Bibliothek

Notizen: Abstract: Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jprsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10-kDa PLP previously reported in jprsh mice on an Rb(1.3) 1Bnr background. High-performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jprsh myelin, further substantiating that PLP is not a component of this junctional complex. X-raydiffraction measurements on unfixed optic nerves showed that the jprsh period is 5–10 Å larger than normal. Moreover, jprsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jprsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jprsh mutant correlate with a wider periodicity and less stable packing of the myelin.

Notizen: Protein zero (P0), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P0 (P0-ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N-linked glycosylation. The x-ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 Å, 50 Å (native), and 90 Å between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x-ray diffraction data on the orientation of P0-ED, and we propose how this immunoglobulin-like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that β-strand predictions for P0-ED are virtually superimposable with those of the VH domain of the phosphocholine-binding immunoglobulin M603 of mouse, which has a similar number of residues as P0-ED and a structure that has been solved crystallographically. The dimensions of P0-ED from the space-filling model, developed using PC- based molecular modeling software, were found to be 44 Å× 25 Å× 23 Å. On the assumption that neither the shape nor the orientation of P0-ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P0-ED from apposed membranes if the molecules were oriented so that the β- strands were approximately perpendicular to the membrane surface. The apposed P0-EDs would fully overlap at the closest apposition of the membranes, partially overlap in the native state, and align end to end in the incompletely swollen state. The P0-ED regions analogous to the complementarity-determining regions of immunoglobulins can account for the recognition of P0-ED from apposed membranes in the incompletely swollen state. Two of the faces of P0-ED that show charge complementarity could account for the homophilic interactions of P0-ED from apposed membranes in the native state. This association can be stabilized further by hydrophobic interactions. The N- linked nonasaccharide after energy minimization fit into a cavity, which was at the base of P0-ED and which was lined with three positively charged residues. Thus, the carbohydrate may help to maintain the orientation of P0 at the membrane surface. Our model shows how the single immunoglobulin-like domain of P0 can account for distinct structural states of myelin membrane packing by homophilic interactions.

Notizen: Abstract: We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4–9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02–0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20–30% higher than normal; non-hydroxycerebroside and sphingomyelin are 15–20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.

Notizen: Abstract: The radial component is a junctional complex that is believed to stabilize the apposition of myelin membranes in the internode of CNS myelin. Based on our previous finding that the radial component of compact myelin retains its structure in tissue treated with the detergent Triton X-100, we have attempted to isolate the junctional complex from spinal cord myelin treated with this detergent. Using 0.5% Triton X-100, our procedures yielded a fraction of isolated myelin that was enriched in well-preserved radial component. This fraction that contained morphologically well-defined radial component was examined by sodium dodecyl sulfate-polyacrylamide gel electropho-resis and immunoblotting, and TLC, and was found to be significantly and consistently enriched in the 21.5-kDa and 17-kDa isoforms of myelin basic protein, and in cerebro-sides, hydroxy sulfatide, and sphingomyelin. In addition, the myelin-associated enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase, tubulin, and actin tended to be resistant to Triton extraction. The fraction of isolated myelin that contained radial component was deficient in proteolipid protein and DM-20, the 18.5-and 14-kDa isoforms of myelin basic proteins, and in the major phospholipids, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine. Our data indicate that the radial component can be isolated and that certain myelin and cytoskeletal proteins and lipids are closely associated with it.

