Biomedical subjects

J Sedzik

Publications and source records attributed to J Sedzik.

13 recordsLinked to original sources

PubMed1992

Three-dimensional reconstruction of bovine intradural spinal root myelin by electron microscope tomography.

Electron microscope tomography was used to reconstruct three-dimensionally the configuration of heavy metal staining in bovine intradural spinal root myelin. Samples were fixed with glutaraldehyde, exposed to osmium tetroxide, embedded, thin sectioned, and finally stained with uranyl-acetate and lead citrate. Reconstructions up to 4.2 nm resolution showed a non-uniform distribution of stain in the planes of individual cytoplasmic appositions (major dense lines). In each reconstructed major dense line the stain was distributed in striated, well-defined structures. Those structures appear to be nearly parallel between neighboring major dense lines. The distribution of stain in the Schmidt-Lanterman cleft did not resemble the distribution of stain in the major dense line; however, weak striations were present. Evidence that the striated structures are not an artifact due to image calculation is discussed.

Animals↗

PubMed1992

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used convention strategies in our search for a suitable composition of a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization - homogeneity of tertiary structure--is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.

Ammonium Sulfate↗

PubMed1988

The three-dimensional structure of P2 myelin protein.

The three-dimensional structure of P2 protein from peripheral nervous system myelin has been determined at 2.7 A resolution by X-ray crystallography. The single isomorphous replacement/anomalous map was interpreted using skeletonized electron density on a computer graphics system. An atomic model was built using fragment fitting. The structure forms a compact 10-stranded up-and-down beta-barrel which encapsulates residual electron density that we interpret as a fatty acid molecule. This beta-barrel shows some similarity to, but is different from, the retinol binding protein family of structures. The relationship of the P2 structure to a family of cytoplasmic, lipid binding proteins is described.

Amino Acid Sequence↗

PubMed1988

Bovine P2 myelin basic protein crystallizes in three different forms.

P2 protein is a minor component of the myelin membrane. We have crystallized this protein for high-resolution crystallographic study. Three crystal morphologies are available. Two of them are from ammonium sulfate, and one is from polyethyleneglycol (PEG). The unit cell of the most suitable crystals from PEG 4000 has the dimensions a = 91.3 A, b = 99.8 A, c = 56.0 A; is of space group P2(1)2(1)2(1); and contains up to four molecules per asymmetric unit. The limit of resolution is 2.7 A.

Ammonium Sulfate↗

PubMed1987

Crystallization of P2 myelin protein.

Single crystals of bovine P2 myelin protein have been grown in polyethylene glycol 4000 by the hanging-drop vapor diffusion method. Crystals belonging to space group P2(1)2(1)2(1) with cell dimensions a = 91.8 A, b = 99.5 A, c = 56.5 A (1 A = 0.1 nm). The diffraction pattern extends to better than 2.3 A resolution.

Amino Acid Sequence↗

PubMed1985

Reconstituted P2/myelin-lipid multilayers.

A complex forms when bovine P2 protein is added to single-bilayer vesicles created by sonicating myelin lipids. The complex was studied by biochemical analysis, freeze-fracture (FF) and thin-section electron microscopy (EM), and by X-ray diffraction. Smaller amounts of P2 cause the vesicles to aggregate and fuse whereas larger amounts (greater than or equal to 4 wt%) cause multilayers to form. Binding saturates at 15 wt% P2. FF EM shows that large, flat multilayers form within 15 min of addition of P2. Only smooth fracture faces are seen, as expected for a peripheral membrane protein. X-ray diffraction shows a constant repeating distance in the multilayers: 86.0 +/- 0.7 A between the centers of bilayers in the range 4 wt% less than or equal to P2/(P2 + lipid) less than or equal to 15 wt%. Assuming a 53 A-thick bilayer, the space between bilayers is 33 A wide. This is a wider space than for myelin basic protein (MBP) (20-25 A wide). The respective widths are consistent with a compact, globular structure for P2 and a flattened shape for MBP. Calculated electron-density profiles of the lipids with and without P2 reveal the protein largely in the interbilayer spaces, with a small part possibly inserted into the lipid headgroup layers. The different proportions of P2 in the sciatic nerve of various species are tentatively correlated with the different average widths observed by X-ray diffraction for the cytoplasmic space (major period line) between bilayers in the respective sciatic myelins.

Animals↗

PubMed1978

Myelin lipids in the brain after transplacental intoxication with ethylnitrosourea.

At day 15 of gestation pregnant mice were injected with a single intravenous dose of ethylnitrosourea (ENU) (80 mg/kg of body weight). The lipid composition of the myelin fraction isolated by means of differential centrifugation from brains of their offsprings was studied. The transplacentally intoxicated mice developed severe changes in the lipid composition of their central myelin sheaths, such as a markedly decreased galactolipid (both cerebroside and sulfatide) as well as plasmalogen contents. The results show, that besides being a well known carcinogenic agent, ENU is capable of affecting the lipid metabolism of the developing central nervous system. The resultant effect is the formation of a chemically defective myelin sheath.

Animals↗

PubMed1978

The effect of transplacental intoxication with ethylnitrosourea on myelin proteins.

Pregnant mice at the 15th day of gestation were injected with a single intravenous dose of ENU/80 mg/kg of body weight). The protein composition of the myelin fraction isolated by means of differential centrifugation from brains of the offsprings was studied. The results obtained lead to the following conclusions: 1. The myelin protein spectrum of transplacentally intoxicated animals aged 40 days postnatal, shows decreased percentages of the Wolfgram protein and of the low molecular weight basic protein, whereas the proportions of the high molecular weight component of the myelin basic protein as well that of the Agrawal's protein are elevated. 2. The alterations in the profile of myelin proteins obtained from experimental mice aged 70 days postnatal are less severe and consist in a decline of the percentage of Wolfgrams protein and elevation of the Agrawal's protein content. 3. The intraplacental intoxication of mouse fetuses leads to development of animals defective with respect to the protein composition of the central myelin.

Age Factors↗

PubMed1976