardial infarction.
Assuntos
Água Corporal/fisiologia , Coração/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Animais , Vasos Coronários/fisiologia , Difusão , Coração/fisiologia , Técnicas In Vitro , Imageamento por Ressonância Magnética , Masculino , Modelos Cardiovasculares , Miocárdio/patologia , CoelhosRESUMO
Nonmonoexponential MR diffusion decay behavior has been observed at high diffusion-weighting strengths for cell aggregates and tissues, including the myocardium; however, implications for myocardial MR diffusion tensor imaging are largely unknown. In this study, a slow-exchange-limit, two-component diffusion tensor model was fitted to diffusion-weighted images obtained in isolated, perfused rat hearts. Results indicate that there are at least two distinct components of anisotropic diffusion, characterized by a "fast" component whose principal diffusivity is comparable to that of the perfusate, and a highly anisotropic "slow" component. It is speculated that the two components correspond to tissue compartments and have a general agreement with the orientations of anisotropy, or fiber orientations, in the myocardium. Moreover, consideration of previous studies of myocardial diffusion suggests that the presently observed fast component may likely be dominated by diffusion in the vascular space, whereas the slow component may include the intracellular and interstitial compartments. The implications of the results for myocardial fiber orientation mapping and limitations of the current two-component model used are also discussed.
Assuntos
Aumento da Imagem , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Miocárdio/patologia , Animais , Anisotropia , Difusão , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Teleost embryos have not been successfully cryopreserved. To formulate successful cryopreservation protocols, the distribution and cellular permeability to water must be understood. In this paper, the zebrafish (Brachydanio rerio) was used as a model for basic studies of the distribution to permeability to water. These embryos are a complex multi-compartmental system composed of two membrane-limited compartments, a large yolk (surrounded by the yolk syncytial layer) and differentiating blastoderm cells (each surrounded by a plasma membrane). Due to the complexity of this system, a variety of techniques, including magnetic resonance microscopy and electron spin resonance, was used to measure the water in these compartments. Cellular water was distributed unequally in each compartment. At the 6-somite stage, the percent water (V/V) was distributed as follows: total in embryo = 74%, total in yolk = 42%, and total in blastoderm = 82%. A one-compartment model was used to analyze kinetic, osmotic shrinkage data and determine a phenomenological water permeability parameter, Lp, assuming intracellular isosmotic compartments of either 40 or 300 mosm. This analysis revealed that the membrane permeability changed (P < 0.05) during development. During the 75% epiboly to 3-somite stage, the mean membrane permeability remained constant (Lp = 0.022 +/- 0.002 micron x min-1atm-1 [mean +/- S.E.M.] assuming isosmotic is 40 mosm or Lp = 0.049 +/- 0.008 micron x min-1atm-1 assuming isosmotic is 300 mosm). However, at the 6-somite stage, Lp increased twofold (Lp = 0.040 +/- 0.004 micron x min-1atm-1 assuming isosmotic is 40 mosm or Lp = 0.100 +/- 0.017 micron x min-1atm-1 assuming isosmotic is 300 mosm). Therefore, the low permeability of the zebrafish embryo coupled with its large size (and consequent low area to volume ratio) led to a very slow osmotic response that should be considered before formulating cryopreservation protocols.
Assuntos
Água Corporal/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Embrião não Mamífero/fisiologia , Peixe-Zebra/embriologia , Animais , Blastoderma/metabolismo , Criopreservação , Gema de Ovo/metabolismo , Imageamento por Ressonância Magnética/métodos , Osmose/fisiologiaRESUMO
A modified spin-echo pulse sequence is described that enables accurate T2 measurements to be made in NMR microimaging experiments. The modified sequence eliminates cumulative diffusion losses that lead to an underestimation of the T2 relaxation time using conventional spin-echo pulse sequences. The approach is theoretically justified and confirmed in comparative experiments on phantoms.
Assuntos
Espectroscopia de Ressonância Magnética , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Imagens de FantasmasRESUMO
Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.