Notizen: Abstract: To model the possible involvement of sulfated proteoglycans in amyloidogenesis, we examined the influence of sulfate ions, heparan, and Congo red on the conformation and morphology of peptides derived from the Alzheimer β/A4 amyloid protein. The peptides included residues 11–28, 13–28, 15–28, and 11–25 of β/A4. Negative-stain electron microscopy revealed a sulfate-specific tendency of the preformed peptide fibrillar assemblies of β(11–28), β(13–28), and β(11–25), but not β(15–28), to undergo extensive lateral aggregation and axial growth into “macrofibers” that were ∼0.1–0.2 μm wide by ∼20–30 μm long. Such effects were observed at low sulfate concentrations (e.g., 5–50 mM) and could not be reproduced under comparable conditions with Na2HPO4, Na2SeO4, or NaCl. Macrofibers in NaCl were only observed at 1,000 mM. At physiological ionic strength of NaCl, fibril aggregation was observed only with addition of sulfate ions at 5–50 mM. Selenate ions, by contrast with sulfate ions, induced only axial and not substantial lateral aggregation of fibrils. X-ray diffraction indicated that the original cross-β peptide conformation remained unchanged; however, sulfate binding did produce an intense ∼65 Å meridional reflection not recorded with control peptides. This new reflection probably arises from the periodic deposition of the electron-dense sulfate along the (long) axis of the fibril. The sulfate binding could provide sites for the binding of additional fibrils that generate the observed lateral and axial aggregation. The binding of heparan to β(11–28) also produced extensive aggregation, suggesting that in vivo sulfated compounds can promote macrofibers. The amyloid-specific, sulfonated dye Congo red, even in the presence of sulfate ions, produced limited aggregation and reduced axial growth of the fibrils. Therefore, electrostatic interactions are important in the binding of exogenous compounds to amyloid fibrils. Our findings suggest that the sulfate moieties of certain molecules, such as glycosaminoglycans, may affect the aggregation and deposition of amyloid fibrils that are observed as extensive deposits in senile plaques and cerebrovascular amyloid.

Notizen: Abstract: We have correlated myelin membrane structure with biochemical composition in the CNS and PNS of a phylogenetic series of animals, including elasmobranchs, teleosts, amphibians, and mammals. X-ray diffraction patterns were recorded from freshly dissected, unfixed tissue and used to determine the thicknesses of the lipid bilayer and the widths of the spaces between membranes at their cytoplasmic and extracellular appositions. The lipid and protein compositions of myelinated tissue from selected animals were determined by TLC and sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis/immunoblotting, respectively. We found that(l) there were considerable differences in lipid (particularly gly-colipid) composition, but no apparent phylogenetic trends; (2) the lipid composition did not seem to affect either the bilayer thickness, which was relatively constant, or the membrane separation; (3) the CNS of elasmobranch and teleost and the PNS of all four classes contained polypeptides that were recognized by antibodies against myelin Po glycoprotein; (4) antibodies against proteolipid protein (PLP) were recognized only by amphibian and mammalian CNS; (5) wide extracellular spaces (ranging from 36 to 48 Å) always correlated with the presence of Po-immunoreactive protein; (6) the narrowest extracellular spaces (∼31 Å) were observed only in PLP-containing myelin; (7) the cytoplasmic space in PLP-containing myelin (∼31 Å) averaged ∼5Å less than that in Po-containing myelin; (8) even narrower cytoplasmic spaces (∼24Å) were measured when both Po and 11–13-kilodalton basic protein were detected; (9) proteins immu-noreactive to antibodies against myelin P2 basic protein were present in elasmobranch and teleost CNS and/or PNS, and in mammalian PNS, but not in amphibian tissues; and (10) among mammalian PNS myelins, the major difference in structure was a variation in membrane separation at the cytoplasmic apposition. These findings demonstrate which features of myelin structure have remained constant and which have become specifically altered as myelin composition changed during evolutionary development.

Notizen: Isolated myelin has been used for determinations of membrane surface charge density and topographical mapping of components in the membrane. To determine how similar such myelin is to myelin of intact tissue, we have used x-ray diffraction to compare their intermembrane interactions. The interactions were monitored by measuring the myelin period in samples treated with distilled water, buffered saline at pH 4–9 and ionic strength 0.06–0.18, and saline containing HgCl2 or triethyl tin sulfate. Myelin was isolated from whole brains and sciatic nerves of mice by conventional methods involving sucrose gradient centrifugation and osmotic shock. Consistent with previous findings, electron microscopy showed that the multilamellar morphology, staining, and repeat periods of isolated myelin were essentially like those of intact myelin; however, the membrane stacks were less extensive than those in whole tissue. X-ray diffraction revealed that isolated CNS myelin was like intact myelin in showing reversible compaction in acidic media and in distilled water. However, unlike the myelin in whole tissue, isolated CNS myelin did not swell in hypotonic or alkaline media, or in the presence of HgCl2-saline or triethyl tin. The altered membrane interactions could result from an increase in adhesiveness of the apposed membrane surfaces. Reorganization of proteolipid protein and/or a reduction of surface charge could account for the change in surface properties of isolated CNS myelin. Isolated PNS myelin, like the membranes in whole tissue, showed both compaction and swelling; however, the membrane pairs were disordered in the swollen structure. This irregular membrane swelling could result from charge variation in the extracellular surfaces.

