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Biomedical subjects

D C Pease

Publications and source records attributed to D C Pease.

At least 19 recordsLinked to original sources
PubMed1983

Supramolecular aggregation and organization in peripheral nerve myelin.

Under certain preparative conditions the lipid bilayers of glutaraldehyde-fixed, PNS myelin demonstrate a marked compartmentalization, which can be augmented by lipid extraction following sectioning. The results are interpreted as indicating a supramolecular domain pattern of arrangement centered upon the transmembrane protein (P0) molecules. The latter are thought to be surrounded by annuli of substantially immobilized phospholipids. In the lamellar planes particular lipids are considered to have a nonrandom distribution. The visualization of bilayer compartmentalization was seen only in negatively stained sections obtained from unembedded or glutaraldehyde-urea-embedded myelin. Lipids were unextracted in the basic preparations except in so far as some unfixed, amphipathic molecules escaped at the trough-fluid interface at the time of sectioning, an observed phenomenon which probably aided in the visualization of the compartmentalization. Visualization was also augmented by surface tension expanding section fragments as they floated on the trough fluid. All stages of transition between well-ordered myelin and dispersed globular units were commonly to be found. Deliberately delipidated myelin exposed more sharply defined and smaller globular units in bilayer regions, but even these are regarded as being supramolecular aggregates including residual lipid annuli around the transmembrane proteins. The addition of cadmium ions as a "fixative" for lecithin seemed to improve the preservation of glutaraldehyde-urea-embedded myelin but was not strictly necessary to reveal its domain structure. A secondary tannic acid fixation was required to process unembedded myelin so as to reveal the fundamental compartmentalization of its lipid bilayers.

Animals↗
PubMed1983

Localization of antibody binding sites in ultrathin sections of unembedded frog retinal tissue.

Much ultrastructural detail is retained in tissue fixed only with aldehydes and subsequently air-dried after suspension in a polyvinyl acetate emulsion. The latter provides an external support only, but permits ultrathin sectioning; thus, an exposure of intracellular contents for potential immunocytochemical reactions is achieved. Sections of unembedded frog retina so prepared have been studied with success. The tissue was incubated first with a rabbit antiserum prepared against gradient purified bovine rod outer segments. Following incubation, reacted sites were labeled with ferritin-conjugated goat anti-rabbit IgG and stained with phosphotungstic acid. Intense labeling of the rod outer segments was clearly achieved, whereas the cone outer segments were without label. Other parts of the retina, including the ellipsoid region of both rods and cones, were also without significant label. These regions provided an intrinsic control for the specificity of the antiserum and established the validity of the general technique.

Animals↗
PubMed1977

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes.

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.

Animals↗
PubMed1977

The probable role of phosphatidyl cholines in the tannic acid enhancement of cytomembrane electron contrast.

Unsaturated natural and synthetic phosphatidyl cholines (PCs), when treated with tannic acid and OsO4, demonstrated a substantial increase in contrast as compared to PC treated only with OsO4. This was not observed when phosphatidyl ethanolamine (PEA) was similarly exposed to tannic acid. The increased electron density observed in the lamellar organization of the PC phospholipids was limited to the hydrophilic layers corresponding to the polar regions of the phospholipid molecules. The repeating periods of lamellae were identical in PC, treated with both tannic acid and OsO4, and when treated only with OsO4. In each case, this approximated 45 A. The enhancement of membrane contrast by tannic acid in the presence of OsO4 is interpreted as being at least in part due to its multivalent capacity, binding to reactive sites on choline, as well as with OsO4.

Cell Membrane↗
PubMed1976

Polysaccharides in lung alveoli.

Rat lung alveolar surfaces and contents were studied after using concanavalin A as a bifunctional agent to link exposed sugars to horseradish peroxidase, in accordance with a technique developed by Bernhard and Avrameas ('71). The Graham and Karnovsky ('69) diaminobenzidine procedure then was used to provide and electron-dense reaction product so as to define the distribution of complex carbohydrates in alveoli. A layer of very dense raction product was intimately associated with the outer leaflets of the luminal plasma membranes of type I and II pneumocytes. Masses of generally less dense reaction product extended irregularly into the alveolar coated by reaction product. When highly ordered tubular myelin bodies were seen, the reaction product filled all the "gutters" created by the intersentions of the membranes. Reasons are presented for believing that the intrinsic periodicity of the tubular myelin may be created and maintained by the domains ol technique. The distribution of autologous albumin, demonstrated by antibody staining by Bignon et al. ('75), apparently coincides with the carbohydrate pattern, suggesting both may be associated as a glycoprotein. The ultimate relationships between carbohydrate moieties and phospholipid membrane systems proved to be of such complexity that we believe it justifiable to think that they also may be truly complexed together.

Albumins↗
PubMed1975

Substructure in rod photoreceptor membranes.

Frog retinae, fixed only in buffered glutaraldehyde, were embedded for sectioning in glutaraldehyde polymerized with urea. In suitably thin sections globular substructures were seen in negative contrast after ionic staining with uranyl acetate and lead citrate, or after staining with neutralized phosphotungstic acid. Efforts to extract at least some of the lipid from sections before ionic staining enhanced the visualization of the "globules". Exposure to KMnO4 solution, used as an oxidative section stain, also outlined globular substructure in negative contrast, but with the additional feature that positively stained surface "leaflets" associated with the aqueous compartment were well defined. Staining sections with OSO4 vapor resulted in positively stained membranes, but without any evident substructure. However, when sections which previously had been exposed to OSO4 vapor were secondarily stained with uranyl acetate and/or lead citrate, positively stained globular substructures then were revealed. The globular substructures always were centered in the hydrophobic core region of the disc membranes, and symmetrically spanned the full thickness of this layer. The diameter of individual particles approximated 50-55 A. Reasons are presented for the supposition that the evident globules incorporate at least hydrophobic components of rhodopsin molecules. Findings are discussed in relation to various models of disc membrane organization that have been proposed in recent years.

Animals↗