Assuntos
Blastoderma , Criopreservação , Crioprotetores , Embrião não Mamífero , Animais , Blastoderma/citologia , Blastoderma/fisiologia , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Dimetil Sulfóxido , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Cinética , Espectroscopia de Ressonância Magnética/métodos , Propilenoglicol , Propilenoglicóis , Fatores de Tempo , Saco Vitelino/citologia , Saco Vitelino/fisiologia , Peixe-ZebraRESUMO
Functional properties of the myocardium are mediated by the tissue structure. Consequently, proper physiological studies and modeling necessitate a precise knowledge of the fiber orientation. Magnetic resonance (MR) diffusion tensor imaging techniques have been used as a nondestructive means to characterize tissue fiber structure; however, the descriptions so far have been mostly qualitative. This study presents a direct, quantitative comparison of high-resolution MR fiber mapping and histology measurements in a block of excised canine myocardium. Results show an excellent correspondence of the measured fiber angles not only on a point-by-point basis (average difference of -2.30 +/- 0.98 degrees, n = 239) but also in the transmural rotation of the helix angles (average correlation coefficient of 0.942 +/- 0.008 with average false-positive probability of 0.004 +/- 0.001, n = 24). These data strongly support the hypothesis that the eigenvector of the largest MR diffusion tensor eigenvalue coincides with the orientation of the local myocardial fibers and underscore the potential of MR imaging as a noninvasive, three-dimensional modality to characterize tissue fiber architecture.
Assuntos
Coração/anatomia & histologia , Modelos Biológicos , Modelos Teóricos , Fibras Musculares Esqueléticas/citologia , Miocárdio/citologia , Animais , Cães , Imageamento por Ressonância MagnéticaRESUMO
NMR microimages of single neural cells were acquired at 500 MHz using a conventional spin echo pulse sequence and a line-narrowing sequence that eliminates susceptibility effects. The data show that any contribution to the measured T2 relaxation rate arising from diffusion in local field inhomogeneities using spin echo sequences at high fields and high spatial resolution is relatively small. We conclude that the measured T2 difference between the nucleus and cytoplasm in these cells represents primarily a true T2 relaxation effect arising from the interactions of water with macromolecules in the two compartments and does not result from microsusceptibility differences. These observations have implications regarding water compartmentation in single cells and the interpretation of the MR characteristics of tissues in vivo.
Assuntos
Espectroscopia de Ressonância Magnética , Neurônios/citologia , Animais , Aplysia , Processamento de Imagem Assistida por Computador , Espectroscopia de Ressonância Magnética/métodos , MicroscopiaRESUMO
Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrine mode. We employed a mammalian expression system to synthesize recombinant human KGF and isolated two preparations, KGF-a and KGF-b, from medium conditioned by Chinese hamster ovary cells. On an SDS-PAGE gel, KGF-a migrates as two bands near 25-29 kDa and contains both N- and O-linked sugar moieties attached near the N-terminus. Detailed structural characterization confirms that KGF-a contains a single amino acid sequence predicted from cDNA sequence and the molecule has two intramolecular disulfide bridges, Cys1-Cys15 and Cys102-Cys106. An additional Cys at position 40 is free and resides in a solvent-inaccessible environment. Mass spectrometric analyses of KGF-a peptides verify the occurrence of several post-translational modifications in the molecule, including partial oxidation at Met28, partial sulfation at Tyr27, and glycosylation at Asn14 and Thr22. The Asn-linked carbohydrate structures are heterogeneous, which include biantennary, triantennary, and tetraantennary structures with none or up to four sialic acids attached to various structures, while the Thr-linked carbohydrates contain typical mucin-type structures. KGF-b is an N-terminally truncated form of KGF-a posttranslationally processed at Arg23 and is not glycosylated. Both KGF-a and KGF-b forms are capable of stimulating DNA synthesis in quiescent Balb/MK mouse epidermal keratinocytes.