Malnutrition and myelin structure: an X-ray scattering study of rat sciatic and optic nerves

Taking advantage of the fast and accurate X-ray scattering techniques recently developed in our laboratory, we tackled the study of the structural alterations induced in myelin by malnutrition. Our work was performed on sciatic and optic nerves dissected from rats fed with either a normal or a low-protein caloric diet, as a function of age (from birth to 60 days). By way of electrophysiological controls we also measured (on the sciatic nerves) the height and velocity of the compound action potential. Malnutrition was found to decrease the amount of myelin and to impair the packing order of the membranes in the sheaths.     

An X-ray study of the condensed and separated states of sciatic nerve myelin

Low-angle X-ray diffraction patterns of peripheral nerve myelin after modification by either rehydration in various solutions or by chemical treatment have been recorded. These X-ray patterns and the previously reported modified nerve myelin patterns demonstrate that nerve myelin has at least five different states: the normal state, condensed state I and II and separated state I and II. There are two membranes per unit cell in the normal state and in states II whereas there is one membrane per unit cell in states I. Under certain conditions normal nerve can go reversibly into either of states II. With continued treatment the nerve myelin structure moves irreversibly from state II to state I and, once in state I, the nerve myelin layers cannot return to the normal state. Our results demonstrate that there is a reversible transformation between condensed state I and separated state I. Fourier profiles of nerve myelin in the normal state, condensed state I and separated state I are presented.     

An X-ray study of the effect of enzymes on frog sciatic nerve myelin

Low-angle X-ray diffraction patterns have been recorded from frog sciatic nerve after digestion with trypsin and Pronase. Reproducible X-ray patterns were obtained by swelling the nerves in distilled water before treatment with enzymes. The X-ray patterns of enzyme-treated nerves are distinctly different from the X-ray pattern of normal (live) nerve. It would appear that the normal asymmetric nerve myelin membrane becomes symmetric about its center after treatment with enzymes as a result of proteolytic cleavage and a subsequent redistribution of protein components.     

A spin-label study of sciatic nerves from quaking,jimpy and trembler mice

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant ($\text{T}{}_6$) between 0°C and 50°C. Within this range of temperatures, we obtained identical values of 2 $\text{T}{}_6$ for nerves from normal and jimpy mice, whereas 2 $\text{T}{}_6$ was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice.The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.     

A spin-label study of sciatic nerves from quaking, jimpy and trembler mice.

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant (T) between 0 degrees C and 50 degrees C. Within this range of temperatures, we obtained identical values of 2 T for nerves from normal and jimpy mice, whereas 2 T was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice. The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.     

Developmental changes at the node and paranode in human sural nerves: morphometric and fine-structural evaluation

Developmental alterations of paranodal fiber segments have not been investigated systematically in human nerve fibers at the light- and electron-microscopic level. We have therefore analyzed developmental changes in the fine structure of the paranode in 43 human sural nerves during the axonal growth period up to 5 years of age, and during the subsequent myelin development up to 20 years and thereafter. The nodal, internodal, and paranodal axon diameters reach their adult values at 4–5 years of age. The ratio between internodal and paranodal axon diameters remains constant at 1.8–2.0. Despite a considerable increase in myelin sheath thickness, the length of the paranodal myelin sheath attachment zone at the axon does not increase correspondingly, because of attenuation, separation from the axolemma, and piling up of myelin loops in the paranode. Separation of variable numbers of terminal myelin loops from the underlying axolemma results in the formation of bracelets of Nageotte, whereas the transverse bands of these loops disappear. The adaptation of the paranodal myelin sheath to axonal expansion during development probably occurs by uneven gliding of the paranodal myelin loops simultaneously with internodal slippage of myelin lamellae. Since mechanically stabilizing structures (tight junctions and desmosomes between adjacent paranodal myelin processes; transverse bands between myelin loops and paranodal axolemma) are unevenly arranged, especially during rapid axonal growth, paranodal axonal growth with simultaneous adaptation of the myelin sheath is probably discontinuous with time.Presented in part at the 10th Biennial Meeting of the Peripheral Nerve Study Group at Arden House, Harriman, New York, USA, June 30th–July 3rd, 1991, and as a doctoral thesis (M. Bertram) at the RWTH Aachen in 1991     

A study of an infected bone tissue (osteomyelitis) by small-angle X-ray scattering

The small-angle X-ray scattering method has been applied to evaluate various macromolecular parameters such as the specific inner surface, the transversal lengths, the length of crystallinity, the range of amorphous zone and the percentage of porosity in pure human bone and osteomyelitis, an infection of bone tissue. The hydroxyapatite crystals of bone being uniformly dispersed throughout the hydrated collagenous matrix creating a large mineral matrix interface, we found the bone samples to behave as a densely packed two phase system. The theories of Kratky and Porod have been utilized to evaluate the macromolecular parameters. These findings may shed light on tertiary structural deformation of human bone when it is infected.     

