X射线衍射法在研究蛋白质动态过程中的应用

介绍了X射线衍射技术在研究蛋白质动态过程中的应用.首先介绍了用常规X射线衍射法和劳埃X射线衍射法等数据采样法研究反应时间为几分钟的蛋白质催化反应.然后介绍了通过选择不匹配底物,不适宜酸度,选择温度和酸度的跳跃,金属和光化学瞬时激发达到反应的同步来研究反应时间为几秒钟的蛋白质催化反应.     

X-Ray Diffraction Studies of the Sulfur Globules Accumulated by Chromatium Species

Isolated wet and dried sulfur globules, obtained by osmotic lysis of lysozyme-ethylenediaminetetraacetic acid prepared spheroplasts of Chromatium okenii, C. weissei, and C. warmingii, were studied by polarizing microscopy and X-ray diffraction. When viewed through crossed Nicol prisms, the sulfur globules, whether in the cell or isolated in a pure, wet state, had a characteristic maltese cross appearance. The observation that rotation of the mount did not change the orientation of the arms suggested a symmetrical radial arrangement of the birefringent units. X-ray diffraction patterns of freshly isolated, wet sulfur globules gave two broad and diffuse diffraction rings with maxima at 0.36 and 0.52 nm. This pattern closely resembled the diffraction pattern of liquid sulfur. When allowed to stand in the wet state, the sulfur globules eventually converted into crystalline orthorhombic sulfur after passing through an unstable crystalline phase not previously described by X-ray diffraction. Vacuum drying of the sulfur globules accelerated the change into crystalline orthorhombic sulfur.     

Note on the Theory of X-Ray Diffraction by Spherical Shell Structures

An equation is derived, which connects two functions P(r) and g(x), the first being related to the scattering intensity by a simple transformation, the second to the elctron density distribution of a spherically symmetric structure. This relation seems to be a suitable starting point for an analysis of shell structures from diffraction patterns.     

Co2+-Tyr配合物的合成及X射线衍射分析

研究了Co2+-Tyr配合物的合成方法,探讨了合成工艺的主要影响因素,确定了最佳反应时间、温度、pH值及原料配比,采用X射线衍射光谱及元素分析对配合物结构进行了分析鉴定,结果表明Co2+能与Tyr形成配合物。     

Direct Determination of the Structure of Barium Stearate Multilayers by X-Ray Diffraction

Diffraction of X-rays is recorded from barium stearate multilayer systems with from 2 to 60 double layers or unit cells. The generalized Patterson function P′(x) is calculated by an integral Fourier transform of observed intensity data from a specimen containing only two unit cells. The Patterson function P₀(x) of a single unit cell is determined from P′(x) and the electron density distribution of a bimolecular leaflet is obtained by a deconvolution procedure of P₀(x) after Hosemann and Bagchi. The electron density distribution is also calculated independently by a conventional Fourier synthesis with an experimentally established set of phases. The results of the two methods are consistent and fit a physical model of the bimolecular leaflet. A direct analysis, therefore, can be performed if diffraction is observed from multilayer systems with a small number of unit cells.     

Expression and Localization of Lung Surfactant Proteins in Human Testis

Background

Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions.

Objective

The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated.

Results

Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig.

Conclusion

Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP''s was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor.     

Reprecipitation during the Preparation of Demineralized Sections I. Histological and X-Ray Diffraction Observations

The histological features of reprecipitation artefacts occasionally present in demineralized sections of teeth and bone are described. X-ray diffraction analysis indicates, that they are aystalline and composed of secondary calcium phosphates.     

Isolation and Characterization of Proteins Associated with the Lung Surfactant System

Rabbit lung washings and purified lung surfactant were delipidated without precipitation or loss of protein. This enabled effective study of the proteins by electrophoretic and immunoelectrophoretlc techniques. The lung washings contained secretory immunoglobulin A and several serum proteins. The protein composition of purified lung surfactant was the same as the unfractionated lung washings confirming our previous study which indicated that there is no specific protein associated with surfactant phospholipids obtained by alveolar lavage with isotonic saline.     

Surfactant Lipidomics in Healthy Children and Childhood Interstitial Lung Disease

BackgroundLipids account for the majority of pulmonary surfactant, which is essential for normal breathing. We asked if interstitial lung diseases (ILD) in children may disrupt alveolar surfactant and give clues for disease categorization.MethodsComprehensive lipidomics profiles of broncho-alveolar lavage fluid were generated in 115 children by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Two reference populations were compared to a broad range of children with ILD.ResultsClass and species composition in healthy children did not differ from that in children with ILD related to diffuse developmental disorders, chronic tachypnoe of infancy, ILD related to lung vessels and the heart, and ILD related to reactive lymphoid lesions. As groups, ILDs related to the alveolar surfactant region, ILD related to unclear respiratory distress syndrome in the mature neonate, or in part ILD related to growth abnormalities reflecting deficient alveolarisation, had significant alterations of some surfactant specific phospholipids. Additionally, lipids derived from inflammatory processes were identified and differentiated. In children with ABCA3-deficiency from two ILD causing mutations saturated and monounsaturated phosphatidylcholine species with 30 and 32 carbons and almost all phosphatidylglycerol species were severely reduced. In other alveolar disorders lipidomic profiles may be of less diagnostic value, but nevertheless may substantiate lack of significant involvement of mechanisms related to surfactant lipid metabolism.ConclusionsLipidomic profiling may identify specific forms of ILD in children with surfactant alterations and characterized the molecular species pattern likely to be transported by ABCA3 in vivo.     

