Comparative study of crystallins from the nucleus and cortex of the bovine ocular lens by the gel filtration and x-ray diffraction methods

Water--soluble proteins (alpha-, beta H-, beta L- and gamma-crystallins) from the bovine lens nucleus and cortex were fractionated and compared by gel filtration on Sephadex G-200. X-ray diffraction patterns from concentrated gels of these proteins were obtained. It allowed to compare qualitatively the structures of different crystallins and also to identify the maxima on X-ray diffraction patterns of the lens intact tissue.     

Absence of small-angle maximums on the x-ray images of ocular lens tissue

X-ray small-angle scattering study of bovine lens tissue was carried out. It was shown that X-ray patterns of lens cortical and nuclear native tissues did not contain the small-angle maxima. The maximum in the range of 15-20 nm Bragg distance appeared as a result of the lens tissue partial dehydration. Earlier such maximum was considered by some authors as the evidence of crystallin proteins short-range order in the native lens. Now it is confirmed to be a preparative artefact of dehydration. It was shown also that similar maximum in 15-20 range existed in the case of concentrated crystallin solutions. This indicates that supramolecular organization of crystallins in the native lens is not similar to that in the concentrated solution.     

An x-ray study of the subcutaneous tissue using an artificial method of providing contrast]

The paper is concerned with the main methodological principles and potentialities of a radiocontrast study of subcutaneous fat to detect pathological changes in it. The proposed method consists in slow administration in the middle fat layers of 20-25% solution of verografin from not less than two points, and subsequent roentgenography in a necessary projection. The method was used in 15 patients with various orthopedic diseases for diagnosis of inflammatory, tumorous and degenerative-dystrophic diseases.     

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.     

X-ray diffraction in the intact isolated frog ocular lens

X-ray diffraction method has been applied for investigating ocular lens native tissue of the frog. X-ray diffraction patterns of intact lenses, their nuclei and cortices are similar and contain a set of concentric diffuse diffraction maxima. The most intensive of these maxima corresponding to the Bragg-spacings of 14.6, 9.1 and 4.6 A are presumably associated with intramolecular structure of lens proteins--crystallins. Intensive small-angle X-ray scattering and diffraction patterns isotropy indicates unavailability of crystallin molecule ordering or orientation in the lens. The shift of 14.6 A maximum up to 12.8 A being the result of nuclei drying shows the necessity of aqueous surrounding for these protein native structure maintenance.     

Packing of ganglioside-phospholipid monolayers: an x-ray diffraction and reflectivity study

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structure of mixed ganglioside-phospholipid monolayers was investigated at the air-water interface. Mixed monolayers of 0, 5, 10, 20, and 100 mol% ganglioside GM(1) and the phospholipid dipalmitoylphosphatidylethanolamine (DPPE) were studied in the solid phase at 23 degrees C and a surface pressure of 45 mN/m. At these concentrations and conditions the two components do not phase-separate and no evidence for domain formation was observed. X-ray scattering measurements reveal that GM(1) is accommodated within the host DPPE monolayer and does not distort the hexagonal in-plane unit cell or out-of-plane two-dimensional (2-D) packing compared with a pure DPPE monolayer. The oligosaccharide headgroups were found to extend normally from the monolayer surface, and the incorporation of these glycolipids into DPPE monolayers did not affect hydrocarbon tail packing (fluidization or condensation of the hydrocarbon region). This is in contrast to previous investigations of lipopolymer-lipid mixtures, where the packing structure of phospholipid monolayers was greatly altered by the inclusion of lipids bearing hydrophilic polymer groups. Indeed, the lack of packing disruptions by the oligosaccharide groups indicates that protein-GM(1) interactions, including binding, insertion, chain fluidization, and domain formation (lipid rafts), can be studied in 2-D monolayers using scattering techniques.     

