scholarly journals Light-induced structural changes of cytoskeleton in squid photoreceptor microvilli detected by rapid-freeze method.

1988 ◽  
Vol 106 (4) ◽  
pp. 1151-1160 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
G Matsumoto
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Physiological Role ◽  
Freeze Substitution ◽  
Photoreceptor Cells ◽  
Rapid Freezing ◽  
Light Stimulation

The cytoskeleton in squid photoreceptor microvilli was studied by freeze-substitution electron microscopy combined with rapid freezing using liquid helium, under dark-adapted and light-illuminated conditions. In the dark-adapted microvilli, actin filaments were regularly associated with granular structures on their surface; these granular structures were cross-linked to the rhodopsin-bearing plasma membranes through slender strands. Upon exposure to light, the granular components detached from the actin filaments, which then appeared to be fragmented and/or depolymerized. These observations have led us to conclude that light stimulation triggers the breakdown of the microvillar actin filament complex in squid photoreceptor cells. The results are discussed with special reference to the physiological role of actin filaments in photoreception.

scholarly journals Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes.

1981 ◽  
Vol 90 (1) ◽  
pp. 40-54 ◽  
Author(s):  
R L Ornberg ◽  
T S Reese
Keyword(s):  
Structural Changes ◽  
Secretory Granules ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Secretory Cells ◽  
Rapid Freezing ◽  
Cytoplasmic Filaments ◽  
Granule Membrane ◽  
Plasmalemma Invaginations

Structural changes underlying exocytosis evoked by the application of endotoxin to Limulus amebocytes were studied at the level of detail afforded by freeze-fracture and freeze-substitution techniques combined with the time resolution of direct rapid-freezing. The results with amebocytes prepared in this manner differed from those with other secretory cells prepared by conventional means. Exocytosis begins within seconds of endotoxin treatment when the plasmalemma invaginates to form pedestallike appositions with peripheral secretory granules. The juxtaposed membranes at these pedestal appositions form several punctate pentalaminar contacts, but examination of freeze-fractured pedestals failed to reveal any corresponding changes in the intramembrane particle distribution. Small secretory granule openings or pores, which are very infrequent, appear within the first 5 s after endotoxin treatment. These pores rapidly widen and this widening is immediately followed by the sequential dissolution of the granule contents, which then move into the surrounding extracellular space. Cytoplasmic filaments connecting the plasmalemma with the granule membrane are suitably deployed to be responsible for the plasmalemma invaginations. How pores begin is not certain, but the appearance of clear spaces between the granule core and the granule membrane at this point in exocytosis supports the possibility of a role of osmotic forces.

scholarly journals Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase.

1989 ◽  
Vol 37 (12) ◽  
pp. 1893-1898 ◽  
Author(s):  
H F Zoellner ◽  
N Hunter
Keyword(s):  
Alkaline Phosphatase ◽  
Physiological Role ◽  
Freeze Substitution ◽  
Differential Sensitivity ◽  
Rat Tissues ◽  
Microvascular Endothelium ◽  
Membrane Bound ◽  
Specific Inhibitors

Alkaline phosphatase (AP) is a widely studied membrane bound ecto-enzyme with an extensive distribution in nature. Three major human isoenzymes have been defined and can be distinguished on the basis of their differential sensitivity to specific inhibitors. Despite the voluminous literature describing AP, the physiological role of this enzyme is unclear. Microvascular endothelium is strongly AP positive and may provide a convenient model for study of the role of AP in vitro. This report describes the use of freeze-substitution and high-resolution plastic embedding techniques to identify the isoenzyme of endothelial AP by quantitative analysis of the relative inhibition by specific inhibitors of AP, using human gingival tissues and a number of rat tissues. Endothelial AP is found to be the liver/bone/kidney isoenzyme, indicating kidney as a credible source of enzyme for further experimental work investigating the role of AP.

Bacterial envelope profiles revealed by freeze-substitution

1992 ◽  
Vol 50 (1) ◽  
pp. 868-869
Author(s):  
L.L. Graham ◽  
T.J. Beveridge
Keyword(s):  
Electron Microscopy ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Chemical Fixation ◽  
Diverse Range ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Conventional Methods ◽  
Section Electron Microscopy ◽  
Better Than

Traditional methods of processing bacteria for thin section electron microscopy rely on chemical fixation and dehydration under conditions which maximize specimen deterioration. Cryotechniques, however, use physical fixation (rapid freezing) and are slowly being recognized as a superior alternative to the more conventional methods. Freeze-substitution is a cryotechnique which combines cryofixation with a gentle chemical fixation and dehydration regimen, yielding specimens amenable to standard embedment procedures and ultramicrotomy. Previous study has shown that freeze-substitution retains the molecular composition of eubacteria better than conventional methods of processing. In this study we extend our observations and show that a simple freeze-substitution protocol reliably preserves the ultrastructure of a diverse range of microorganisms including archaeobacteria and anaerobic eubacteria.