Notizen: Abstract: The serine protease inhibitor α1-antichymotrypsin (ACT) consistently colocalizes with amyloid deposits of Alzheimer's disease (AD) and may contribute to the generation of amyloid proteins and/or physically affect fibril assembly. AD amyloid fibrils are composed primarily of Aβ, which is a proteolytic fragment of the larger β-amyloid precursor protein. Using negative-stain and immunochemical electron microscopy, we have investigated the binding of ACT to the fibrils formed by four synthetic Aβ analogues corresponding to the wild-type human 1–40 sequence [HWt(1–40)], a 1–40 peptide [HDu(1–40)] containing the Glu22→ Gln mutation found in hereditary cerebral hemorrhage with amyloidosis of the Dutch type, the N-terminal 1–28 residues [β(1–28)], and an internal fragment of Aβ containing residues 11–28 [β(11–28)]. Each of these peptide analogues assembled into 70–90-Å-diameter fibrils resembling native amyloid and, except for β(11–28), bound ACT, as indicated by the appearance of 80–100-Å globular particles that adhered to preformed fibrils and that could be decorated with anti-ACT antibodies. Under the conditions used, ACT binding destabilized the in vitro fibrils and produced a gradual dissolution of the macromolecular assemblies into constituent filaments and shorter fragments. The internal fragment (11–28) did not exhibit ACT binding or any structural changes. These results suggest that a specific sequence likely contained within the N-terminal 10 residues of Aβ is responsible for the formation of the ACT-amyloid complex. Although the observed fibril disassembly is surprising in view of the notion that ACT contributes directly to the physical process involved in amyloid fibril formation, the induced structural changes may expose new domains in Aβ for additional proteolysis or for interactions with cell-surface receptors.

Notizen: Ultrathin films of 3d-transition metal alloys, and in particular FeCo alloys, currently receive considerable interest because of their potential technological application and the possibility to adjust magnetic properties via the variation of composition and structure. To study magnetic and structural properties of the otherwise unstable fcc phase of FeCo, this structural phase was stabilized by epitaxial growth on Cu(001). Ultrathin FexCo1−x films were deposited at room temperature by coevaporation from two separate Knudsen cells, operated under stabilized conditions. The film thickness was varied between 2 and 9 monolayers (ML) and the Fe concentration between x=0.2 and x=0.95. The growth process was monitored by medium energy electron diffraction (MEED). Auger electron spectroscopy and low energy electron diffraction (LEED) were employed to analyze the composition and structure of the films. A nearly perfect layer-by-layer growth up to at least 9 ML, as seen by MEED, is encountered for x≤0.7. For higher Fe concentrations and thicknesses greater than 4 ML, deviations from the layer-by-layer growth are observed, indicating a structural rearrangement. LEED-I(V) curves reveal the coexistence of two structural phases with different interlayer spacings, the relative amount of which depends on the composition. Magnetic properties were characterized by the magneto-optical Kerr effect (MOKE). The remanent magnetization was found to lie within the film plane over the whole range of thicknesses and concentrations investigated.A linear increase of the Kerr signal at saturation magnetization with increasing thickness indicates that practically the whole film is magnetic. As a function of composition, the saturation Kerr signal develops continuously with increasing Fe content. This suggests that in fcc FeCo alloys the contribution of Fe and Co to the total magnetic moment is nearly constant over the whole compositional range. © 1996 American Institute of Physics.