Comparative study of lens proteins of gray squirrel and human

1. The four crystallins of the gray squirrel lens have been characterized using gel filtration chromatography, polyacrylamide gel electrophoresis, and immunoblotting. Alpha, beta-heavy, beta-light, and gamma crystallins of squirrel lenses have been identified immunologically, and they cross-react strongly with rabbit polyclonal antibodies. The gamma-24 crystallin of the squirrel lens also reacts strongly with monoclonal anti-human lens gamma-24, as shown by its inhibition of the ELISA reaction by 85%. 2. The water-insoluble urea soluble proteins represent non-covalently associated species of soluble crystallins and the lens cytoskeletal proteins. The membrane intrinsic protein in the urea insoluble pellet has a mol. wt of 27,000 but other lower and higher mol. wt components are also present, which were removed by washing with 0.1 NaOH. The N-terminal 30 amino acid of squirrel lens gamma crystallin was found to be identical to that of the bovine (and human) lens. 3. Measurements of the distribution and state of SH and SS compounds in the squirrel lens have shown greater similarities to those of primates than those of rodents. The findings show that on the basis of both protein and sulfur chemistry the squirrel lens is a representative model for studies of oxidative lens changes in diurnal animals, including man.     

Urease and hexadecylamine-urease films at the air-water interface: an x-ray reflection and grazing incidence x-ray diffraction study

We report the results of surface x-ray scattering measurements performed on urease and hexadecylamine-urease films at the air-aqueous solution interface. It is demonstrated that although hexadecylamine does not form a stable monolayer on the pure aqueous surface, it does self-assemble into a stable, well-organized structure when spread on top of a urease film at the air-water interface. It is also likely that protein and hexadecylamine domains coexist at the interface.     

Immunoproteasome expression in a nonimmune tissue,the ocular lens

Interferon gamma (IFN gamma) induces the expression of three catalytic subunits of the 20S proteasome that can replace their constitutive homologues to form the "immunoproteasome," named to reflect its antigen presentation function. However, immunoproteasome levels and their modulation in nonimmune tissues remain unknown. A disrupted lens differentiation program observed in transgenic mice that constitutively express IFN gamma in the immune-privileged lens tissue suggests a role for this cytokine in differentiation. We have developed a competitive RT-PCR assay that demonstrates substantially increased levels of immuno subunits and unchanged levels of constitutive subunits in transgenic compared to wild-type lenses. Similar results were observed with IFN gamma treated alpha TN4-1 lens epithelial cells. A comparison of these subunits in different immune and nonimmune mouse tissues revealed unique expression patterns. The presence of immuno subunits in nonimmune tissues such as lens suggests that the immunoproteasome may also have nonimmune functions, such as that in lens differentiation.     

Collagen organization in canine myxomatous mitral valve disease: an x-ray diffraction study

Collagen fibrils, a major component of mitral valve leaflets, play an important role in defining shape and providing mechanical strength and flexibility. Histopathological studies show that collagen fibrils undergo dramatic changes in the course of myxomatous mitral valve disease in both dogs and humans. However, little is known about the detailed organization of collagen in this disease. This study was designed to analyze and compare collagen fibril organization in healthy and lesional areas of myxomatous mitral valves of dogs, using synchrotron small-angle x-ray diffraction. The orientation, density, and alignment of collagen fibrils were mapped across six different valves. The findings reveal a preferred collagen alignment in the main body of the leaflets between two commissures. Qualitative and quantitative analysis of the data showed significant differences between affected and lesion-free areas in terms of collagen content, fibril alignment, and total tissue volume. Regression analysis of the amount of collagen compared to the total tissue content at each point revealed a significant relationship between these two parameters in lesion-free but not in affected areas. This is the first time this technique has been used to map collagen fibrils in cardiac tissue; the findings have important applications to human cardiology.     

Photoaffinity labeling of proteins in bovine testis nuclear extract

A binary system of photoaffinity reagents for selective affinity labeling of DNA polymerases has been developed. The photoreactive probe was formed in nuclear extract, using an end-labeled oligonucleotide containing a synthetic abasic site. This site was incised by apurinic/apyrimidinic endonuclease and then dNMPs carrying a photoreactive adduct were added to the 3(') hydroxyl using base-substituted arylazido derivatives of dUTP or dCTP. This results in the synthesis of photoreactive base excision repair (BER) intermediates. The photoreactive group was then activated, either directly (UV light exposure 320nm) or in the presence of the sensitizer of dTTP analog containing a pyrene group (Pyr-dUTP) under UV light 365nm. DNA polymerase beta was the main target crosslinked by photoreactive BER intermediates in this nuclear extract. In contrast, several proteins were labeled under the conditions of direct activation of arylazido group.     

Crystallization and preliminary x-ray diffraction study of cholera toxin B-subunit

Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600). Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside. The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees. There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit. Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data.