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Biomedical subjects

N J Severs

Publications and source records attributed to N J Severs.

At least 145 records · Page 8Linked to original sources
PubMed1981

Plasma membrane cholesterol in myocardial muscle and capillary endothelial cells. Distribution of filipin-induced deformations in freeze-fracture.

Glutaraldehyde-fixed rabbit myocardium was treated with the polyene antibiotic, filipin, which binds specifically to sterols in membranes, producing distinct 25 nm-diameter deformations. These deformations (lesions) serve as labels for the presence of cholesterol-rich membranes and membrane domains. The distribution and density of filipin-induced lesions was examined in the plasma membranes of cardiac muscle and capillary endothelial cells by freeze-fracture electron microscopy. In the exposed fracture faces of endothelial plasma membranes, more than 99% of the surface area was covered with lesions, whereas about 90% of the plasma membrane area in the muscle cells remained unaffected by the treatment. Muscle cell plasma membranes revealed a patchy response; the lesions occurred in clusters which were separated by interconnecting expanses of unaffected membrane. Endothelial cell plasmalemmal vesicles, and muscle cell caveolae and transverse tubules displayed the same response to filipin as neighbouring areas of plasma membrane. However, gap junctions and desmosomes of the muscle intercalated disc membrane, and coated pits in both cell types were completely resistant to filipin treatment. These results provide evidence that the plasma membranes of the two cell types possess markedly different overall cholesterol levels, and that each has a characteristic pattern in the planar distribution of cholesterol.

Animals↗
PubMed1980

A simple technique for the examination of urothelial surface morphology by transmission electron microscopy: platinum-carbon replicas of critical point dried bladder tissue.

A simple technique is described for the examination of bladder luminal surface morphology by transmission electron microscopy (TEM). Pieces of bladder tissue are critical point dried and replicated with platinum-carbon in the manner standard for freeze-fracture. The large intact areas of replica produced show improved resolution of detail compared with scanning electron microscopy (SEM), and allow the survey of larger expanses of membrane than is possible with standard TEM techniques. Comparison of the structure observed in platinum-carbon replicas of critical point dried tissue with that seen in frozen-surface replicas, freeze-fracture replicas and thin-sections reveals the presence of artefacts in the critical point dried specimens which are not detected by SEM. Similar artefacts are evident in replicas of partially freeze-dried samples. The nature and origin of these artefacts are discussed. It is concluded that replicas of critical point dried specimens provide useful information on surface morphology at magnifications intermediate between those normally used with standard TEM and SEM techniques. The details of surface morphology viewed by this method are discussed with particular reference to the structure of the intercellular boundary.

Animals↗
PubMed1977

Frozen-surface replicas of rat bladder luminal membrane.

A method for preparing replicas of the luminal surface of frozen, unfractured but deep-etched whole bladder tissue using a Bullivant type II device is described. A small piece of glutaraldehyde-fixed (uncryoprotected) rat bladder is rinsed in distilled water, mounted luminal side uppermost on a specimen holder and rapidly frozen by immersion in liquid nitrogen (cooled below its boiling point in a vacuum) or by contact with a copper block at liquid nitrogen temperature. The specimen is processed in the type II device without fracturing and 'deep-etched' by allowing a longer period than usual to elapse before shadowing. The results are assessed with reference to the appearance of the luminal membrane in standard freeze-fracture replicas, and some preliminary observations on the structure of the normal luminal membrane and its counterpart in bladder tumours are presented.

Animals↗
PubMed1977

Nuclear envelope transport capacity and the cell cycle in yeast (Saccharomyces cerevisiae).

Changes in the nuclear envelope transport capacity, as measured by the number of nuclear pore complexes/unit nuclear volume/cell, were followed during the Saccharomyces cerevisiae cell cycle using data obtained by freeze-fracture electron microscopy. Pore number per unit nuclear volume decreased sharply in early G0, remained steady from mid-GO through S to G2, and showed a further slight decrease at M and G1. These periods of decline apparently resulted from nuclear enlargement without sufficient formation of new nuclear pore complexes to maintain the pore number to nuclear volume ratio. However, marked nuclear pore formation did accompany both increases in nuclear volume. The significance of these changes in relation to other events in the cell cycle is discussed. The validity of using nuclear pore number/unit nuclear volume and other pore number data as indices of nuclear envelope transport capacity and cell activity is critically examined.

Biological Transport↗
PubMed1976

Nuclear envelope inclusions demonstrated by freeze-fracture.

Inclusions in the perinuclear space of the nuclear envelope of human diploid (MRC-5) fibroblasts, limpet (Patella vulgata) haemocytes, and yeast (Saccharomyces cerevisiae) as observed with the freeze-fracture technique are described. The significance of these inclusions is discussed and it is tentatively concluded that they represent vesicles engaged in transporting macromolecules between nucleus and cytoplasm. Although the inclusions were infrequently observed, their demonstration in mammalian, invertebrate and lower eukaryotic cell types raises the possibility that this form of nucleocytoplasmic exchange may potentially be adopted under appropriate circumstances by the eukaryotic cell in general.