A novel role of fibroblast growth factor-2 and pentosan polysulfate in the pathogenesis of intestinal bleeding in mice

2007 ◽  
Vol 292 (2) ◽  
pp. H743-H750 ◽  
Author(s):  
Marina Jerebtsova ◽  
Edward Wong ◽  
Ronald Przygodzki ◽  
Pingtao Tang ◽  
Patricio E. Ray
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Structural Changes ◽  
Physiological Role ◽  
Intestinal Bleeding ◽  
Bleeding Disorders ◽  
Fibroblast Growth ◽  
Heparin Binding ◽  
Pentosan Polysulfate

Pentosan polysulfate (PPS) is a heparin-like polysaccharide that can affect the binding interactions of fibroblast growth factor (FGF-2) with its high-affinity receptors. Patients with angiogenic tumors frequently show high levels of FGF-2 in the circulation. Since FGF-2 is a heparin-binding angiogenic growth factor, PPS has been used successfully to block its activity in patients with angiogenic tumors. However, because of its heparin-like activity, the major toxic effect of PPS is the development of bleeding disorders. The role that circulating FGF-2 plays in the pathogenesis of bleeding disorders in patients treated with PPS is currently unknown. Here we hypothesized that FGF-2 might play a physiological role in the pathogenesis of intestinal bleeding induced by PPS. This hypothesis is supported by previous studies showing that PPS is accumulated in the intestine and that circulating FGF-2 specifically binds to and modulates the angiogenic activity of intestinal submucosal endothelial cells. We used recombinant adenoviral vectors carrying a secreted form of FGF-2 and LacZ control vectors to determine whether high levels of circulating FGF-2 facilitate the development of intestinal bleeding disorders in FVB/N and C57BL/6J mice treated with PPS. We found that PPS, acting together with FGF-2, induced structural changes in intestinal vessels leading to the development of lethal intestinal hemorrhages. These findings might have wider clinical implications for the systemic use of PPS and other heparinoids in the treatment of patients with angiogenic diseases associated with high levels of circulating FGF-2.

scholarly journals Rapid Freezing/Freeze Substitution Transmission Electron Microscopy Assessment of Cultured Endothelial Cell Glycocalyx Structure

2011 ◽  
Vol 17 (S2) ◽  
pp. 162-163
Author(s):  
E Ebong ◽  
F Macaluso ◽  
D Spray ◽  
J Tarbell
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endothelial Cell ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Transmission Electron

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

scholarly journals CALCIUM BINDING TO INTESTINAL MEMBRANES

1972 ◽  
Vol 55 (1) ◽  
pp. 58-73 ◽  
Author(s):  
James L. Oschman ◽  
Betty J. Wall
Keyword(s):  
Electron Microscopy ◽  
Calcium Binding ◽  
Plasma Membranes ◽  
Calcium Uptake ◽  
Divalent Cations ◽  
Cell Communication ◽  
Flame Photometry ◽  
Sodium Potassium ◽  
Buffer Solutions

Flame photometry reveals that glutaraldehyde and buffer solutions in routine use for electron microscopy contain varying amounts of calcium. The presence of electron-opaque deposits adjacent to membranes in a variety of tissues can be correlated with the presence of calcium in the fixative. In insect intestine (midgut), deposits occur adjacent to apical and lateral plasma membranes. The deposits are particularly evident in tissues fixed in glutaraldehyde without postosmication. They are also observed in osmicated tissue if calcium is added to wash and osmium solutions. Deposits are absent when calcium-free fixatives are used, but are present when traces of CaCl2 (as low as 5 x 10-5 M) are added. The deposits occur at regular intervals along junctional membranes, providing images strikingly similar to those obtained by other workers who have used pyroantimonate in an effort to localize sodium. Other divalent cations (Mg++, Sr++, Ba++, Mn++, Fe++) appear to substitute for calcium, while sodium, potassium, lanthanum, and mercury do not. After postfixing with osmium with calcium added, the deposits can be resolved as patches along the inner leaflet of apical and lateral plasma membranes. The dense regions may thus localize membrane constituents that bind calcium. The results are discussed in relation to the role of calcium in control of cell-to-cell communication, intestinal calcium uptake, and the pyroantimonate technique for ion localization.

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