Comparison of the structures of the native form of rat liver 5S rRNA and yeast tRNAphe: small-angle and wide-angle X-ray scattering study

The small-angle and wide-angle X-ray scattering of tRNA^phe (yeast) and ribosomal 5S RNA (rat liver) in solution have been analysed and compared. tRNA^phe in solution is folded into a compact L-shaped structure similar to its structure in crystals. The geometry of the secondary structure of the double helical regions is also equivalent to the A-form in the crystalline state. Despite differences between the molar mosses of 5S rRNA (40 000 g mol^?1) and tRNA^phe (25 000 g mol^?1), and the fact that the 5S rRNA molecule is more anisometric than the tRNA^phe molecule, there are many structural similarities. The geometrical parameters of the secondary structure of double helical regions in both RNA molecules are almost identical; the mean rise per base pair is about 0.253–0.28 nm and the mean turn angle is about 32.5–33.5. Identical cross-sectional radii of gyration, R_sq,1 ≈ 1.16 nm and R_sq,2 = 0.92 nm, identical molar mass per unit length, , and a mean thickness of the molecules D ≈ 1.65 nm suggest a similar, nearly coplanar organization of isolated, double helical arms. Furthermore, there are compact regions in the central parts of both molecules, which are the sites of tertiary interactions in the tRNA^phe molecule and are a potential site of tertiary interactions in the SS rRNA molecule for stabilization of the complicated L-shape of the two molecules. Both molecules have a pseudo-twofold axis,w hich may play a role in recognition for binding of specific proteins.     

X-ray scattering studies of a model complex of lipid and basic protein of myelin

Low-angle and wide-angle X-ray scattering data from phosphatidylglycerol complexed with myelin basic protein, poly(L-lysine) and calcium ions are analyzed. The results confirm our earlier report (Brady, G.W., Murthy, N.S., Fein, D.B., Wood, D.D. and Moscarello, M.A. (1981) Biophys. J. 34, 345-350) that the basic protein interacts primarily with the polar headgroups of the lipid; and that at high protein concentrations (greater than 35%) the bilayers aggregate to form multilayers with a repeat period of 68 A, the single bilayer to multilayer transition being a cooperative process. Polylysine and Ca2+ produce multilayers with a smaller repeat of approx. 55 A. Basic protein and polylysine do not change the fluid-like arrangement of the hydrocarbon chains (diffuse 4.6 A peak in the wide-angle pattern), whereas Ca2+ probably induces a two-dimensional order (4.3 A and 3.9 A peak in the wide-angle pattern). Electron density profiles of the lipid and lipid-basic protein vesicles indicate that the basic protein penetrates into the bilayer.     

Order-disorder phenomena in myelinated nerve sheaths. II. The structure of myelin in native and swollen rat sciatic nerves and in the course of myelinogenesis

The algorithm described in the accompanying paper was applied to X-ray scattering experiments performed with rat sciatic nerves, either as a function of the age of the animal (4 to 30 days), or with adult nerves swollen in non-isotonic media. The results were all consistent with the model of disorder used in the theoretical treatment. The algorithm leads, in one step, from the data to the numerical values of the parameters, avoiding all intermediate manipulation. For each experiment a variety of parameters was determined: the average D and the variance sigma 2D of the repeat distance, the average number [N] of motifs per crystallite, the set [idiff(h/D)], which defines the diffuse scattering, the fraction alphaloose of myelin that does not belong to the compact sheaths, and the set [imotif (k/2D)], which suffices to define the continuous intensity curve of the motif imotif(s). Note the remarkable wealth of information, especially by contrast with conventional analyses which, as a rule, only yield the values of D and of the set [imotif(h/D)] (insufficient to determine the function imotif(s]. The function imotif(s) and the parameters D and sigma D (and thus the local structure of the myelin sheaths) were shown to be almost invariant in the course of myelinogenesis; what varies is mainly the total amount of myelin in the nerve and the number of membranes per sheath. Swelling agents have a dramatic influence on the X-ray scattering spectra, but in spite of the conspicuous variation of D, sigma D and [N] the structure of the motif is invariant. The structure of the motif was shown to be quite different in the native and in the swollen samples; the stacking disorder appears to involve mainly the cytoplasmic space in native myelin, the external space in swollen nerves. The very notion of electron density profile, when disorder is present, is discussed. Two criteria were proposed to select the "best" signs of the reflections: two sets came out at almost the same rank, one corresponding to Caspar & Kirschner's the other to Worthington & McIntosh's proposals, neither of which can be ruled out according to the criteria used in this work.     

