Veronica J. James

Use of X-Ray Diffraction in Study of Human Diabetic and Aging Collagen

Extensive investigations of the solubility and fluorescence of collagen fibers in diabetes have revealed that there are significant changes in their physical properties. These changes are associated with increased cross-link formation. We used X-ray diffraction to study these changes in human extensor tendons at a molecular level in relation to both aging and diabetes. Our results indicate that diabetes induces significant alterations in the ultrastructure of collagen in the lateral packing of the molecules and the axial structure of the specimen. These changes can be induced in normal tendon by incubation in ribose and glucose-6-phosphate but are different from those associated with the normal process of aging.

The intermediate filament structure of human hair.

X-ray diffraction studies of hard alpha-keratin have led to a proposed model for the lateral arrangement of molecules within the keratin fibrils of tissues such as hair, nail and claw. Using low-angle synchrotron radiation to examine human scalp hair we have obtained discrete equatorial diffraction maxima which have not been reported previously. These reflections can be divided into three subsets. The first of these reveals the information that the hair fibres consist basically of cylindrical fibrils arranged in a disordered lattice. The mean diameters of these cylinders have been determined, together with their average separation. The diameters of the protofibrils have been determined from the second set. The third set, a set of diffuse arcs, index onto a spacing which is characteristic of the disordered components of the matrix.

Location and effect of procaine on lecithin/cholesterol membranes using X-ray diffraction methods.

X-ray diffraction studies were made on lecithin/cholesterol multilayers with very high water content and containing the local anaesthetic procaine. Narrow-angle diffraction experiments show that the procaine molecules are located with the uncharged aromatic amine group approx. 10 A from the centre of the bilayer. The polar tertiary amine group of these molecules is almost certainly located in the polar head-group region of the membrane. Wide-angle diffraction experiments show that the incorporation of procaine molecules into such lipid membranes produces an approx. 30% increase in the spread of acyl chain separation, although the average spacing between the chains is slightly reduced.

An X-ray crystallographic study of α-d-allopyranosyl α-d-allopyranoside · CaCl2 · 5H2O (a pentadentate complex)

Abstract Three-dimensional, single-crystal, X-ray diffraction methods were used to determine the structure of the calcium chloride complex of α - d -allopyranosyl α - d -allopyranoside. The crystal is monoclinic with cell dimensions: a = 16.262(5), b = 8.345(5), c = 8.298(5)A, β = 98.428(5)A, and z = 2. The space group is P2 1 . The structure was solved by three-dimensional Patterson and Fourier methods, and was refined by least-squares techniques to give a conventional discrepancy factor of R = 0.026; 2435 diffractometer-read reflections were used. The cation was found to be in 9-fold co-ordination to O-1, O-2, O-3, O-2′, O-3′, and four water molecules.

The d-spacing of collagen from mitral heart valves changes with ageing, but not with collagen type III content

A low angle X-ray diffraction study of normal and myxomatous human heart valves has been carried out using a Synchrotron radiation source. This study has shown a slight progressive decrease in the d-spacing of the collagen with age. Despite suggestions reported in the literature, no significant relative change was observed in the d-spacing with the increase in the percentage of collagen type III from 24% in normal to 31% in myxomatous heart valves.

An x-ray crystallographic study of 5-Oacetyl-1,2:3,4-di-O isopropylidene-α-d-galactoseptanose

Abstract Three-dimensional, single-crystal X-ray diffraction methods were used to determine the solid-state structure of 5- O acetyl-1,2:3,4-di- O -isopropylidene-α- d -galactoseptanose. The crystals are tetragonal with cell dimensions: a  b  9.571 (5), c  16.944(5) A, α  β  γ  90°, and z  4. The space group is P41 1 . The structure was solved by direct methods and refined by least-squares techniques to give a conventional discrepancy-factor, R , of 0.040. The seven-membered ring is close to a boat conformation, 1,2,5 B.