The Formation of Otoliths in the Frog Rana esculenta. Scanning Electron Microscopic and X-Ray Diffraction Studies

Two crystal forms of calcium carbonate were observed: calcite (utricle) and aragonite (saccule, lagena, endolymphatic sac). The first step in otolith formation is the appearance of organic structures in the macula. The subsequent step is characterized by fast growing primitive crystals with a prismatic habitus that successively transform into adult or mature crystals. With the metamorphosis, the aragonite crystals of the endolymphatic organ show clear signs of erosion that can be related to a process of CaCO₃ mobilization from such deposits.     

Mature Surfactant Protein-B Expression by Immunohistochemistry as a Marker for Surfactant System Development in the Fetal Sheep Lung

Evaluation of the number of type II alveolar epithelial cells (AECs) is an important measure of the lung’s ability to produce surfactant. Immunohistochemical staining of these cells in lung tissue commonly uses antibodies directed against mature surfactant protein (SP)-C, which is regarded as a reliable SP marker of type II AECs in rodents. There has been no study demonstrating reliable markers for surfactant system maturation by immunohistochemistry in the fetal sheep lung despite being widely used as a model to study lung development. Here we examine staining of a panel of surfactant pro-proteins (pro–SP-B and pro–SP-C) and mature proteins (SP-B and SP-C) in the fetal sheep lung during late gestation in the saccular/alveolar phase of development (120, 130, and 140 days), with term being 150 ± 3 days, to identify the most reliable marker of surfactant producing cells in this species. Results from this study indicate that during late gestation, use of anti-SP-B antibodies in the sheep lung yields significantly higher cell counts in the alveolar epithelium than SP-C antibodies. Furthermore, this study highlights that mature SP-B antibodies are more reliable markers than SP-C antibodies to evaluate surfactant maturation in the fetal sheep lung by immunohistochemistry.     

Dynamics of Thin-Filament Activation in Rabbit Skeletal Muscle Fibers Examined by Time-Resolved X-Ray Diffraction

By using skinned-rabbit skeletal muscle fibers, the time courses of changes of thin filament-based x-ray reflections were followed at a 3.4-ms time resolution during thin-filament activation. To discriminate between the effects of calcium binding and myosin binding on thin-filament activity, measurements were performed after caged-calcium photolysis in fibers with full-filament or no-filament overlap, or during force recovery after a quick release. All three reflections examined, i.e., the second actin layer line (second ALL, reporting the tropomyosin movement), the sixth ALL (reporting actin structural change), and the meridional troponin reflections, exhibited calcium-induced and myosin-induced components, but their rate constants and polarities were different. Generally, calcium-induced components exhibited fast rate constants (>100 s⁻¹). The myosin-induced components of the second ALL had a rate constant similar to that of the force (7-10 s⁻¹), but that of the sixth ALL was apparently faster. The myosin-induced component of troponin reflection was the only one with negative polarity, and was too slow to be analyzed with this protocol. The results suggest that the three regulation-related proteins change their structures with different rate constants, and the significance of these findings is discussed in the context of a cooperative thin-filament activation mechanism.     

Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP₅ 2-K) is an enzyme that catalyses the formation of phytic acid (IP₆) from IP₅ and ATP. In mammals, IP₆ is involved in multiple events such as DNA repair and mRNA edit and it is the precursor of inositol pyrophosphates, emerging compounds shown to have an essential role in apoptosis. In addition, IP₅ 2-K have functions in cells independently of its catalytic activity, for example in rRNA biogenesis. We pursue the structure determination of a mammal IP₅ 2-K by Protein Crystallography. For this purpose, we have designed protocols for recombinant expression and purification of Mus musculus IP₅ 2-K (mIP₅ 2-K). The recombinant protein has been expressed in two different hosts, E. coli and insect cells using the LSLt and GST fusion proteins, respectively. Both macromolecule preparations yielded crystals of similar quality. Best crystals diffracted to 4.3 Å (E. coli expression) and 4.0 Å (insect cells expression) maximum resolution. Both type of crystals belong to space group P2₁2₁2₁ with an estimated solvent content compatible with the presence of two molecules per asymmetric unit. Gel filtration experiments are in agreement with this enzyme being a monomer. Crystallographic data analysis is currently undergoing.     

Freeze-Etching and X-Ray Diffraction of the Isolated Double-Track Layer from the Cell Wall of a Gram-Negative Marine Pseudomonad

The isolated double-track layer of the cell wall of the gram-negative marine pseudomonad studied here contains a cleavage plane. This finding localizes the single cleavage plane of the cell wall and shows that the molecular architecture of this layer provides the lipid-enriched layer which cleaves preferentially in the frozen cell. The observation that the isolated double-track layer of the cell wall is sufficiently ordered at the molecular level to yield a well-defined X-ray diffraction pattern with a d-spacing of 0.44 nm shows that its molecular architecture is very similar to that of true membranes. This specific d-spacing is produced by the highly ordered packing of the hydrophobic portions of phospholipid molecules. Therefore, the double-track layer of the cell wall has been shown, by these two biophysical means, to have a molecular architecture which would allow it to function as the membrane-like “molecular sieve” layer, whose presence has been deduced from physiological data. This layer is important in the retention of cell wall-associated enzymes and in the control of the movement of large molecules through the cell wall.