Animals↗
PubMedDate not supplied

The structure of the contractile apparatus in ultrarapidly frozen smooth muscle: freeze-fracture, deep-etch, and freeze-substitution studies.

The structure of the smooth muscle contractile apparatus was studied using ultrarapid freezing followed by freeze substitution or by longitudinal freeze-fracture, deep-etch, and platinum-carbon replication. Freeze substitution minimises the detrimental effects of chemical fixation and freeze fracture eliminates them entirely whilst revealing the ultrastructure in three dimensions. Unidirectionally shadowed freeze-fracture replicas of ultrarapidly frozen, relaxed, intact smooth muscle showed a well-preserved actin filament structure the 5.5-nm repeat of the actin subunits was clearly observed. In transversely fractured tissue the thick filaments were revealed, with a distribution comparable to that seen in transverse sections of freeze-substituted muscle. Relaxed muscle permeabilised using Triton X-100 showed a similar structure to that of intact tissue after ultrarapid freezing and examination both by freeze fracture and by freeze examination both by freeze fracture and by freeze substitution; the ratios of actin to myosin were also comparable. In permeabilised, rigorised tissue the structure of the actomyosin complex was revealed in detail; this was especially clear in freeze-substituted muscle. A cross-bridge spacing of 38 nm was measured in freeze-fractured, deep-etched tissue. The structural detail revealed is compatible with a side polar model of the actomyosin interaction and with the sliding filament mechanism of muscle contraction.

Actin Cytoskeleton↗
PubMedDate not supplied

Failure of filipin to detect cholesterol-rich domains in smooth muscle plasma membrane.

Using the cholesterol probe filipin and freeze-fracture electron microscopy, Montesano reported an apparent difference in cholesterol content between contiguous zones of the smooth muscle plasma membrane. In this membrane, microinvaginations termed caveolae are organized in band-like domains, which were highly sensitive to filipin (and therefore cholesterol-rich) compared with the surrounding non-caveolar inter-band regions (assumed to be cholesterol-poor). Using another cholesterol probe, the saponin tomatin, applied alone and after filipin treatment, we now present evidence that both the band and inter-band domains of the smooth muscle plasma membrane are cholesterol-rich. This leads to the conclusion that the lack of inter-band response to filipin is a false-negative cytochemical result.

Animals↗
PubMedDate not supplied

Inverted logic.

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Animals↗
PubMedDate not supplied

Gap junction remodeling and cardiac arrhythmogenesis: cause or coincidence?

Gap junctions, clusters of transmembrane channels that link adjoining cells, mediate myocyte-to-myocyte electrical coupling and communication. The component proteins of gap junction channels are termed connexins and, in in vitro expression systems, gap-junctional channels composed of different connexin types exhibit different biophysical properties. In common with other tissues, the heart expresses multiple connexin isoforms. Spatially defined patterns of expression of three connexin isoforms - connexin43, connexin40 and connexin45 - form the cell-to-cell conduction pathways responsible for the orderly spread of current flow that governs the normal cardiac rhythm. Remodeling of gap junction organization and connexin expression is a common feature of human heart disease conditions in which there is an arrhythmic tendency. This remodeling may take the form of disturbances in the distribution of gap junctions and/or quantitative alterations in connexin expression, notably reduced ventricular connexin43 levels. The idea that such changes may contribute to the development of a pro-arrhythmic substrate in the diseased heart has gained ground over the last decade. Recent studies using transgenic mice models have raised new opportunities to explore the significance of gap junction remodeling in the diseased heart.

Animals↗
PubMedDate not supplied

Display of low density lipoprotein receptors is clustered, not dispersed, in fibroblast and hepatocyte plasma membranes.

Although the principal details of low density lipoprotein (LDL) uptake by receptor-mediated endocytosis and its subsequent intracellular fate have been thoroughly investigated, an aspect of this mechanism that continues to provoke controversy concerns the manner of display of LDL receptors upon their initial insertion at the cell surface. While our studies based on electron microscopy of platinum/carbon replicas of gold-labeled cells have previously suggested a clustered display pattern, others have concluded, before and since, that LDL receptors are inserted individually at random widely dispersed sites in the plasma membrane. In this article, we present a series of experiments designed to discriminate between these competing hypotheses. In addition to the use of LDL-colloidal gold complexes, visualized electron microscopically, on cells subjected to a variety of experimental procedures, these experiments include the application of anti-apolipoprotein B-100 antibodies, anti-LDL-receptor antibodies, and direct visualization of native (unlabeled) LDL molecules at the cell surface. All results point to a loose-cluster arrangement, not one involving widely dispersed individual units, as the initial display pattern of newly inserted LDL receptors. A comparison of LDL and beta-very low density lipoprotein receptor distribution in fibroblasts and hepatocytes suggests that this cluster pattern is a characteristic of the LDL (apolipoprotein B/E) receptor across cell types, but that the closely related apolipoprotein E receptor differs in that it is inserted individually in a highly dispersed state, in common with a variety of other receptor types.

Animals↗