Changes of extracellular matrix in a baboon (Papio hamadryas) model of insulin dependent diabetes: studies using electron microscopy and X-ray diffraction techniques

Extracellular matrix plays an important role in many physiological functions and its abnormalities are thought to play a key role in the pathogenesis of diabetic complications. In this paper we used the techniques of electron microscopy, immunostaining and X-ray diffraction to document some of the early events in the changes of extracellular matrix in a model of insulin dependent diabetes in baboons. Our results show that thickening of basement membrane and enlargement of mesangium are demonstrable in the glomeruli of prepubertal diabetic baboons within 2 years from the onset of diabetes. Concomitant with this was the accumulation of type IV collagen and laminin in the mesangium. By contrast, even the very sensitive technique of X-ray diffraction failed to demonstrate changes in the equatorial direction of collagen molecules of the skin and tendon. We conclude that changes of glomerular extracellular matrix are demonstrable early in insulin dependent diabetes even in prepubertal baboons. These can be used as endpoints in evaluating the efficacy of pharmacological agents such as aminoguanidine in preventing diabetic complications.

Intermediate filament structure of α-keratin in baboon hair

Abstract High-quality photographic recording of X-ray diffraction data from the hard α-keratins has mainly been limited to that from porcupine quill. Earlier diffraction patterns of hair have shown very few reflections. In the present study, extensive sets of high-quality data have been obtained using a synchrotron source and an image plate detector, revealing information about the internal structure, both axial and lateral. The diffraction patterns of hair from six different baboons have been examined. The meridional axial diffraction data reveal evidence of the superposition of three and possibly four separate lattices. Despite considerable overlapping, sufficient reflections unique to each lattice have been observed. These lattices comprise an infinite one of 46.76 nm, together with finite lattices of 19.67, 27.20 and 7.27 nm. These patterns reveal information regarding the fibrillar structure of the hair fibres, namely that it consists basically of cylindrical fibrils arranged in a disordered lattice. The mean diameters of these cylinders have been determined, together with their average separation. There is also an indication of the presence of another set of cylinders of smaller diameter, possibly comprising the microfibrils of keratin.

Intermediate Filament Packing in α-Keratin of Echidna Quill

The swelling and associated physical behavior of wool α-keratin fibers when im mersed in a solution of formic acid in water have given rise to a detailed model for hexagonal packing of the intermediate filaments in hard α-keratin. Low-angle syn chrotron x-ray diffraction studies of the α-keratin fibers of dry and fully hydrated echidna quills not only provide extremely accurate measurements of the radii of the intermediate filaments and their center-to-center spacing, but also reveal the superpos ition of two separate infinite lattices in the meridional direction, one the accepted infinite repeat length of α-keratin, 47.0 ± 0.2 nm, and the other 62.6 ± 0.2 nm. Despite considerable overlapping, there are sufficient reflections unique to each lattice. These results and the equatorial data are consistent with the hexagonal packing suggested from the swelling experiments.

Molecular structural changes in human fetal tissue during the early stages of embryogenesis

Low-angle synchrotron X-ray diffraction studies of human fetal extensor tendon and skin collagen, centred in time about the period of first major movement by the fetus, indicate that alignment of the tendon collagen fibrils occurs about this time. At this stage there appears to be no detectable structural difference between tendon and dermis. By three weeks post-partum, marked differences between these tissues can be detected. The distribution of the intermediate filament diameters for all fetal tendons investigated was unimodal (mean 41.2 +/- 0.4 nm) in contrast with that for post-partum tendon which is multimodal. Equatorial periodicities of 353 +/- 3 nm and 32.1 +/- 0.1 nm, consistent with the presence of type IV collagen, were obtained from all fetal samples examined. Neither of these periodicities were observed in post-partum normal tendon and only the larger were observed in post-partum normal skin. The consistency of the results suggest that low-angle X-ray diffraction could be used for the identification of fetal-like tissues found in pathological tissues.