X-ray small angle scattering of the human transferrin protein aggregates. A fractal study.

X-ray small angle scattering experiments, using a pin hole SAXS camera with Synchrotron radiation source, have been performed to study the conformational changes of lyophilized samples of Apo-, Mono-, and Diferric- human transferrin. We report the experimental evidence that the analysis of the scattered intensity through the fractal theory may give information on the particle size and its variation upon iron binding.     

Head-to-tail and side-by-side oligomerization of human carbonic anhydrase II: a small angle X-ray scattering study

Solvent-induced directional aggregation of human carbonic anhydrase II (hCA) was studied by small angle X-ray scattering and fluorescence and fourth-derivative ultraviolet absorption spectroscopy. We propose that hCA at 5 mg ml(-1) in pure water forms head-to-tail oligomers built up, on average, by four to five monomers. At higher protein concentrations, the oligomers associate pair-wise and side-by-side. Spectroscopic evidence suggests that the subunits forming the aggregates are tightly folded, but with a structure that differs, at least locally, from the native state. A more complex aggregation pattern was observed under solvent conditions that favor the removal of zinc from the enzyme-active site, conditions under which the subunits are significantly less compact than in water. hCA may provide a useful model to investigate the effects of additives and genetic manipulation on protein aggregation.     

A small-angle X-ray solution scattering study of bovine alpha-crystallin.

The native high molecular mass form of alpha-crystallin, the most important soluble protein in the eye lens, and its low molecular mass form obtained at 37 degrees C in dilute solutions were investigated by synchrotron radiation small-angle X-ray scattering. The alpha-crystallin solutions are polydisperse and good fits to the experimental data can be obtained using distributions of spheres with radii varying between about 5 and 10 nm. In spite of the polydispersity, two different ab initio methods were used to retrieve low resolution shapes from the scattering data. These shapes correspond to the z-average structure of the oligomers. In the absence of any symmetry constraints, the scattering curves of the two forms of alpha-crystallin yield bean-like shapes. The shape corresponding to the low molecular mass form has about 20% less mass at the periphery. Imposing tetrahedral symmetry on the average structures worsens the fit to the experimental data. We emphasized the apparent contradiction between hydrodynamic and molecular properties of alpha-crystallin. An explanation was put forward based on the presence of solvent-exposed flexible C-terminal extensions. We present two bead models ('hollow globule with tentacles' and 'bean with tentacles') based on NMR and cryo-electron microscopy studies and discuss how well they correspond with our data from X-ray scattering, light scattering and analytical ultracentrifugation.     

Reconstruction of protein form with X-ray solution scattering and a genetic algorithm

We have reconstructed, from experimental approximately 2 nm resolution X-ray solution scattering profiles, the corresponding shapes and sizes of myoglobin, troponin C, spermadhesin PSP-I/PSP-II, chymotrypsinogen A, superoxide dismutase, ovalbumin, tubulin, nitrite reductase, catalase, the structural change of troponin C upon dissociation of the two high affinity Ca(2+), and the solution model structure of a tandem pair of fibronectin type III cytoplasmic domains of integrin alpha6beta4 before determination of its crystal structure. To this purpose we have designed a new genetic algorithm which gradually explores a discrete search space and evolves convergent models made of several hundred beads (down to 0.3 nm radius) best fitting the scattering profile upon Debye calculation, without geometrical constraints or penalty for loose beads. This is a procedure of effective numerical transformation of the one-dimensional scattering profiles into three-dimensional model structures. The number of beads in models is correlated with the protein molecular mass (with one exception). The shape and approximate dimensions of each protein have been retrieved by a set of ten solution models, essentially superimposable with the available crystal structures.